A MODIFIED YEAST ONE-HYBRID SYSTEM TO INVESTIGATE …...A modified yeast one-hybrid (MY1H) system has been developed for . in vivo. investigation of simultaneous protein-protein and

A MODIFIED YEAST ONE-HYBRID SYSTEM TO INVESTIGATE PROTEIN-PROTEIN AND PROTEIN:DNA

INTERACTIONS

by

Gang Chen

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Graduate Department of Chemistry University of Toronto

© Copyright by Gang Chen 2008

ii

A MODIFIED YEAST ONE-HYBRID SYSTEM TO INVESTIGATE

PROTEIN-PROTEIN AND PROTEIN:DNA INTERACTIONS

Gang Chen

Doctor of Philosophy

Graduate Department of Chemistry University of Toronto

2008

Abstract A modified yeast one-hybrid (MY1H) system has been developed for in vivo

investigation of simultaneous protein-protein and protein:DNA interactions. The traditional yeast

one-hybrid assay (Y1H) permits examination of one expressed protein targeting one DNA site,

whereas our MY1H allows coexpression of two different proteins and examination of their

activity at the DNA target. This single-plasmid based MY1H was validated by use of the DNA-

binding protein p53 and its inhibitory partners, large T antigen (LTAg) and 53BP2. The MY1H

system could be used to examine proteins that contribute inhibitory, repressive, coactivational or

bridging functions to the protein under investigation, as well as potential extension toward

library screening for identification of novel accessory proteins.

After development and validation of the MY1H with the p53/LTAg/53BP2 system, we

applied the MY1H system to investigate the DNA binding activities of heterodimeric proteins,

the bHLH/PAS domains of AhR and Arnt that target the xenobiotic response element (XRE).

The AhR/Arnt:XRE interaction, which served as our positive control for heterodimeric protein

binding of the XRE DNA site, showed negative signals in initial MY1H experiments. These false

negative observations were turned into true positives by increasing the number of DNA target

iii

sites upstream of the reporter genes and increasing the number of activator domains fused to the

two monomers. This methodology may help trouble-shooting false negatives stemming from

unproductive transcription in yeast genetic assays, which can be a common problem.

In the study of XRE-binding proteins, two bHLHZ-like hybrid proteins, AhRJunD and

ArntFos were designed and coexpressed in the MY1H and yeast two-hybrid (Y2H) systems;

these proteins comprise the bHLH domains of AhR and Arnt fused to the leucine zipper (LZ)

elements from bZIP proteins JunD and Fos, respectively. The in vivo assays revealed that in the

absence of the XRE DNA site, heterodimers and homodimers formed, but in the presence of the

nonpalindromic XRE, only heterodimers bound to the XRE and activated reporter transcription.

The present results provide valuable information on DNA-mediated protein heterodimerization

and specific DNA binding, as well as the relationship between protein structure and DNA-

binding function.

iv

Acknowledgments First and Foremost I would like to express my sincere appreciation to Dr. Jumi Shin for

giving me the right amount of freedom and guidance during my graduate studies at UofT. Your

dedication, encouragement and enthusiasm served as a constant source of inspiration.

I would like to express my gratitude to the rest of my Supervisory Committee Dr.

Deborah Zamble and Dr. Ulrich Krull for their involvement in my research and their advice that

helped to shape my research skills. I am very thankful to Dr. Erica Golemis (University of

Pennsylvania) for being my external reviewer, and Dr. Andrew Woolley, Dr. Patrica Harper, and

Dr. Mark Nitz for sitting on my Examination Committee, critical reading of my thesis and

offering useful suggestions.

I would like to thank Dr. Kuniyoshi Iwabuchi (Kanazawa Medical University, Japan) and

Dr. Stanley Fields (University of Washington) for kindly providing human 53BP2 cDNA, and Dr.

Patricia Harper and Dr. Allan Okey (University of Toronto) for kindly providing human AhR

and Arnt cDNA. These gifts greatly helped the progress of my thesis work and eventually made

my thesis possible.

I would like to thank all the past and present members of Shin group for providing

invaluable help, for both experimental work and discussion, in my thesis work. They are Dr.

Adrian Schwartz, Dr. Jing Xu, Ms. Lisa Denboer, Ms. Antonia De Jong, Ms. Joanna Chow, Dr.

Christopher Damaso, Ms. Cassie Ho, Dr. Anna Fedorova, Ms. Alevtina Pavlenco, Ms. I-San

Chan, and Mr. Hesam Shahravan. Special thanks to Dr. Adrian Schwartz, who introduced me to

the magic world of yeast genetic reporter systems, and to Ms. Antonia De Jong, who is now

working on the downstream work of Chapter 5 as well as some interesting ideas I would

otherwise love to try.

v

The thesis work was funded by National Institutes of Health (R01 GM069041), Premier's

Research Excellence Award (PREA), Canadian Foundation for Innovation/Ontario Innovation

Trust (CFI/OIT), Natural Sciences and Engineering Research Council of Canada (NSERC)

Discovery Grant, and the University of Toronto.

I would like to express my earnest gratitude to my parents for their constant support and

love, without which any of my achievements would not have been possible. I am deeply indebted

to my dear wife Ying for her love and understanding, as well as her unconditional support right

behind me. Finally thanks to our son, Bernie, for the joy and the happiness he brings to us all the

time. Sharing my life with them has been a wonderful experience and I look forward to our many

more years to come.

Toronto, August 2008

Gang Chen

vi

Table of Contents Abstract ........................................................................................................................................... ii

Acknowledgments.......................................................................................................................... iv

vi

xii

xiii

xvi

1

1

1

4

6

7

9

14

15

17

19

20

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Table of Contents...........................................................................................................................

List of Tables ................................................................................................................................

List of Figures ..............................................................................................................................

List of Abbreviations ...................................................................................................................

Chapter 1 Introduction ....................................................................................................................

1.1 Yeast genetic reporter assays ..............................................................................................

1.1.1 Yeast two- and one-hybrid systems ........................................................................

1.1.2 Modified yeast genetic reporter systems for detection of simultaneous protein-

protein and protein:DNA interactions..................................................................... 3

1.1.3 Advantages and disadvantages of yeast genetic reporter systems ..........................

1.2 Transcription factors ...........................................................................................................

1.2.1 The bZIP family......................................................................................................

1.2.2 The bHLH superfamily ...........................................................................................

1.2.3 Heterodimeric transcription factors increase regulatory diversity........................

1.3 Studies of bHLH superfamily proteins in yeast genetic reporter systems ........................

1.4 Scope.................................................................................................................................

Chapter 2 Design of a single plasmid-based modified yeast one-hybrid system for

investigation of in vivo protein-protein and protein:DNA interactions ...................................

2.1 Abstract .............................................................................................................................

2.2 Introduction.......................................................................................................................

vii

2.3 Materials and Methods...................................................................................................... 23

23

24

24

27

28

28

28

29

33

33

34

37

Chapter 3 AhR/Arnt:XRE interaction: turning false negatives into true positives in the

modified yeast one-hybrid assay.............................................................................................. 38

39

40

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42

42

43

2.3.1 Bacterial and yeast strains.....................................................................................

2.3.2 Transformation, DNA preparation, and plasmid rescue .......................................

2.3.3 Plasmid Construction ............................................................................................

2.3.4 3-AT titration analysis ..........................................................................................

2.3.5 X-gal colony-lift filter assay and ONPG liquid assay ..........................................

2.4 Results...............................................................................................................................

2.4.1 Design of protein expression vectors....................................................................

2.4.2 Coexpression of LTAg or 53BP2 decreases the transactivation potential of

GAL4AD-p53 in the MY1H.................................................................................

2.5 Discussion .........................................................................................................................

2.5.1 The DNA-binding activity of p53 and its interaction with LTAg or 53BP2:

comparison of MY1H observations with earlier studies.......................................

2.5.2 Different types of interactions between two proteins and a DNA target can be

examined in our MY1H system ............................................................................

2.6 Supplemental Information ................................................................................................

3.1 Abstract .............................................................................................................................

3.2 Introduction.......................................................................................................................

3.3 Materials and methods ......................................................................................................


3.3.2 Construction of reporter strains ............................................................................


viii

3.3.4 Plasmid Construction ............................................................................................ 43

45

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3.3.5 HIS3 reporter assay ...............................................................................................

3.3.6 LacZ reporter assay ...............................................................................................

3.4 Results...............................................................................................................................

3.4.1 Initial trials with pCETT: fusion of the GAL4 AD to NAhR only.......................

3.4.2 Next generation trials with pCETT2: fusion of the GAL4 AD to both NAhR

and NArnt..............................................................................................................

3.5 Discussion .........................................................................................................................

3.5.1 Are false negatives due to unproductive transcription?........................................

3.5.2 Copy number of XRE target sites .........................................................................

3.5.3 Double AD system................................................................................................

3.5.4 Synergistic activation is due to both increase of XRE target sites and doubling

of AD ....................................................................................................................


Chapter 4 Forced protein heterodimerization and specific DNA binding to a nonpalindromic

DNA sequence in vivo and in vitro: bHLHZ-like hybrid heterodimers of bHLH/PAS

proteins AhR and Arnt and bZIP proteins JunD and Fos as a model ......................................

4.1 Abstract .............................................................................................................................

4.2 Introduction.......................................................................................................................

4.3 Materials and methods ......................................................................................................



4.3.3 Plasmid Construction ............................................................................................

4.3.4 HIS3 reporter assay ...............................................................................................

ix

4.3.5 LacZ reporter assay ............................................................................................... 64

64

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4.3.6 Protein-protein interactions in the Y2H system....................................................

4.4 Results...............................................................................................................................

4.4.1 The AhRJunD/ArntFos and AhR(ΔL)JunD/ArntFos heterodimers bind the

XRE site in the MY1H..........................................................................................

4.4.2 Heterodimerization between two monomers confirmed in the Y2H assay ..........

4.5 Discussion .........................................................................................................................

4.5.1 Fusion of the JunD and Fos LZ domains to the Arnt and AhR bHLH domains

reconstitutes the heterodimeric structure and specific DNA-binding function

of the bHLH/PAS domains of AhR and Arnt .......................................................

4.5.2 The nonpalindromic XRE DNA target mediates the heterodimerized structure

of AhRJunD and ArntFos, resulting in DNA-binding function............................

4.5.3 What causes the negative signal in XRE binding by the bHLH domains of

AhR and Arnt in the MY1H?................................................................................

4.5.4 Deletion of one leucine residue in the JunD LZ negatively affects

heterodimerization but not DNA binding .............................................................

4.5.5 Our domain swapping experiments support the hypothesis of domain shuffling

in the evolutionary pathway of the bHLH superfamily ........................................


Chapter 5 Summary and future work............................................................................................

5.1 Summary ...........................................................................................................................

5.2 Future work.......................................................................................................................

5.2.1 Downstream work on the AhRJunD/ArntFos:XRE interaction............................

5.2.2 Improvement on the MY1H system......................................................................

x

5.2.3 Other DNA-binding hybrid proteins of interest.................................................... 89

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Chapter 6 Materials and Methods .................................................................................................

6.1 General ..............................................................................................................................

6.1.1 Two reporter assays used in the MY1H................................................................

6.1.2 Examination of protein-protein interactions in the Y2H system ..........................

6.2 Modified Y1H for identification of protein-protein/protein:DNA interactions................

6.2.1 Construction of p53 target-reporter strains ...........................................................

6.2.2 Plasmid construction.............................................................................................

6.2.3 3-AT titration analysis ..........................................................................................

6.3 Turning false negatives into true positives in the MY1H.................................................

6.3.1 Construction of XRE target-reporter strains .........................................................


6.4 DNA binding forces heterodimerization of hybrids of bHLH/PAS and bZIP..................


References.....................................................................................................................................

Appendix A Design of a single plasmid-based modified yeast one-hybrid system for

investigation of in vivo protein-protein and protein:DNA interactions .................................

A.1 Western blot analysis ......................................................................................................

A.2 Expression levels of LTAg and 53BP2 in the MY1H system ........................................

A.3 Expression of the AD-fusion protein from MCS I is not affected by expression from

MCS II ............................................................................................................................

Appendix B AhR/Arnt:XRE interaction: turning false negatives into true positives in the

modified yeast one-hybrid assay............................................................................................

B.1 Construction of reporter strains ......................................................................................

xi

B.1.1 Three-copy strains............................................................................................... 112

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B.1.2 Six-copy strains...................................................................................................

B.2 Construction of AhR6-436 (NAhR) fragment ................................................................

B.3 Construction of Arnt82-464 (NArnt) fragment...............................................................

Appendix C Forced protein heterodimerization and specific DNA binding to a

nonpalindromic DNA sequence in vivo and in vitro: bHLHZ-like hybrid heterodimers of

bHLH/PAS proteins AhR and Arnt and bZIP proteins JunD and Fos as a model.................

C.1 Plasmid Construction ......................................................................................................

C.1.1 pCETT2/AhRbHLH/ArntbHLH.........................................................................

C.1.2 pCETT/AhRJunD/ArntFos and pCETT2/AhRJunD/ArntFos ............................

C.1.3 pCETT2/AhR(ΔL)JunD/ArntFos........................................................................

C.1.4 pGBKT7/AhRbHLH and pGADT7/AhRbHLH.................................................

C.1.5 pGBKT7/ArntbHLH and pGADT7/ArntbHLH .................................................

C.1.6 pGBKT7/AhRJunD and pGADT7/AhRJunD.....................................................

C.1.7 pGBKT7/AhR(ΔL)JunD and pGADT7/AhR(ΔL)JunD .....................................

C.1.8 pGBKT7/ArntFos and pGADT7/ArntFos ..........................................................

C.2 Expression of GAL4AD-AhRJunD and ArntFos by use of pCETT in the MY1H ........

Appendix D Table of Oligonucleotides...................................................................................

xii

List of Tables Table 2.1 Oligonucleotides used in this study............................................................................. 25

74

109

112

116

122

Table 4.1 Dimerization of AhR- and Arnt-hybrid proteins.........................................................

Table A.1 Reporter activation of GAL4AD fusions of p53 or Max expressed from different

vectors .........................................................................................................................................

Table B.1 Oligonucleotides used in this study..........................................................................

Table C.1 Oligonucleotides used in this study..........................................................................

Table D.1 Oligonucleotides used in this thesis .........................................................................

xiii

List of Figures Figure 1.1 Schematic representation of Y2H and Y1H systems.................................................... 1

8

11

12

21

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Figure 1.2 Crystal structure of the GCN4 bZIP homodimer bound to the AP-1 DNA site ..........

Figure 1.3 Crystal structure of the MyoD:DNA complex (A) and Max:DNA complex (B) ......

Figure 1.4 Ribbon presentation of the Drosophila Period dimer................................................

Figure 2.1 Five different types of interactions between proteins and DNA can be detected with

the MY1H system .........................................................................................................................

Figure 2.2 Plasmids pCETT and pCETF were constructed for coexpression of two proteins in a

yeast model system .......................................................................................................................

Figure 2.3 3-AT titrations reveal that the survival rates of transformants decreases when the

inhibitory proteins are expressed ..................................................................................................

Figure 2.4 Colony-lift filter assay indicates LTAg and 53BP2 inhibit DNA binding of p53 to

different extents ............................................................................................................................

Figure 2.5 Histogram comparing the effects of different expression levels of inhibitory proteins

LTAg and 53BP2 on DNA binding by p53 ..................................................................................

Figure 3.1 Plasmid pCETT2 for coexpression of two AD fusion proteins in the MY1H system

.......................................................................................................................................................

Figure 3.2 HIS3 reporter assay for detection of NAhR/NArnt:XRE interaction from protein

expression vector pCETT or pCETT2 in YM4271[pHISi-1/XRE-3] (A and C) and

YM4271[pHISi-1/XRE-6] (B and D) strains................................................................................

Figure 3.3 Colony-lift filter assay for detection of NAhR/NArnt:XRE interaction from protein


YM4271[pHISi-1/XRE-6] (B and D) strains................................................................................

xiv

Figure 3.4 ONPG assay for detection of NAhR/NArnt:XRE interaction from protein expression

vector pCETT or pCETT2 in YM4271[pHISi-1/XRE-3] (A and C) and YM4271[pHISi-1/XRE-6]

(B and D) strains ........................................................................................................................... 48

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68

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73

105

107

115

Figure 4.1 Primary sequence alignment of the native Max bHLHZ domain and bHLH•LZ

hybrids...........................................................................................................................................

Figure 4.2 HIS3 reporter assay for detection of interactions between heterodimers and the XRE

cognate sequence ..........................................................................................................................

Figure 4.3 HIS3 reporter spot titration assay for comparison of the relative strengths of

AhRJunD/ArntFos:XRE and AhR(ΔL)JunD/ArntFos:XRE interactions.....................................

Figure 4.4 Colony-lift filter assay for the detection of interactions between heterodimers and the

XRE cognate sequence .................................................................................................................

Figure 4.5 ONPG measurements for detection of AhRJunD/ArntFos:XRE and

AhR(ΔL)JunD/ArntFos:XRE interactions by use of protein expression vector pCETT2 in strain

YM4271[pHISi-1/XRE-6] ............................................................................................................

Figure 4.6 Y2H assay of homo- and heterodimerization of AhR and Arnt hybrid proteins.......

Figure A.1 Plasmids pCETT (truncated ADH1 promoter) and pCETF (full-length ADH1

promoter) were constructed for coexpression of two proteins in a yeast model system ............

Figure A.2 Promoter length dictates differential expression levels of inhibitory proteins when

YM4271[p53HIS] cells were transformed with the indicated plasmids and grown to exponential

phase in YPDA media.................................................................................................................

Figure B.1 Colony-lift filter assay for detection of NAhR/NArnt:XRE interaction from protein


YM4271[pHISi-1/XRE-6] (B and D) strains..............................................................................

xv

Figure C.1 The HIS3 reporter assay for detection of the AhRJunD/ArntFos:XRE interaction by

use of protein expression vector pCETT in strain YM4271[pHISi-1/XRE-6] ........................... 120

120

121

Figure C.2 The X-gal colony-lift filter assay............................................................................

Figure C.3 ONPG measurements for detection of AhRJunD/ArntFos:XRE interactions by use

of protein expression vector pCETT in strain YM4271[pHISi-1/XRE-6] .................................

xvi

List of Abbreviations Abbreviation Full Name

3-AT 3-amino-1,2,4-triazole

AD activation domain

AhR aryl hydrocarbon receptor

AhRJunD AhRbHLH20-86-JunD296-332 fusion

AhR(ΔL)JunD AhRbHLH20-86-JunD297-332 fusion

Arnt AhR nuclear translocator

ArntFos ArntbHLH82-148-Fos165-201 fusion

ARRE2 antigen receptor response element

bHLH basic helix-loop-helix

bHLH/PAS basic helix-loop-helix/Per-Arnt-Sim

bHLHZ basic helix-loop-helix/leucine zipper

bZIP basic region/leucine zipper

CD circular dichroism

DBD DNA binding domain

DBP DNA-binding protein

DR dioxin receptor

DRE dioxin response element

E-box enhancer box

GAL4AD GAL4 activation domain

GAL4DBD GAL4 DNA-binding domain

GFP green fluorescent protein

HIF hypoxia-inducible factor

Hsp90 heat shock protein 90

HTH helix-turn-helix

Kd dissociation constant

LTAg large T antigen

LZ leucine zipper

mAb monoclonal antibody

xvii

Abbreviation Full Name

Max Myc-associated factor X

MCS multiple cloning site

MRF muscle regulatory factor

MY1H modified yeast one-hybrid

NAhR human AhR6-436 fragment

NArnt human Arnt82-464 fragment

NMR nuclear magnetic resonance

ONPG ortho-nitrophenyl-galactoside

PAS Per-Arnt-Sim

PCB polychlorinated biphenyl

Per Period protein

PMSF phenylmethylsulfonyl fluoride

SD minimal synthetic dropout medium

SEM standard error measurement

Sim single-minded protein

S/N signal-to-noise ratio

SRE serum response element

SRF serum response factor

SURE Stop Unwanted Rearrangement Events

SV40 Simian Virus 40

TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin

VDR vitamin D receptor

XRE xenobiotic response element

Y1H yeast one-hybrid

Y2H yeast two-hybrid

1

Chap 1ter Introduction

1.1 Yeast genetic reporter assays

1.1.1 Yeast two- and one-hybrid systems Yeast two-hybrid (Y2H) systems are genetic assays that allow identification of novel

protein-protein interactions, confirmation of suspected interactions, and definition and mapping

of interacting domains (1-4). Y2H systems, as well as variant yeast one-hybrid (Y1H) systems,

exploit the modular nature of transcription factors, which typically comprise discrete DNA

binding domains (DBD) and activation domains (AD) (1). In Y2H systems, one protein of

interest “X” is expressed as a fusion with the DBD, which serves as the bait, while the other

protein “Y” is expressed as a fusion to the AD serving as the prey. When both chimeric proteins

are coexpressed and localized to the nucleus, interaction between the bait and the prey brings the

DBD and AD into proximity, thereby reconstituting the functional transcription factor, and

reporter gene expression is activated (Figure 1.1A).

Figure 1.1 Schematic representation of Y2H and Y1H systems. A) In Y2H systems, the bait “X” is fused to a DBD, which targets specific DNA binding sites in the promoter region upstream of reporter genes such as HIS3 and lacZ. The prey “Y” is fused to an AD. B) In Y1H systems, the DNA element E placed upstream of reporter genes serves as the bait; the DNA-binding protein (DBP), being the prey, is expressed as a fusion with the AD. Reporter gene expression is activated when the AD-fused DBP interacts with the specific DNA element E.

Y1H systems are genetic assays for isolation of novel genes encoding proteins that bind

to a target, cis-acting regulatory element and for further characterization of known protein:DNA

2

interactions (4-6). In Y1H systems, the bait is not a protein, but a DNA element placed upstream

of reporter genes such as lacZ and HIS3; the protein of interest, expressed as a fusion with an AD,

serves as the prey. Reporter gene expression is activated when the AD-fused protein interacts

with the specific DNA element upstream of the reporter gene (Figure 1.1B).

Over the years of development, yeast genetic reporter systems have undergone

considerable modifications and modernizations to improve the overall performance of their

utilization, therefore allowing greater robustness and flexibility, and/or greatly expanding their

applicable scope. Classic yeast genetic reporter systems commonly utilize a DBD derived from

GAL4 or LexA, and AD from GAL4, B42, or the stronger VP16. The earliest systems developed

were usually based on one reporter gene, lacZ; subsequently, growth selection markers, such as

HIS3, LEU2, and URA3 were introduced. In order to increase the sensitivity of these systems,

multiple reporter genes under the control of different promoters were implemented into one

single system, which has been proven to enable detection of weak interactions and efficiently

eliminate false positives (7,8). In addition to the aforementioned traditional reporter genes,

counter-selectable reporters such as CYH2 or URA3 can also be used to detect de novo

autoactivators to minimize false positive interactions or for functional analysis by dissociation of

protein-protein or protein:DNA interactions (8-10). Other than lacZ, the reporter green

fluorescent protein (GFP) protein was also applied in both the Y2H and Y1H systems (11-13).

The application of GFP in yeast genetic reporter systems simplifies and accelerates the

identification and screening process of putative interactions, as well as increases the detection

sensitivity.

Moreover, several systems have been developed to detect protein-protein and/or

protein:DNA interactions occurring outside the yeast nucleus. For instance, split-GFP systems, in

which the two halves of GFP are fused to the two proteins of interest and upon interaction,

3

reconstitute functional GFP, were applied to Y2H systems to localize interactions in living cells,

which might be used to detect the compartment where the protein-protein interaction occurs in

yeast (14). In order to circumvent problems associated with the analysis of membrane proteins in

classic Y2H systems, split-ubiquitin systems were developed which allow the detection of

interactions of membrane proteins (15,16). In the split-ubiquitin systems, a functional ubiquitin

is reconstituted by the interaction between the two proteins of interest fused to two halves of

ubiquitin, respectively, and then recognized by endogenous ubiquitin-specific proteases, leading

to release of the originally attached artificial transcription factor. The released transcription

factor thereby enters the nucleus and activates reporter transcription.

In addition to straightforward protein-protein and protein:DNA interactions, yeast genetic

reporter systems have been modified to explore much broader interactions such as multiprotein

interactions, multiprotein:DNA interactions, protein:RNA interactions, and interactions between

proteins and small molecules (for a recent comprehensive review on yeast hybrid approaches by

Golemis and coworkers, see ref. (17)). Because of the scope of this thesis, we will only discuss

in detail the study of simultaneous protein-protein and protein:DNA interactions by use of yeast

genetic reporter systems.

1.1.2 Modified yeast genetic reporter systems for detection of simultaneous protein-protein and protein:DNA interactions

Several approaches that combine features of the Y1H and Y2H have been reported for

investigation of simultaneous protein-protein and protein:DNA interactions. These approaches

enable identification of a protein in complex with a known partner, thereby forming a

heterodimeric complex that binds DNA; these complexes may include one partner lacking

intrinsic DNA-binding capability that is only enabled in the presence of an accessory protein. A

modified Y1H system was developed to identify SAP-1, which binds the c-fos serum response

4

element (SRE) only when complexed with serum response factor (SRF) (18). This system was

later used to isolate BETA2, a novel member of the basic helix-loop-helix (bHLH) superfamily

that heterodimerizes with the ubiquitous bHLH protein E47, where the heterodimer binds to the

insulin enhancer box (E-box) sequence with high affinity and is important for the regulation of

insulin genes (19). The same concept was applied to investigate the trimer complex

NFAT/Jun/Fos, which targets the upstream antigen receptor response element (ARRE2) located

in an enhancer region that controls antigen-dependent induction of the interleukin 2 gene (20,21).

Another approach was developed to simultaneously identify proteins that bind to the ftz proximal

enhancer element and cofactors that directly interact with the Ftz protein (22). This approach was

also used to isolate BAF60a, a mammalian protein capable of interacting with the Vitamin D

receptor (VDR) heterodimer complex while driving expression from a repressor VDR element

(23).

1.1.3 Advantages and disadvantages of yeast genetic reporter systems There are several advantages of using yeast genetic reporter systems to investigate

protein-protein or protein:DNA interactions. First, an obvious advantage of using yeast genetic

reporter systems over classic biochemical or other genetic methods is that they are in vivo

techniques that use the yeast cell as a living test tube. The eukaryotic yeast host bears a greater

resemblance to higher eukaryotic systems than does bacteria, while less likely to have

endogenous interference issues than higher eukaryotic hosts. Second, the yeast machinery can

provide post-translational modifications required by eukaryotic protein-protein or protein:DNA

interactions. Third, yeast genetic reporter systems can be used to detect weak or transient

interactions important in signaling pathways. In some cases, transient interactions not stable

under the in vitro conditions of immunoprecipitation are able to display a transcriptional

response in vivo in yeast genetic reporter systems (24). Fourth, yeast assays do not require the

5

purification of the proteins under investigation as only the cDNA of the gene of interest is

needed, which is in contrast to classic biochemical approaches that require either high quantities

of purified proteins and/or good quality antibodies. Last, although these yeast genetic assays do

not provide direct, quantitative measurement of the strength of interactions under investigation,

the transcriptional readouts of reporter activation generally correlate with the strength of the

interactions as determined in vitro, therefore permitting discrimination of interactions with

different affinities (25).

However, various factors lead to the observation of false positives and false negatives,

which are commonly observed in yeast genetic reporter assays. Interference from endogenous

proteins may cause either false positives or false negatives. Autoactivator and some “sticky” or

“promiscuous” proteins are common sources of false positives. In addition, as the proteins under

investigation must be fused to the DBD or AD in the reporter systems, some physically

interactive, yet physiologically irrelevant, interactions might also be detected (26).

Recent explorations of protein-protein and protein:DNA interactions at genome- or

pathway-wide scale by use of yeast reporter systems have shown the frequent occurrence of false

negatives that had otherwise been severely underestimated (26,27). Factors that lead to false

negatives generally include weak interactions beyond detection limitations, proteins unstably

expressed and/or improperly folded in the host cell and/or unable to localize to the nucleus,

toxicity caused by heterologous expression of some hybrid proteins, and structural hindrance by

fused domains or epitope tags (26,28-30). Such issues with in vivo systems necessitate

confirmation of results by other independent methods.

Although there are certain disadvantages involved in the use of yeast genetic reporter

systems, the relative ease of manipulation, high efficiency and superior efficacy of these systems

have already made their use widespread. These genetic systems will surely continue to be useful

6

methods for investigation of the assembly and specificity of a variety of protein-protein and

protein:DNA interactions that provide the basis for further understanding of cell function.

1.2 Transcription factors Transcription factors are a large and diverse class of DNA-binding proteins that

specifically target DNA for transcriptional regulation of gene expression. They play a critical

role in the control of important physiological functions including cell development, growth, and

differentiation. Thus, aberrant expression or incorrect processing of transcription factors

contributes to the progression of a variety of diseases, including developmental abnormalities

and cancer (31).

Transcription factors are typically composed of an autonomous DBD responsible for

directing the protein to a specific DNA target site, and an effector domain mediating activation

or repression of targeted genes. In general, transcription factors function by binding to specific

DNA sequences located upstream of the gene promoter region in chromatin and induce or

repress gene expression by recruitment of appropriate global regulatory complexes including

RNA polymerase II, basal transcription factors, and other cofactors bound at the gene promoter

(31,32).

Because the DBD of transcription factors determines which promoter sequences are

bound and therefore, which genes are regulated, much attention has been focused on deciphering

the basis of protein:DNA recognition at the molecular level. Tremendously valuable information

on how different transcription factors bind their cognate DNA targets has been revealed with

advances in genetic and biochemical techniques. Particularly, the structures of protein:DNA

complexes determined by both X-ray and nuclear magnetic resonance (NMR) provide clues for

understanding the mechanism of protein:DNA recognition. However, analysis of the available

7

functional and structural data on DNA binding by transcription factors has shown that there is no

clear “recognition code” that underlies protein:DNA interactions (33-35).

Although no general rules exist in protein:DNA recognition, transcription factors can still

be grouped into families based on sequence and structural homologies within their DBDs. These

families include helix-turn-helix (HTH), zinc finger, basic region/leucine zipper (bZIP), bHLH,

and homeodomain (36,37). Of all these DNA binding proteins, the simple α-helix scaffold is

usually the common secondary structure untilized to bind to the DNA major groove (38). Several

excellent reviews summarize the general scaffolds that proteins use to recognize DNA and

discuss in detail the diversity of ways in which proteins can contact DNA (32,37-39).

Three classes of transcription factors are the focus of our interest: the bZIP family and

two families within the bHLH superfamily: the basic helix-loop-helix/leucine zipper (bHLHZ)

and basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS). All these motifs utilize a dimer of α-

helices to bind specific DNA sequences in the major groove. The following is a brief

introduction to these three classes of transcription factors.

1.2.1 The bZIP family The bZIP transcription factors comprise a large family of DNA binding proteins involved

in the regulation of DNA transcription. Fifty-three and sixty-seven bZIP proteins were identified

in the genomes of Homo sapiens (40) and Arabidopsis thaliana (41), respectively. All of these

factors bind to specific DNA sequences as homo- or heterodimers with the DNA interaction

surface, which is achieved by a region of basic amino acids that immediately precedes the

leucine zipper domain in the primary protein sequence. Members of the bZIP family are

exclusively eukaryotic nucleoproteins that are widely expressed in diverse cell types and tissues

and responsible for regulation of a variety of cellular processes including cell development,

proliferation, differentiation, apoptosis, and oncogenesis (42).

8

The α-helical bZIP motif, which forms hetero- and/or homodimers of α-helices of 60-80

residues, is the smallest and simplest protein structure that recognizes specific DNA sites with

high affinity (44,45). The classic domain swapping experiments between GCN4 and C/EBP

confirmed that sequence-specific DNA-binding activity resides in the basic region, and that

dimerization specificity is determined by the leucine zipper (46). The crystal structures of several

bZIP domains bound to their cognate sequences, including GCN4 bound either to the AP-1 (43)

or CRE sites (47,48), the Jun/Fos heterodimer (49), CREB (50), and PAP1 (51), provide clearer

images of DNA recognition by bZIP proteins.

Figure 1.2 Crystal structure of the GCN4 bZIP homodimer bound to the AP-1 DNA site (43). The double-stranded DNA is in light gray and the bZIP α-helices are in dark gray with the leucines in the fourth position of the heptads shown in the gray ball model. The basic region and the leucine zipper are labeled. The carboxyl-terminal leucine zippers of the two monomers pack together as a coiled coil, which gradually diverges to allow the amino-terminal basic region to follow the major groove of either DNA half site. (PDB ID: 1YSA)

Figure 1.2 presents the structure of the GCN4 homodimer bound to the AP-1 DNA site,

5'-TGACTCA (43). The entire structure is a dimer of continuous bipartite α-helices. In each

monomer, the N-terminal basic region, rich in positively charged residues, interacts with the

DNA site in a sequence-specific manner. Comparisons of DNA-binding basic regions show a

9

high degree of sequence similarity (51). Each basic region of the two monomers contacts a half-

site in the DNA major groove (45,52). A unique aspect of the basic region of bZIP proteins is

that it undergoes a coil-to-helix transition to form an α-helical extension of the leucine zipper

upon sequence-specific DNA binding (53-55); hence, bZIP transcription factors are not stable

and fully folded until binding to DNA.

The C-terminal leucine zipper region, which typically contains a leucine residue (or

occasionally other hydrophobic residues such as Met, Ile or Val) every seven amino acids, is

responsible for regulating dimerization stability and specificity. The two leucine zippers, one

from each monomer, dimerize to generate a parallel amphipathic coiled-coil to position the basic

region for specific DNA binding. The leucine zippers contain various heptad repeats, each of

which is composed of two α-helical turns or seven amino acids (56). The residues at the

dimerization interface, occupying the first and fourth positions of the heptad, are typically

hydrophobic, and the remaining positions are mostly polar or charged. The residues at the

dimerization interface dictate partner specificity together with neighboring residues near the

leucine zipper interface (40).

1.2.2 The bHLH superfamily The bHLH proteins form an important and versatile superfamily of eukaryotic

transcription factors found in organisms from yeast to human and are involved in diverse

fundamental biological processes, including cell cycle and developmental regulation, apoptosis

and homeostasis, and stress response pathways (57-60).

Compared to bZIP proteins, bHLH proteins utilize a similar mode of DNA binding: a

highly conserved structural motif organized into a DNA-binding basic region and a dimerization

domain. Same as for bZIP proteins, the N-terminal basic region of bHLH proteins, adjacent to

the dimerization domain, contacts the DNA recognition site. In contrast to the leucine zipper in

10

bZIP proteins, the dimerization domain of bHLH proteins is composed of two amphipathic

helices separated by a nonconserved loop typically 5-12 residues in length, forming a compact

hydrophobic four-helix bundle that positions the contiguous basic regions for DNA binding. The

loop separating the two helices leads to more flexibility in positioning the basic region on the

DNA site (61,62). In addition to the basic region that determines specificity of DNA recognition,

the loop and Helix 2 have been observed to make DNA contacts in some bHLH:DNA complexes

(57,61,63). Both the basic region and HLH dimerization domain are required for formation of

functional DNA binding complexes.

In addition to those bHLH proteins containing the bHLH domain only, there are two

other families of bHLH proteins: bHLHZ and bHLH/PAS proteins. Members of these two

families contain an additional structural motif contiguous with the bHLH domain: a leucine

zipper or PAS domain, which additionally regulates dimerization (65,66).

The bHLH proteins typically associate as homo- or heterodimers that recognize the

hexameric E-box DNA site (5'-CANNTG) (58,65). Figure 1.3 shows the crystal structure of

bHLH protein MyoD and bHLHZ protein Max (Myc-associated factor X) bound to their

corresponding DNA target sites, respectively. MyoD is a mammalian protein involved in

myogenesis (67), whereas Max is a member of the Myc/Max/Mad network of transcription

factors involved in cell proliferation, differentiation, and death (65,68). In the MyoD:DNA

complex, Helices 1 and 2 from each monomer are connected by an eight-residue loop and

participate in forming a parallel, left-handed, four-helix bundle, which allows the basic region

contiguous with Helix 1 to contact the DNA major groove, and the N-terminal basic region and

Helix 1 form a long uninterrupted α-helix (64). In contrast, in addition to the bHLH domain,

bHLHZ protein Max contains an additional secondary leucine zipper dimerization domain that is

contiguous with the C-terminus of Helix 2 in each monomer, forming a long seamless α-helix.

11

The two leucine zippers form a parallel, left-handed coiled coil in a similar manner to the leucine

zipper in the bZIP motif (61).

Figure 1.3 Crystal structure of the MyoD:DNA complex (64) (A) and Max:DNA complex (61) (B). Both MyoD and Max form a four-helix bundle with Helix 1 and Helix 2 from each monomer, allowing the basic region contiguous with Helix 1 to contact both sides of the DNA major groove. Max contains a secondary dimerization domain, the leucine zipper, which forms a long continuous α-helix with the preceding Helix 2 in each monomer. (A: PDB ID: 1MDY and B: PDB ID: 1AN2)

Similar to the basic region of the bZIP motif, the disordered basic regions of both bHLH

and bHLHZ proteins display an induced α-helical structure upon DNA binding. Furthermore, in

addition to contributing to the overall stability of bHLHZ proteins by adding substantial buried

surface area to the dimerization interface, the leucine zipper, not the HLH domain of bHLHZ

proteins, acts as the determinant of dimerization specificity (69,70), which is different from

bHLH proteins where the HLH domain determines dimerization preferences. This also indicates

12

the intrinsic subtle structural differences between the HLH domains of bHLH and bHLHZ

proteins (71).

Figure 1.4 Ribbon presentation of the Drosophila Period dimer. Each monomer comprises two tandemly organized PAS domains (PAS A and PAS B) and two additional C-terminal α helices (74). The two monomers are shown in dark gray and light gray, respectively. The disordered regions are depicted as dotted lines. (PDB ID: 1WA9)

In comparison, bHLH/PAS proteins contain structurally and functionally more

complicated PAS domain, which typically consists of 250–350 amino acids in conjunction with

the HLH domain and contains two adjacent, highly degenerate 50 amino-acid subdomains

termed PAS A and PAS B. The PAS domain is a well conserved signaling module that responds

to environmental and developmental stimulus. In addition, the PAS domain is also involved in

protein dimerization and specification of heterodimerization partner (72,73). Figure 1.4 presents

the crystal structure of an N-terminal Drosophila Period (Per) fragment comprising PAS A and

PAS B domains as well as two additional C-terminal α helices. Structural Analysis revealed a

noncrystallographic dimer mediated by intermolecular interactions of PAS A with PAS B and C-

terminal α helix (74). Unlike bHLH and bHLHZ families, in which quite a few high resolution

structures of protein:DNA complexes have been solved, including MyoD (64), E47 (75), USF

(76), Max homodimers (61,77) and heterodimers with Myc (78) and Mad (70), the structural data

13

of the complete DNA-binding and dimerization regions of any bHLH/PAS family member is still

unavailable. However, the bHLH domains of bHLH/PAS proteins are predicted to be structurally

similar and adopt a similar DNA-binding mode as does the closely related bHLH and bHLHZ

proteins based on their homologous primary sequences and DNA binding specificities (59).

Typical members of the bHLH/PAS family include the aromatic hydrocarbon receptor

(AhR, also know as dioxin receptor, DR), a mammalian protein that regulates xenobiotic

metabolizing enzymes in response to environmental contaminants such as prototypical 2,3,7,8-

tetrachlorodibenzo-p-dioxin (TCDD); hypoxia-inducible factor (HIF) 1α, a protein involved in

mediating cellular responses to hypoxia; the AhR nuclear translocator (Arnt), a central regulator

that functions by associating with AhR or HIF; and single-minded (Sim) and Period proteins,

Drosophila proteins that are involved in central nervous system midline development and

circadian rhythms, respectively (59,65).

In addition to their significant signal transduction activity under physiological conditions,

there are several unique characteristics involved in DNA recognition by the AhR/Arnt

heterodimer. First, biochemical and genetics studies have determined that the AhR/Arnt

heterodimer functions by binding the asymmetric xenobiotic response element (XRE, also

known as dioxin response element, DRE) site, 5'-TNGCGTG (79,80), which is in contrast to

bHLH and bHLHZ proteins that usually target the consensus hexameric E-box DNA site. AhR

targets the 5'-TNGC half site, and Arnt the 5'-GTG half site. Arnt is able to homodimerize and

target the symmetric E-box site; the amino acids critical for E-box recognition by Max and USF

are conserved in Arnt (81). However, AhR is unable to form homodimers and can only function

through heterodimerization with Arnt. Given the fact that the basic region of AhR is proline rich,

AhR probably does not undergo the characteristic coil-to-helix transition upon DNA binding (63).

14

Therefore, AhR and Arnt provide interesting targets for exploration of heterodimeric recognition

of DNA.

1.2.3 Heterodimeric transcription factors increase regulatory diversity All three types of DNA binding proteins discussed above function by forming homo- or

heterodimers. As a matter of fact, many prokaryotic and eukaryotic transcriptional factors are

dimeric DNA-binding proteins. This fact, from the perspective of evolution, probably indicates

that dimerization capability of transcriptional factors might lead to the regulation of biological

processes more specific and efficient while genomically economical.

Heterodimeric formation provides several potential advantages over monomer and

homodimer counterparts. First, heterodimeric formation of two proteins that have distinct DNA-

binding specificities in their homodimeric form could create a complex with a novel binding

specificity, therefore expanding the repertoire of potential DNA sequences that a family of

factors can bind (82). Second, heterodimeric formation may also form more subtle interactions

within gene regulatory regions, thereby generating diverse transcriptional control from a limited

number of transcription factors (82). For instance, protein dimers within one family can bind to

DNA sites containing common half-site sequences that differ in polarity and inter-half-site

separation (32). This provides a fine tuning of gene expression by competition of different

complexes able to bind the same or similar DNA target sequences. Third, heterodimeric

formation can increase specificity of gene regulation. Some tissue-specific DNA-binding

proteins are incapable of forming homodimers and preferentially heterodimerize to exert

function with a constitutively expressed DNA-binding protein capable of forming either homo-

or heterodimers. In this way, the transcriptional regulation is tightly and specifically controlled.

Finally, different combinations of activation and/or repression domains brought together by

heterodimeric formation at regulatory DNA sequences also allows the change of regulatory

15

properties of the specific protein bound at that fixed DNA site (36). This change of regulation

would not be possible if the DNA-binding proteins functioned only as monomers or homodimers.

Therefore, the mechanisms that control the formation of heterodimerzation of DNA-

binding proteins at the molecular level may help us further understand the relationship between

protein structure and its resultant functional activity and may potentially guide the development

of useful tools to promote or repress the corresponding function.

1.3 Studies of bHLH superfamily proteins in yeast genetic reporter systems Yeast genetic reporter systems were utilized to study bZIP and bHLH family proteins

shortly after the development of the Y2H and Y1H. Because transcriptional activation studies of

human oncoproteins and other transcription factors in mammalian cells have often been hindered

by the presence of endogenous proteins, the study of these proteins in yeast provides an obvious

advantage, as the chance of endogenous interference by their homologous counterparts in yeast is

greatly decreased.

Moreover, a more open chromatin structure in yeast than that in higher eukaryotes means

a higher level of DNA accessibility, a critical point as gene activation relies on the binding of

transcription factors to their accessible consensus sites (83,84). Analysis of the crystal structure

of the yeast nucleosome core particle reveals that the overall principles of DNA organization in

the nucleosome are conserved between lower and higher eukaryotes. However, yeast

nucleosomes are likely to be subtly destabilized as compared with nucleosomes from higher

eukaryotes. As a result, much of the yeast genome is constitutively open for transcription, as

opposed to the small percentage of actively transcribed genes at any given time in the cells of

higher eukaryotes (83). In addition, the activation of gene expression in yeast genetic reporter

systems is achieved through the use of activation domains such as GAL4 or VP16 AD. These

16

domains have been shown to interact with factors implicated in the perturbation of chromatin

structure, leading to transcriptional activation (85). Therefore, yeast genetic reporter systems

provide a simplified model for the study of DNA-binding function of mammalian transcription

factors, as complications stemming from the contribution of chromatin structure to gene

regulation is not a major concern in yeast.

In addition to the studies mentioned in Section 1.1.2 of this Chapter, there are also a

number of studies on mammalian transcription factors using yeast genetic assays. For instance,

the activities of mammalian bHLH proteins MyoD and muscle regulatory factor (MRF) were

examined in yeast, demonstrating that in vivo yeast systems can be a useful approach to facilitate

functional studies of bHLH transcription factor regulation (86). This work was followed by the

study of regulation of bHLHZ proteins Myc/Max-mediated transactivation in yeast (87). Another

elegant use of the yeast genetic system was to decipher the interplay between two mammalian

bHLHZ proteins, Mad1 and Max, and the yeast protein Sin3. The study revealed that Mad1 and

Max form a complex with inhibitory Sin3 and therefore, are unable to activate reporter gene

expression in yeast, as the expression of both proteins in a SIN3-knockout yeast strain activated

reporter gene expression. These experiments provided direct evidence that yeast protein Sin3 can

repress transcription through interaction with DNA-binding proteins by the same repression

mechanism as its mammalian counterpart (88).

Several yeast genetic reporter systems have been developed to study signal transduction

by bHLH/PAS transcription factors AhR and Arnt. A study of transcription factor AhR in a yeast

model system provided the first genetic evidence that heat shock protein 90 (Hsp90) is critical to

AhR signaling (89), which was confirmed by Poellinger et al. in a similar yeast reporter system

(90). A cDNA library screening study revealed that Arnt was able to interact with the AhR

bHLH/PAS in yeast (91). This work was followed by two similar systems developed to

17

investigate the AhR/Arnt heterodimer's (full-length or bHLH/PAS domain) response to different

AhR ligands in the Y1H system (92,93).

1.4 Scope The thesis is organized as follows. Chapters 2-4 are entire manuscripts that comprise

three different projects. With the intention of maintaining the integrity of each manuscript as a

published entity, only necessary changes for each chapter, such as bibliographic referencing and

figure formatting, have been made for thesis organization purposes. Therefore, some unavoidable

redundancy will appear in the text. In Chapter 2, a modified yeast one-hybrid (MY1H) system is

described that was developed for in vivo investigation of protein-protein and protein:DNA

interactions. This single-plasmid expression system was validated by use of the well-

characterized DNA-binding protein p53 and its inhibitory partners, large T antigen (LTAg) and

53BP2 (in press in BioTechniques). Chapter 3 focuses on turning a false negative protein

heterodimer:DNA interaction into a true positive control in the MY1H system. Negative signals

were observed when we initially coexpressed the heterodimeric bHLH/PAS domains of AhR and

Arnt that target the cognate XRE sequence as a positive control in the study of XRE-binding

proteins in the MY1H system. By increasing the number of DNA target sites upstream of the

reporter genes and increasing the number of activator domains fused to the proteins of interest,

false negative results were salvaged into true positives (in press in Analytical Biochemistry).

Analysis of how a pair of bHLHZ-like hybrid proteins, AhRJunD and ArntFos, undergoes

target sequence-mediated heterodimerization upon specific DNA binding in MY1H is described

in Chapter 4. This current manuscript focuses on the in vivo yeast assays, and the final

manuscript will be submitted upon completion of in vitro fluorescence anisotropy titrations and

western blotting assays; these bacterially expressed proteins are currently being expressed and

purified. Although this work is not yet completed, Chapter 4 maintains the format of the previous

18

two chapters that are in press. Chapter 5 summarizes the thesis results and recommends future

directions for research. Chapter 6 comprehensively discusses all the materials and methods that

are used in this thesis. Appendices A-C comprise the supplemental materials supplied with the

manuscripts presented in Chapters 2-4. Appendix D lists and describes all the oligonucleotides

used in the thesis.

It is noted that the MY1H system developed in this thesis could be particularly useful for

testing the effects of a new protein, or mutant versions of a protein, on the DNA-binding activity

of a transcription factor. Our lab applied this system to explore the repression of the Max:E-box

DNA site interaction with several competitor mutants of Max. The results revealed different

repression capabilities of these designed Max mutants on the Max:E-box interaction in vivo. This

manuscript is in preparation, and I am the second author.

19

Chap 2ter

Design of a single plasmid-based modified yeast one-hybrid system for investigation of in vivo protein-protein and protein:DNA

interactions Gang Chen, Lisa M. DenBoer, and Jumi A. Shin

(Short running title: Modified Y1H for identification of protein-protein/protein:DNA interactions)

Contributions:

I initiated this project. I performed part of the plasmid construction, part of the 3-AT

titration assays, and all of the X-gal colony-lift assays and ONPG assays. Lisa DenBoer

performed most of the plasmid construction and 3-AT titration assays. I completed the data

interpretation. Lisa DenBoer provided intellectual input and editing to the manuscript. Jumi Shin

provided supervision and intellectual input to the project and manuscript.

This chapter is adapted from the original manuscript accepted by BioTechniques, with

necessary changes for thesis organization purposes.

Authors retain copyright of manuscript published in BioTechniques, as per publisher's

agreement.

20

2.1 Abstract We have developed a modified yeast one-hybrid system (MY1H) useful for in vivo

investigation of protein-protein and protein:DNA interactions. Our single-plasmid expression

system is capable of differential protein expression levels; in addition to a GAL4 activation

domain (AD) fusion protein, a second protein can be coexpressed at either comparable or higher

transcriptional levels from expression vectors pCETT or pCETF, respectively. This second

protein can play a structural, modifying, or inhibitory role that restores or blocks reporter gene

expression. Our MY1H was validated by use of the well-characterized DNA-binding protein p53

and its inhibitory partners, large T antigen (LTAg) and 53BP2. By coexpressing LTAg or 53BP2

at comparable or higher levels than the GAL4AD-p53 fusion in the MY1H, we show that DNA

binding of p53 decreases by different, measurable extents dependent on the expression level of

inhibitory partner. As with the traditional Y1H, our system could also be used to investigate

proteins that provide coactivational or bridging functions and to identify novel protein- or DNA-

binding partners through library screening. Our MY1H provides a system for investigation of

simultaneous protein-protein and protein:DNA interactions, and thus, is a useful addition to

current methods for in vivo investigation of such interactions.

21

2.2 Introduction

Figure 2.1 Five different types of interactions between proteins and DNA can be detected with the MY1H system. Top: If P1 is able to target the binding element E, the second protein P2 can be expressed to block the DNA binding region of P1, directly bind to binding element E, or recruit repressors to P1, thereby inhibiting reporter gene expression. Bottom: If P1 is unable to target the binding element E, P2 can be expressed as a bridging protein or coregulatory accessory protein, or it can modify the structure of P1 to enable DNA binding, thereby restoring reporter gene expression. As a coregulatory protein, P2 may function with or without making direct contact with P1.

Gene expression is a sophisticated, finely tuned process that involves the regulated

interactions of multiple proteins with promoter and enhancer elements. A variety of approaches

are currently employed in the study of these interactions, including phage display and yeast-

based assays, as well as other biophysical and biochemical methods (94). The yeast one-hybrid

22

system (Y1H), a variant of the yeast two-hybrid system (Y2H) (1), is a powerful and commonly

used in vivo genetic assay for identification of protein:DNA interactions. The Y1H is useful for

isolation of genes encoding proteins that bind to cis-acting regulatory elements and for further

characterization of known protein:DNA interactions, whereas the Y2H allows detection of

protein-protein interactions (1-3).

In many cases, protein-protein and protein:DNA interactions are intertwined in vivo:

DNA-binding proteins are often modulated by the recruitment of accessory proteins that cannot

bind DNA directly but rather serve to repress or coactivate transcription through the formation of

transcriptional complexes (95,96). Most bZIP and bHLH families, such as Jun-Fos (97), Myc-

Max (98), and the classic bacteriophage λ repressor and Cro proteins, belong to this class of

transcription factors. A number of yeast genetic approaches have been reported for investigation

of a protein in complex with a known partner, thereby forming a heterodimeric complex that

binds DNA; these complexes may include one partner lacking intrinsic DNA-binding capability

enabled by dimerization with an accessory protein (18-23). These studies have traditionally used

two separate plasmids for expression of the two different proteins.

We have developed a single plasmid-based modified Y1H system (MY1H) useful for

examination of both protein-protein and protein:DNA interactions in vivo. In addition to an AD

fusion protein, a second protein is coexpressed at either comparable or excess levels. The

interaction of this second protein with the AD fusion via cooperative oligomerization, structural

modification, or inhibition, can restore or block reporter gene expression (Figure 2.1). We chose

to validate our MY1H using the extensively studied interactions of DNA-binding protein p53

and its inhibitory partners, Simian Virus 40 (SV40) LTAg and 53BP2 (99-103). Both LTAg and

53BP2 inhibit wild-type p53 function through a protein-protein interaction at the DNA-binding

domain of p53, thereby preventing p53 from binding to its consensus DNA target site (also

23

known as the p53 cis-acting DNA target element) (102-106). It was reported that the p53-53BP2

complex forms with a dissociation constant (Kd) of about 30 nM as determined by surface

plasmon resonance (106). However, isothermal titration calorimetry revealed that p53 interacted

with 53BP2 with a Kd of 2.2 μM (107), so there is significant discrepancy in these quantitative

measurements. No quantitative data is available regarding the relative affinity of the LTAg-p53

interaction, to the best of our knowledge. The well-characterized p53-LTAg and p53-53BP2

interactions—protein-protein interactions that modulate DNA-binding ability—provide an ideal

system for validation of our MY1H.

Our MY1H combines the features of the Y1H and Y2H systems, and also extends their

scopes, such that simultaneous protein-protein and protein:DNA interactions can be investigated;

hence, this MY1H is speculated to have broad utility (Figure 2.1) and may provide a widely

applicable approach for investigation of various types of interactions, including heterodimer-

DNA interactions or the effects of different protein modifiers on the DNA-binding capability of a

transcription factor.

2.3 Materials and Methods Reagents were purchased from BioShop Canada (Burlington, ON), enzymes were

purchased from New England Biolabs (Pickering, ON), and oligonucleotides were synthesized

by Operon Biotechnologies (Huntsville, AL) unless otherwise stated.

2.3.1 Bacterial and yeast strains Escherichia coli DH5α (Stratagene, La Jolla, CA) or dam-/dcm- C2925H (New England

Biolabs) was used for standard cloning and for rescue of plasmids from yeast cells.

Saccharomyces cerevisiae YM4271 [MATa, ura3-52, his3-200, ade2-101, lys2-801, leu2-3, 112,

trp1-901, tyr1-501, gal4-∆512, gal80-∆538, ade5::hisG] was used for plasmid construction via

homologous recombination and reporter strain construction. Two yeast reporter strains,

24

YM4271[p53HIS] and YM4271[p53BLUE], were created according to the Matchmaker™ One-

hybrid System User Manual (Clontech, Palo Alto, CA) for reporter assay analysis in the MY1H.

These two strains contain three tandem copies of the consensus p53 binding site upstream of the

HIS3 and lacZ reporter genes, respectively.

2.3.2 Transformation, DNA preparation, and plasmid rescue Recombinant plasmids were transformed into E. coli by the standard TSS procedure

(108). Plasmids were isolated from bacteria using the Wizard® Plus SV Miniprep DNA

Purification System (Promega, Madison, WI). Yeast transformations were performed using

either the standard lithium acetate method (Yeast Protocols Handbook, Clontech) or the Frozen-

EZ Yeast Transformation II™ Kit (Zymo Research, Orange, CA). Transformants were selected

by leucine prototrophy. Isolation of yeast plasmids was performed using the Zymoprep™ II

Yeast Plasmid Miniprep Kit (Zymo Research). PCR reactions were performed using Phusion™

high-fidelity DNA polymerase (New England Biolabs). PCR products and DNA fragments for

cloning were purified using the QIAquick Spin Kits or MinElute Kits (Qiagen, Mississauga, ON).

2.3.3 Plasmid Construction All new constructs were confirmed by dideoxynucleotide DNA sequencing on an ABI

(Applied Biosystems) 3730XL 96 capillary sequencer at the DNA Sequencing Facility in the

Centre for Applied Genomics, Hospital for Sick Children (Toronto, ON).

2.3.3.1 pGAD424-MCS I and pGAD424-MCS II pGAD424-MCS I and pGAD424-MCS II were constructed by homologous

recombination (109) in YM4271 to replace the original multiple cloning site (MCS) in

pGAD424 (110) using the 6.6 kb EcoR I/Pst I pGAD424 fragment along with the CE4MCS

fragment and 679 bp BstZ17 I/Mlu I pGADT7 fragment, respectively. The CE4MCS fragment

25

was assembled by self-priming PCR (111) using oligonucleotides 2-1 to 2-6 (Table 2.1; all

oligonucleotides discussed in Chapter 2 are listed in Table 2.1 and also described in Table D.1 of

Appendix D); the fragment contains a T7 promoter, a c-Myc epitope tag, and a multiple cloning

site (MCS I) with recognition sequences for five restriction enzymes (Sac II, Sal I, BssH II, Xba I,

and Bcl I). Similarly, the BstZ17 I/Mlu I pGADT7 fragment contains a T7 promoter, a HA

epitope tag, and a multiple cloning site (MCS II) with recognition sequences for six restriction

enzymes (EcoR I, Sma I, BamH I, Sac I, Xho I, and Pst I).

Table 2.1 Oligonucleotides used in this study No Sequence

2-1 ACTATCTATTCGATGATGAAGATACCCCACCAAACCCAAA

2-2 GGCGCTCGCCCTATAGTGAGTCGTATTAAAGATCTCTTTTTTTGGGTTTGGTGGGGTATC

2-3 CTCACTATAGGGCGAGCGCCGCCATCATGGAGGAGCAGAAGCTGATCTCAGAGGAGGACC

2-4 GCGCGCACCTTGTCGACCGCGGCCTCCATGGCCATATGCAGGTCCTCCTCTGAGATCAGC

2-5 GCGGTCGACAAGGTGCGCGCTCTAGATGATCATGAATCGTAGATACTGAAAAACCCCGCA

2-6 ATGCACAGTTGAAGTGAACTTGCGGGGTTTTTCAGTATCT

2-7 AGAAAGGTCGAATTGGGTACCGCCGCCAATAAAGAGATCTTTAAT

2-8 CTCGCCCTATAGTGAGTCGTATTAAAGATCTCTTTATTGGCGGCG

2-9 AAAGACGTCGCATGCAACTTCTTTTCTTT

2-10 ATTGACGTCAAGCTTGCATGCCGGTAGAGGT

2-11 AAAGACGTCCCTGCAGGTCGAGATCCGGGA

2-12 AAAAGTCGACCCTGTCACCGAGACCCCTGG

2-13 ACGCTCTAGATCAGTCTGAGTCAGGCCCCA

2-14 AAAGAATTCGGAACTGATGAATGGGAGCAG

2-15 AAAGGATCCTTATGTTTCAGGTTCAGGGGGAG

2-16 AAAGAATTCCCGCCTGAAATCACCGGGCAG

2-17 AAAGGATCCTCAGGCCAAGCTCCTTTGTCTT

*Oligonucleotide sequences are shown in 5’ to 3’ direction. Restriction sites used for cloning are in bold.

26

2.3.3.2 pGAD424-MCS IIΔAD and pGADT7ΔAD In pGAD424-MCS IIΔAD and pGADT7ΔAD, the GAL4AD was deleted while the open

reading frame was maintained. To create these two recombinant plasmids, the FINALREC

fragment was assembled by mutually primed synthesis (112) using oligonucleotides 2-7 and 2-8.

The 5’ and 3’ ends of the FINALREC fragment contain 30 and 35 bp homology, respectively, to

both Bgl II-linearized pGAD424-MCS II and Bgl II-linearized pGADT7. YM4271 was

cotransformed with either Bgl II-linearized pGAD424-MCS II or Bgl II-linearized pGADT7 and

the FINALREC fragment to give rise to pGAD424-MCS IIΔAD and pGADT7ΔAD, respectively.

2.3.3.3 pCETT and pCETF The T2 fragment was amplified with oligonucleotides 2-9 and 2-10 from pGAD424-MCS

IIΔAD. The F2 fragment was amplified with oligonucleotides 2-10 and 2-11 from pGADT7ΔAD.

The amplified fragments T2 and F2 were digested with Aat II, treated with alkaline phosphatase,

and then ligated into the Aat II site of pGAD424-MCS I to generate pCETT and pCETF (Figure

2.2), respectively.

2.3.3.4 pCETT/53 and pCETF/53 The sequence encoding amino acids 72-390 of the murine p53 gene was amplified from

pGAD53m (Clontech) using oligonucleotides 2-12 and 2-13. This fragment was inserted

between the Sal I and Xba I sites of pCETT and pCETF to construct pCETT/53 and pCETF/53,

respectively.

2.3.3.5 pCETT/53/T and pCETF/53/T The fragment encoding amino acids 87-708 of SV40 LTAg was amplified from

pGADT7/T (Clontech) with oligonucleotides 2-14 and 2-15. This fragment was inserted into the

27

EcoR I and BamH I sites of pCETT/53 and pCETF/53 to construct pCETT/53/T and

pCETF/53/T, respectively.

2.3.3.6 pCETT/53/BP2 and pCETF/53/BP2 The cDNA plasmid pGBT9, which contains the human 53BP2 sequence, was kindly

given to us by Dr. Kuniyoshi Iwabuchi (Kanazawa Medical University, Japan) (103). The

sequence encoding amino acids 768-1005 of 53BP2 was amplified with oligonucleotides 2-16

and 2-17 and inserted between the EcoR I and BamH I sites of pCETT/53 and pCETF/53 to

generate pCETT/53/BP2 and pCETF/53/BP2, respectively.

2.3.4 3-AT titration analysis HIS3 gene expression was measured by growing transformed yeast cells on selective

media lacking leucine, uracil, and histidine. Activity of the HIS3 reporter was quantified as

survival rates of yeast transformants on plates containing increasing amounts of 3-aminotriazole

(3-AT), a competitive inhibitor of the His3 protein.

Transformed yeast cells were initially grown at 30 °C with shaking in SD/-L media for 2

days or until OD600 >1.5 was reached, and then used to inoculate a fresh culture of SD/-L media.

This secondary culture was grown overnight until OD600 1.0-1.3 was reached. An aliquot of the

secondary culture was resuspended in YPDA to give a starting OD600 ~0.2. The YPDA culture

was then grown for 3-5 hrs until OD600 0.60-0.65 was reached. The culture was then diluted by a

factor of 4000 and 100 μl of the diluent was plated on SD/-H/-L/-U plates containing increasing

3-AT concentrations ranging from 0 to 80 mM. Individual colonies were counted after 5 days

growth at 30 °C. The survival rate of a specific transformant was calculated as the number of

colonies on the SD/-H/-L/-U plate containing a specific 3-AT concentration divided by the

28

number of the colonies on the control SD/-L/-U plate. This assay was performed in triplicate and

independently repeated at least 3 times in order to ensure reproducibility.

2.3.5 X-gal colony-lift filter assay and ONPG liquid assay The X-gal colony-lift filter assay and ortho-nitrophenyl-galactoside (ONPG) liquid assay

were performed according to the protocols provided in the Yeast Protocols Handbook (Clontech)

with the following modifications: in the X-gal assay, the cells were subjected to two cycles of

freeze/thaw in order to lyse the cells. In the ONPG assay, yeast cells were grown as described

above for the 3-AT titration assay before harvesting for lysis. Results are presented as mean

values ± S.E.M. of 3-4 independent experiments, each performed in triplicate.

2.4 Results

2.4.1 Design of protein expression vectors In order to examine protein-protein and protein:DNA interactions simultaneously in a

single yeast genetic system, two GAL4AD fusion vectors, pCETT and pCETF, were constructed

based on pGAD424, the protein expression plasmid provided in the Matchmaker™ One-Hybrid

System (Clontech). In both pCETT and pCETF, the gene encoding the AD fusion protein is

inserted into MCS I where transcription is under the control of a truncated ADH1 promoter,

leading to low protein expression levels (113,114). A second gene inserted into the second

multiple cloning site, MCS II, can also be expressed from the same plasmid under the control of

the truncated ADH1 promoter identical to that in MCS I. In pCETF, MCS II is under the control

of the full-length ADH1 promoter, leading to higher transcription levels (114,115).

Our plasmid design provides the option of allowing the second protein to be expressed at

a comparable level with the AD fusion protein (pCETT; truncated ADH1 promoters in MCS I

and II) or in excess (pCETF; truncated ADH1 promoter in MCS I, full-length ADH1 in MCS II).

In addition, these plasmids were designed with different epitope tag-coding sequences upstream

29

from each multiple cloning site, and a T7 promoter upstream of both multiple cloning sites to

allow in vitro transcription and translation.

Figure 2.2 Plasmids pCETT and pCETF were constructed for coexpression of two proteins in a yeast model system. The two vectors have unique restriction sites located in both the MCS I and MCS II regions. MCS I is at the 3'-end of the open reading frame for the GAL4 AD sequence allowing a fusion protein combining amino acids 768–881 of the GAL4 AD and the cloned protein of interest to be expressed at low levels from a truncated constitutive ADH1 promoter. The expression of genes inserted into MCS II is controlled by either the truncated ADH1 promoter (pCETT) or full-length ADH1 promoter (pCETF). Therefore, a second protein can be coexpressed at either low levels (pCETT) or high levels (pCETF). Both vectors also contain a T7 promoter at both MCS regions, a c-Myc epitope tag at MCS I, and an HA epitope tag at MCS II.

2.4.2 Coexpression of LTAg or 53BP2 decreases the transactivation potential of GAL4AD-p53 in the MY1H

In our MY1H, we used three assays to measure the inhibitory strengths of LTAg and

53BP2 on binding of the p53 DNA consensus site by GAL4AD-p53, including titration on

inhibitory 3-AT-containing media (HIS3 assay) and two colorimetric lacZ reporter-based assays:

qualitative X-gal colony-lift filter assay and quantitative ONPG liquid assay. All three assays

showed that the ability of GAL4AD-p53 to activate transcription of reporter genes was adversely

affected when coexpressed with LTAg or 53BP2 in the MY1H.

30

Figure 2.3 3-AT titrations reveal that the survival rates of transformants decreases when the inhibitory proteins are expressed. YM4271[p53HIS] cells were transformed with A) pCETT (□), pCETT/53 (◊), pCETT/53/T (○) or pCETT/53/BP2 (Δ) or B) pCETF (□), pCETF/53 (◊), pCETF/53/T (○) or pCETF/53/BP2 (Δ). Key for labels as follows. pCETT and pCETF: negative controls, no protein expression. pCETT/53 and pCETF/53: positive controls, expression of GAL4AD-p53 from MCS I only. pCETT/53/T and pCETF/53/T: low-level expression of GAL4AD-p53 from MCS I, low- or high-level expression of LTAg from MCS II. pCETT/53/BP2 and pCETF/53/BP2: low-level expression of GAL4AD-p53 from MCS I, low- or high-level expression of 53BP2 from MCS II. Cells were grown to exponential phase in YPDA media and plated at equal densities on selective media containing increasing concentrations of 3-AT. Individual colonies were counted after 5 days growth at 30 °C. The survival rate of a specific transformant was calculated as the number of colonies on the SD/-H/-L/-U plate containing a specific 3-AT concentration divided by the number of the colonies on the control SD/-L/-U plate. The assay was conducted in triplicate and independently repeated at least 3 times. Data is presented as average values ± standard error measurement.

We titrated the yeast transformants on SD/-H/-L plates containing 0 to 80 mM 3-AT (3-

amino-1,2,4-triazole) and plotted the survival rates of each transformant at increasing 3-AT

concentrations (Figure 2.3). Survival rates in the presence of 3-AT correlate with transcriptional

activity (116). Therefore, lower survival of transformants on 3-AT-containing plates indicates

stronger inhibitory strengths of LTAg or 53BP2 on the ability of GAL4AD-p53 to bind the p53

consensus DNA site. As expected, the survival rates of yeast cells transformed with pCETT or

pCETF decrease sharply as 3-AT concentration increases and reach zero at 20 mM 3-AT, while

cells transformed with pCETT/p53 or pCETF/p53 only begin to show decreased survival at 3-AT

concentrations over 70 mM. In contrast, when LTAg or 53BP2 is coexpressed with GAL4AD-

p53, an immediate decrease in cell survival is observed at 10-20 mM 3-AT and continues to

31

decline at higher 3-AT concentrations, consistent with the inhibitory role both LTAg and 53BP2

play in the DNA-binding ability of p53.

The sensitive X-gal colony-lift filter assay corroborates the 3-AT titration results in the

HIS3 assay (Figure 2.4). In the colony-lift assay, reporter gene activation is visualized by the

blue chromophore released by the action of β-galactosidase encoded by the lacZ gene.

Transformants expressing only GAL4AD-p53 become blue very quickly (within 10 min, Figure

2.4B and F), and continue to increase in intensity, becoming vivid blue after 45 minutes. When

LTAg or 53BP2 is coexpressed at low expression levels from pCETT (Figure 2.4C and D), blue

color only begins to appear after 15 minutes and is considerably less intense after 45 minutes,

indicating discernible inhibition of reporter gene expression. Higher expression of either LTAg

or 53BP2 from pCETF (Figure 2.4G and H) inhibits lacZ transcription to a much greater extent,

as only a faint blue color is achieved after 45 minutes.

Figure 2.4 Colony-lift filter assay indicates LTAg and 53BP2 inhibit DNA binding of p53 to different extents. YM4271[p53BLUE] cells were transformed with A) pCETT; B) pCETT/53; C) pCETT/53/T; D) pCETT/53/BP2; E) pCETF; F) pCETF/53; G) pCETF/53/T; or H) pCETF/53/BP2 and plated on SD/-L/-U plates. The X-gal colony-lift assay was performed after 4 days growth at 30 °C. Photos were taken after 45 min incubation at 30 °C. See caption for Figure 2.3 for the key to labels.

32

The quantitative ONPG assay further corroborates the qualitative results obtained from

both the 3-AT titration and the colony-lift assays. Expression of GAL4AD-p53 from either

pCETT or pCETF leads to comparable β-galactosidase activities (Figure 2.5), demonstrating that

expression from MCS I is not affected by the different ADH1 promoters (truncated vs. full-length)

that control the transcription of genes cloned into MCS II. When no gene is cloned into MCS II,

a nonsense protein (76 residues) from the cloning vector is expressed. We further tested the

effect of this nonsense protein by assay of β-galactosidase activity and compared these results to

those obtained when using singly transformed yeast expressing GAL4AD-p53 from the original

pGAD53m plasmid supplied by Clontech. The results of this assay are not statistically different,

suggesting that this nonsense protein expressed from pCETT or pCETF, regardless of its high or

low expression levels, does not affect GAL4AD-p53 in either an enhancing or inhibitory fashion,

nor does its expression adversely affect normal yeast growth (data not shown).

Figure 2.5 Histogram comparing the effects of different expression levels of inhibitory proteins LTAg and 53BP2 on DNA binding by p53. Quantitative assessment of β-galactosidase activity of the GAL4AD-p53 fusion in yeast strain YM4271[p53BLUE] in the presence/absence of high/low expression of 53BP2 or LTAg. Vertical axis indicates the mean values of β-galactosidase units. Error bars represent standard error from at least three independent trials conducted in triplicate. See caption for Figure 2.3 for the key to histogram labels.

33

Low level coexpression from pCETT of GAL4AD-p53 and LTAg (pCETT/53/T, Figure

2.5) gives a ~3.5-fold decrease in β-galactosidase activity compared with the same cells

expressing GAL4AD-p53 alone (pCETT/53, Figure 2.5), compared to a ~26-fold decrease when

LTAg is expressed from the full-length ADH1 promoter in pCETF (pCETF/53/T, Figure 2.5).

Similar results were observed for the coexpression of GAL4AD-p53 and 53BP2: a minimal

decrease in β-galactosidase activity was observed when 53BP2 was expressed from pCETT

(pCETT/53/BP2, Figure 2.5), while a 36-fold decrease was observed when using pCETF

(pCETF/53/BP2, Figure 2.5). As expected, expression of either LTAg or 53BP2 alone does not

result in reporter gene activation (data not shown).

2.5 Discussion

2.5.1 The DNA-binding activity of p53 and its interaction with LTAg or 53BP2: comparison of MY1H observations with earlier studies

In our MY1H system, the decrease in positive signal from transactivation of GAL4AD-

p53 requires the interaction of LTAg or 53BP2 with GAL4AD-p53: LTAg and 53BP2 prevent

the DNA binding of p53 in vivo, thereby leading to decreased activation potential. This

conclusion is supported both by our experimental data and by evidence gained from previously

published studies. Firstly, as discussed above, previous studies have already proven that LTAg

and 53BP2 inhibit DNA binding of p53 by binding to and masking p53's DNA-binding domain

(102-106). Second, we observed by the HIS3 reporter assay that neither LTAg nor 53BP2

interacts with the p53 consensus DNA site (data not shown), which excludes the possibility that

LTAg or 53BP2 inhibits transcription of GAL4AD-p53 through occupying the p53 DNA

consensus target. Third, there is no evidence that LTAg or 53BP2 can interact with GAL4AD,

indicating the unlikelihood of LTAg or 53BP2 interference with GAL4AD function. Fourth, no

abnormal growth of yeast upon expression of either LTAg or 53BP2 was observed in our

34

experiments, and none was reported by other labs in their experiments on p53 interactions with

either LTAg or 53BP2 (103,104,117). In our MY1H, all three assays provide convincing in vivo

evidence that either LTAg or 53BP2 excludes p53 from binding to its DNA consensus sequence.

Interestingly, all three assays show that at low expression levels, the inhibitory activity of

53BP2 is not vastly different from the positive control expressing only GAL4AD-p53, whereas

LTAg shows strong inhibition of reporter gene activation (Figures 2.3-2.5). At high expression

levels, both 53BP2 and LTAg efficiently inhibit transcription potential of the reporter gene,

leading to comparable minimal levels of reporter gene activity; the 3-AT titration assay shows,

however, that LTAg still appears to be a more effective inhibitor at low and high expression

levels (Figure 2.3). Although these observations suggest that LTAg may be a more capable

inhibitor of transcription potency than 53BP2, they do not necessarily prove that LTAg inhibits

the binding of p53 to its consensus site more efficiently than does 53BP2 due to the complexity

of the in vivo system and the inherent differences between LTAg and 53BP2, including size,

ability to permeate the yeast nucleus, and potential differences in cellular concentrations. Given

such differences between dissimilar proteins in the in vivo environment and that truly

quantitative comparisons of data obtained from in vivo genetic systems is impractical (25), a

more appropriate and reliable use of our MY1H would be, for example, the in vivo comparison

of the effect of several mutant versions of a targeted protein on the DNA-binding ability of a

transcription factor, with potential for correlation of in vivo data with in vitro measurements.

2.5.2 Different types of interactions between two proteins and a DNA target can be examined in our MY1H system

By expressing a second protein in our modified system, different interactions can be

investigated (Figure 2.1). If P1, which is expressed as a fusion to GAL4AD, is able to target

DNA element E, the second protein P2 can interact with the DNA-binding domain of P1

35

(protein-protein interaction) or directly bind to element E (protein:DNA interaction), or P2 can

recruit repressors to P1: either of these scenarios can result in blockage or decrease of reporter

gene activation. If P1 itself is unable to target element E, the second protein can serve to rescue

or restore reporter gene expression by serving as a bridge between P1 and element E, by

dimerizing with P1 to enable binding at element E, or by modifying the structure of P1 to allow

DNA binding.

As demonstrated here, our MY1H system might be particularly useful for testing the

effects of a new protein, or mutant versions of a protein, on the DNA-binding activity of a

transcription factor. As we study the effects of a particular protein on the DNA-binding activity

of a transcription factor, this protein can be transcribed at low or high levels from MCS II, which

is a considerable advantage during the study of inhibitors of a DNA-binding protein or DNA-

binding competition assays. The activities of strong inhibitors can be evaluated with pCETT,

while the activities of weaker inhibitors can be distinguished with pCETF. Such a combined use

of the two plasmids allows reliable qualitative assessment of the strength of the protein:DNA or

protein-protein interaction of interest. For example, we have successfully applied this MY1H

system to examine two other protein:DNA systems (unpublished results). In the first case, the

MY1H was used to test the repression of Max-DNA interactions with several mutants of Max

such that the competitive binding of two proteins vying for the same DNA target was examined:

hence, a protein:DNA interaction was assayed, as shown in Figure 2.1 where P2 serves as a

repressor. In the second case, we utilized the MY1H for examination of mutants of AhR and

Arnt that must heterodimerize in order to bind to a specific DNA target site: hence, a protein

heterodimer-DNA interaction was assayed, as shown in Figure 2.1 where P2 acts as a

coregulatory protein. In the p53/LTAg/53BP2 system presented here, a protein-protein

interaction was assayed, and hence, P2 serves as a blocker.

36

Moreover, investigation of cooperative heterodimer-DNA interactions benefits from

expression of both proteins from the same plasmid. The dimeric complex comprises two

different monomers at equimolar ratio, and such expression is more controllable when plasmid

copy number is not a complicating factor. In the MY1H system, the two genes inserted into

pCETT are transcribed at the same level, resulting in comparable protein concentrations within

the cell. (We note that although both proteins encoded in each plasmid should be expressed

comparably, their actual concentrations in the cell can depend on other factors including protein

size, stability, and post-translational processing.) In contrast, previously published yeast genetic

approaches for investigation of multiprotein:DNA interactions utilize two separate plasmids to

express two different proteins, commonly controlled by different promoters (18-23). These two-

plasmid systems inherently lack the ability to control the expression of both proteins at

comparable levels, as fluctuations in plasmid copy numbers and differential promoter strengths

can lead to variable and unpredictable expression levels of the two different proteins. Our one-

plasmid system, therefore, eliminates the issue of variable protein expression levels stemming

from differential copy numbers between plasmids within the same cell.

Although not shown in this report, our system could also be used to examine proteins that

contribute coactivational or bridging functions to the protein under investigation (Figure 2.1).

Furthermore, with some prior knowledge of possible target proteins, this system could

potentially be extended toward library screening for identification of novel accessory proteins

that rescue DNA-binding capability of a target protein that is otherwise incapable of specific

DNA binding.

In summary, we have developed a modified Y1H system that can be used to detect both

protein-protein and protein:DNA interactions in vivo. The system was validated by use of DNA

binding protein p53 and inhibitors LTAg and 53BP2. This MY1H system should be particularly

37

useful in the investigation of the effects of a regulatory protein on the target transcription factor-

DNA interaction, and should complement current methods available for identifying and

investigating novel protein-protein or protein:DNA interactions in yeast Saccharomyces

cerevisiae.

2.6 Supplemental Information Supplemental Information associated with this manuscript is provided in Appendix A.

38

Chap 3ter

AhR/Arnt:XRE interaction: turning false negatives into true positives in the modified yeast one-hybrid assay

Gang Chen and Jumi A. Shin

(Short running title: Turning false negatives into true positives in modified Y1H)

Contributions:

I performed all the experiments and completed the data interpretation. Jumi Shin

provided supervision and intellectual input to the project and manuscript.

This chapter is adapted from the original manuscript accepted by Analytical Biochemistry,

with necessary changes for thesis organization purposes.

Reproduced with permission from Analytical Biochemistry. Copyright (2008) Elsevier

Inc.

39

3.1 Abstract Given the frequent occurrence of false negatives in yeast genetic assays, it is both

interesting and practical to address the possible mechanisms of false negatives, and more

importantly, turn false negatives to true positives. We recently developed a modified yeast one-

hybrid system (MY1H) useful for investigation of simultaneous protein-protein and protein:DNA

interactions in vivo. We coexpressed the bHLH/PAS domains of AhR and Arnt—namely NAhR

and NArnt, respetively—which are known to form heterodimers and bind the cognate XRE

sequence both in vitro and in vivo, as a positive control in the study of XRE-binding proteins in

the MY1H system. However, we observed negative results, i.e. no positive signal detected from

binding of the NAhR/NArnt heterodimer and XRE site. We demonstrate that by increasing the

copy number of XRE sites integrated into the yeast genome and using double GAL4 activation

domains, the NAhR/NArnt heterodimer forms and specifically binds the cognate XRE sequence,

an interaction that is now clearly detectable in the MY1H. This methodology may be helpful in

trouble-shooting and correcting false negatives that arise from unproductive transcription in

yeast genetic assays.

40

3.2 Introduction Since their introduction almost two decades ago, the yeast two-hybrid (Y2H) (1) and

yeast one-hybrid (Y1H) (4) in vivo genetic assays have been widely utilized for identification

and characterization of protein-protein and protein:DNA interactions. With the arrival of the

post-genome era, these two systems, together with their mammalian and bacterial counterparts

(118,119), are being extensively applied to search networks of interactions in pathways and

genomes (28,120,121).

A general pitfall of genetic assays is the all-too-common observation of false positives

and false negatives in yeast reporter assays (122). Endogenous proteins can affect the protein-

protein or protein:DNA interactions under investigation either positively (false positive) or

negatively (false negative). Additionally, some activation domain (AD) fusions auto-activate

transcription without protein- or DNA-binding as a prerequisite, and some AD fusions can bind

or regulate promoter sequences by themselves without interaction with the DNA-binding domain

(DBD) fusion in the Y2H or designated DNA target element in the Y1H; in the case of the Y2H,

the DBD fusion may also activate transcription independent of the AD fusion (10,29,110). In

addition, there exist a relatively small number of “sticky” or “promiscuous” proteins frequently

detected using multiple baits. Moreover, some physically true, yet physiologically irrelevant,

interactions may also be categorized as one class of false positives (26).

Compared with false positives, false negatives have received much less attention.

However, as researchers shift their focus from detecting any protein that interacts with their

protein or DNA targets of interest toward mapping all of the protein-protein and protein:DNA

interactions on a genome- or pathway-wide scale, the issue of false negatives becomes more

important (26). False negatives stem from various factors, including weak interactions beyond

the detection limitations of a yeast-based genetic system, proteins not stably expressed or folded

41

improperly in the host cell, proteins not localizing to the nucleus, and post-translational

modifications not provided by the host cell's machinery. Additionally, high expression levels of

some hybrid proteins can be toxic to the host cell, and eukaryotic regulatory proteins may

interfere with the function of their yeast homologs. Also, fused domains or epitope tags can have

unintended effects by perturbing protein structure or occluding the site of interaction.

Furthermore, false negatives can simply be due to unproductive transcription of the reporter gene,

caused by an inappropriately positioned DNA target in the promoter and/or insufficient AD

potency (26,28,30,110). In some cases, inappropriate intersite spacing between multiple binding

sites can also lead to unproductive gene transcription, especially for a weak activator (123).

We examined the human aryl hydrocarbon receptor (AhR) and its partner aryl

hydrocarbon receptor nuclear translocator (Arnt), which are bHLH/PAS (basic helix-loop-

helix/Per-Arnt-Sim) proteins that regulate genes involved in the metabolism of carcinogens, such

as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyls (PCBs). As AhR

alone is incapable of DNA binding, Arnt regulates the transcriptional activity of AhR by

heterodimerization; thus, the AhR/Arnt heterodimer targets the cognate xenobiotic response

element (XRE), 5'-TNGCGTG, also known as the dioxin response element (DRE) (91-93,124-

126).

In this paper, we demonstrate that a false negative interaction between the AhR/Arnt

heterodimer and cognate XRE DNA site can be turned into a positive control in our modified

Y1H system (MY1H) developed to investigate simultaneous protein-protein and protein:DNA

interactions in vivo (127). The methodology presented may assist in trouble-shooting and

correcting false negatives that arise from unproductive transcription in yeast genetic assays.

42

3.3 Materials and methods Reagents were purchased from BioShop Canada (Burlington, ON), enzymes were


by Operon Biotechnologies (Huntsville, AL) unless otherwise stated.

3.3.1 Bacterial and yeast strains Escherichia coli SURE® strain (Stop Unwanted Rearrangement Events, Stratagene, La

Jolla, CA) or dam-/dcm- C2925 (New England Biolabs) was used for standard cloning and rescue

of plasmids from yeast cells. SURE cells were used for routine cloning of DNA with secondary

structures likely to be rearranged or deleted in conventional strains. C2925 is a

methyltransferase-deficient E. coli strain used for growth and purification of plasmids free of

dam and dcm methylation; this allows cloning to be performed with dam- or dcm-sensitive

restriction sites. Saccharomyces cerevisiae YM4271 [MATa, ura3-52, his3-200, ade2-101, lys2-

801, leu2-3, 112, trp1-901, tyr1-501, gal4-∆512, gal80-∆538, ade5::hisG] was purchased from

Clontech (Palo Alto, CA) and used for plasmid construction via homologous recombination and

reporter-strain construction.

3.3.2 Construction of reporter strains Four yeast reporter strains were classified into two sets: three-copy strains and six-copy

strains were created according to the Matchmaker™ One-hybrid System User Manual (Clontech)

for reporter assay analysis in the MY1H. Three-copy strains YM4271[pHISi-1/XRE-3] and

YM4271[pLacZi/XRE-3] contain three tandem copies of the consensus XRE (5’-TTGCGTG)

(128) upstream of the HIS3 and lacZ reporter genes, respectively. Similarly, the six-copy strains

YM4271[pHISi-1/XRE-6] and YM4271[pLacZi/XRE-6] contain six tandem copies of the

consensus XRE upstream of the HIS3 and lacZ reporters, respectively. 3-amino-1,2,4-triazole (3-

AT) titration assay revealed that 30 mM 3-AT, necessary for inhibition of background HIS3

43

expression, was sufficient to suppress background growth from both YM4271[pHISi-1/XRE-3]

and YM4271[pHISi-1/XRE-6] on SD/–His medium (minimal synthetic dropout medium lacking

histidine). Details of reporter strain construction are provided in the Supplemental Information.

3.3.3 Transformation, DNA preparation, and plasmid rescue Recombinant plasmids were transformed into E. coli by the standard TSS procedure

(108). Plasmids were isolated using the Wizard® Plus SV Minipreps DNA Purification System

(Promega, Madison, WI). Yeast transformations were performed using either the standard

lithium acetate method (Yeast Protocols Handbook, Clontech) or the transformation procedure

developed by Dohmen et al. (129). Transformants were selected by leucine prototrophy.

Isolation of yeast plasmids was performed using the Zymoprep™ II Yeast Plasmid Miniprep Kit

(ZymoResearch). PCR reactions were performed with Phusion™ high-fidelity DNA polymerase

(New England Biolabs). PCR products and DNA fragments for cloning were purified using

QIAquick Spin Kit, MinElute Kit, or QIAEX II Gel Extraction Kit (Qiagen, Mississauga, ON).

3.3.4 Plasmid Construction All new constructs were confirmed by DNA sequencing on an ABI (Applied Biosystems)

3730XL 96 capillary sequencer at the DNA Sequencing Facility in the Centre for Applied

Genomics, Hospital for Sick Children (Toronto, ON).

3.3.4.1 pCETT2 Plasmid pCETT2 (Figure 3.1) was constructed by homologous recombination in

YM4271 by insertion of a GAL4 AD gene upsteam of the MCS II site (multiple cloning site) in

pCETT (127). This was achieved by use of Sma I-linearized pCETT and the T2AD fragment

amplified by mutually primed synthesis (112) from pGAD424-MCS II (127) with

oligonucleotides 2-9 and 2-10 (Table 2.1 in Chapter 2 and Table D.1 in Appendix D). The

44

homology between the T2AD fragment and both ends of Sma I-linearized pCETT allows

generation of pCETT2. In pCETT2, the gene encoding amino acids 768–881 of the GAL4 AD is

inserted between PADH1(T) and PT7 upstream of both MCS I and MCS II (in pCETT, this gene is

inserted between PADH1(T) and PT7 upstream of MCS I only). Therefore, both monomers

expressed from pCETT2 are expressed as GAL4 AD fusion proteins.

Figure 3.1 Plasmid pCETT2 for coexpression of two AD fusion proteins in the MY1H system. The plasmid contains unique restriction sites located in the MCS I and MCS II regions. Both MCS I and MCS II are at the 3'-end of the open reading frame of the GAL4 AD sequence, allowing two fusion proteins, each of which combines the GAL4 AD and cloned protein of interest, to be expressed at low levels from a truncated constitutive ADH1 promoter.

3.3.4.2 pCETT/NAhR/NArnt and pCETT2/NAhR/NArnt Both human AhR (pRc-CMV/AhR) and human Arnt variant 3 (pRc-CMV/Arnt) cDNA

(130) were generously provided by Patricia Harper and Allan Okey (Department of

Pharmacology and Toxicology, University of Toronto). Details regarding the construction of the

human AhR6-436 fragment (NAhR) and the human Arnt82-464 fragment (NArnt) are provided

in the Supplemental Information. The NAhR and NArnt fragments contain cDNA fragments

45

encoding the bHLH/PAS domains of each protein, respectively. The NAhR fragment was

amplified and inserted into the Sal I and Xba I sites of MCS I in pCETT and pCETT2 to generate

pCETT/NAhR and pCETT2/NAhR, respectively. In order to be consistent with a previously

reported in vitro study where the dissociation constant (Kd) of the XRE DNA complex of the

NAhR/NArnt heterodimer was 2.0 nM as determined by electrophoretic mobility shift assay

(126), the NArnt fragment was based on the sequence of human Arnt variant 1, which we

generated from the human Arnt variant 3 template provided in pRc-CMV/Arnt. The NArnt

fragment was then inserted into the Bam HI and Xho I sites of MCS II in pCETT and pCETT2 to

give pCETT//NArnt and pCETT2//NArnt, respectively. (We note that the recombinant plasmids

pCETT and pCETT2 are named in the format "plasmid/MCS I/MCS II." When a gene is inserted

into MCS I only, the format is "plasmid/MCS I"; when a gene is inserted into MCS II only, the

format is "plasmid//MCS II.") Similarly, the NAhR fragment was inserted into the Sal I and Xba

I sites of pCETT//NArnt and pCETT2//NArnt to generate pCETT/NAhR/NArnt and

pCETT2/NAhR/NArnt, respectively.

3.3.5 HIS3 reporter assay The integrated HIS3 reporter strain was transformed with an AD-fusion plasmid. The

transformants were plated on SD/-H/-L plates. Interactions between the proteins under

investigation and corresponding DNA target element were determined by activation of the HIS3

reporter gene, which was measured by spotting 15 μL diluted fresh cell resuspensions at OD600

~0.01 on SD/-H/-L control plates and SD/-H/-L testing plates that contained 30 mM 3-AT. A

positive interaction was indicated by growth of colonies on both types of plates.

3.3.6 LacZ reporter assay Two commonly used β-galactosidase assays, qualitative X-gal colony-lift filter assay and

quantitative ortho-nitrophenyl-galactoside (ONPG) liquid assay, were performed as described

46

previously (127). In the ONPG assay, results are presented as mean values ± standard error

measurement (SEM) from 3-4 independent experiments, each performed in triplicate.

3.4 Results

3.4.1 Initial trials with pCETT: fusion of the GAL4 AD to NAhR only

Figure 3.2 HIS3 reporter assay for detection of NAhR/NArnt:XRE interaction from protein expression vector pCETT or pCETT2 in YM4271[pHISi-1/XRE-3] (A and C) and YM4271[pHISi-1/XRE-6] (B and D) strains. The transformants that coexpress both GAL4AD-NAhR and NArnt (or GAL4AD-NAhR and GAL4AD-NArnt in the cases of B and D) as well as their corresponding controls were grown as patches on SD/-H/-L control plates (left in each subfigure) that select only for the presence of plasmid; these were replica-plated on SD/-H/-L/30 mM 3-AT testing plates (right in each subfigure) that select for HIS3 reporter activity (30 °C, 5 days). The transformants on the same plate were identified according to the following order. A and B: upper left, pCETT/NAhR/NArnt; upper right, pCETT/NAhR; lower left, pCETT//NArnt; lower right, pCETT. C and D: upper left, pCETT2/NAhR/NArnt; upper right, pCETT2/NAhR; lower left, pCETT2//NArnt; lower right, pCETT2.

In order to construct a positive control in the study of XRE-binding proteins in our

MY1H system, we first tried to coexpress both NAhR and NArnt from pCETT in yeast strain

YM4271[pHISi-1/XRE-3], in which three copies of the XRE site were integrated into the yeast

genome upstream of the HIS3 reporter gene. Arnt can homodimerize, although it preferentially

heterodimerizes (124). In order to minimize the effect of Arnt homodimerization on reporter

activation, NAhR is expressed as the fusion to the GAL4 AD (GAL4AD-NAhR, AD at N

terminus), and NArnt is expressed as the independent protein with no AD fusion. The

transformant expressing GAL4AD-NAhR and NArnt did not grow on SD/-H/-L plates

containing 30 mM 3-AT, nor did the corresponding controls (Figure 3.2A). Because increasing

47

the copy number of XRE sites integrated upstream of the reporter gene may increase the

probability of the NAhR/NArnt heterodimer binding at the promoter and/or optimize the distance

between the promoter and site of transcription initiation, we constructed the six-copy strain

YM4271[pHISi-1/XRE-6], wherein six copies of the XRE were integrated upstream of the

reporter. However, coexpression of GAL4AD-NAhR and NArnt from pCETT in the

YM4271[pHISi-1/XRE-6] strain did not show positive signal by HIS3 assay as well (Figure

3.2B).

Figure 3.3 Colony-lift filter assay for detection of NAhR/NArnt:XRE interaction from protein expression vector pCETT or pCETT2 in YM4271[pHISi-1/XRE-3] (A and C) and YM4271[pHISi-1/XRE-6] (B and D) strains. The X-gal colony-lift assay was performed after 4 days growth of transformants that were transformed with pCETT/NAhR/NArnt (A and B) or pCETT2/NAhR/NArnt (C and D) and plated on SD/-L/-U plates at 30 °C. Photos were taken after 60 min incubation at 30 °C. The colony-lift filter assay results of their corresponding controls are shown in Figure B.1 in the Appendix B.

The X-gal colony-lift filter assay shows similar results to that of the HIS3 assay. After 60

minutes incubation, both three-copy and six-copy transformants that coexpress GAL4AD-NAhR

48

and NArnt proteins developed a faint blue color indistinguishable from their corresponding

controls (Figure 3.3A and B).

Figure 3.4 ONPG assay for detection of NAhR/NArnt:XRE interaction from protein expression vector pCETT or pCETT2 in YM4271[pHISi-1/XRE-3] (A and C) and YM4271[pHISi-1/XRE-6] (B and D) strains. Vertical axis indicates the mean values of β-galactosidase units. Error bars represent standard error measurements (SEM) from at least three independent trials conducted in triplicate.

The quantitative ONPG assay provides more information than the HIS3 and colony-lift

assays (Figure 3.4A and B). When pCETT is utilized as the coexpression vector, the control

transformant that contains vector only with no inserted genes gives background expression at

1.38±0.06 in the three-copy strain strain; in contrast, in the six-copy strain, the background is

4.83±0.30, and therefore, increasing the copy number of DNA target elements increases

49

background expression. When GAL4AD-NAhR and NArnt are coexpressed, the ONPG values

are 3.16±0.37 and 11.31±0.81 in the three- and six-copy strains, respectively. Although the

absolute value increases in the latter case, the actual signal-to-noise ratios (S/N) in both cases are

not statistically different (2.29 vs. 2.34).

3.4.2 Next generation trials with pCETT2: fusion of the GAL4 AD to both NAhR and NArnt

Plasmid pCETT2 is a double AD vector based on pCETT for coexpression of two AD

fusion proteins in the MY1H system (Figure 3.1). The difference between pCETT2 and pCETT

lies in the expression cassette of the second protein; in pCETT, the GAL4 AD is only fused to

the protein expressed from MCS I, while in pCETT2, the GAL4 AD is fused to proteins

expressed from both MCS I and II. In addition, the transcription of both genes is under the

control of the same truncated ADH1 promoter, leading to comparable low levels of protein

expression (113).

From pCETT2, we coexpressed GAL4AD-NAhR and GAL4AD-NArnt in yeast strain

YM4271[pHISi-1/XRE-3]. This time, transformants clearly grew normally on SD/-H/-L test

plates containing 30 mM 3-AT, while the corresponding controls, which expressed either

GAL4AD-NAhR or GAL4AD-NArnt or neither, were still clear on test plates (Figure 3.2C). The

same test was performed in YM4271[pHISi-1/XRE-6] with the same results (Figure 3.2D).

Because the HIS3 reporter assay we performed is qualititative and only shows growth or death,

this assay did not indicate a difference in signal from the three- or six-copy strain.

Results from the X-gal colony-lift filter assay were consistent with the HIS3 reporter

assay. The cells transformed with pCETT2/NAhR/NArnt developed vivid blue color in the filter

assay, regardless of whether the plasmid was transformed into the three- or six-copy strain

(Figure 3.3C and D). The corresponding controls that express only one of the two proteins or

50

neither showed no blue color development even after 60 minutes incubation (Figure B.1C and D

in the Appendix B). Furthermore, when GAL4AD-NAhR and GAL4AD-NArnt were

coexpressed in the six-copy strain, blue color began to show after twenty minutes, while in the

three-copy strain, color started to develop after forty minutes; after one hour incubation, the

former transformant showed blue color much more intense than the latter.

The ONPG assay quantitatively confirmed the results obtained from both HIS3 and

colony-lift assays. When GAL4AD-NAhR and GAL4AD-NArnt were coexpressed in

YM4271[pLacZi/XRE-3], the ONPG value was 3.82±0.13; its corresponding negative control

transformed with pCETT2 gave ONPG value 1.59±0.13 (Figure 3.4C) corresponding to S/N 2.40.

In contrast, when GAL4AD-NAhR and GAL4AD-NArnt were coexpressed in

YM4271[pLacZi/XRE-6], the ONPG value was 62.3±5.9; its corresponding negative control

transformed with pCETT2 gave ONPG value 5.67±0.52 (Figure 3.4D) corresponding to S/N

10.98. In addition, ONPG values from these two negative controls in yeast strains with different

copy numbers of XRE sites indicate again that increasing the copy number of DNA target

elements also increases the background expression (1.59±0.13 for three-copy strain vs. 5.67±0.52

for six-copy strain).

As expected, the controls that express either GAL4AD-NAhR or GAL4AD-NArnt only

give ONPG values close to their corresponding negative controls that express GAL4 AD only.

Although Arnt can homodimerize and target the symmetric sequence 5'-CACGTG (131), we did

not anticipate that the Arnt homodimer would target the asymmetric XRE sequence, 5'-

TTGCGTG, and our data show that GAL4AD-NArnt displays activity indistinguishable from

backgound.

51

3.5 Discussion

3.5.1 Are false negatives due to unproductive transcription? Yeast model systems have already been used to study the protein-protein and

protein:DNA interactions of AhR and Arnt. In 1995, successful isolation of a cDNA encoding

Arnt was reported by using the recombinant AhR bHLH/PAS domain as a probe in cDNA

library screening in the Y2H (91); this work was followed by two similar systems developed to

investigate the AhR/Arnt heterodimer's (full-length or bHLH/PAS domain only) response to

different AhR ligands in the Y1H system (92,93). We therefore believe the factors leading to the

false negative observations in our initial experiments with pCETT may include unproductive

transcription of the reporter gene from inappropriate positioning of XRE sites in the promoter

region, masking effects of endogenous proteins that interact with DNA sites in the promoter

region, steric hindrance from the NAhR/NArnt heterodimer that adversely affect transactivation

of the GAL4 AD, and/or insufficient transactivation potency of a single AD on the heterodimer.

The above possibilities can be classified into two categories: target DNA element and AD.

There are numerous factors that affect levels of gene transcription. From the standpoint of

artificial transcription factor design, by using a DBD that binds its cognate response element

with higher affinity or by using an AD that possesses stronger activation potency, levels of gene

transcription can be increased (132). Alternatively, increasing the potential for the DBD to find

its specific target element by increasing the number of cognate DNA sequences in the promoter

(132,133) or multimerizing the AD on a single transcriptional factor (134) can also produce

similar effects. Although intersite spacing between multiple binding sites can also be a critical

factor for effective gene transcription (123), given that high background was obtained when a

spacer of 4 bp or 6 bp was inserted between adjacent XRE binding sites, tandem copies of

52

multiple XRE binding sites without intersite spacing sequences eventually became our desired

option.

The NAhR/NArnt:XRE interaction in our MY1H initially showed a false negative. In

order to turn this false negative into a true positive, the tactics mentioned above were combined

to increase the levels of reporter gene transcription.

3.5.2 Copy number of XRE target sites Transcription factor binding sites can function as regulated enhancer elements when

multimerized and ligated to a promoter (132,135). Therefore, increasing the copy number of

XRE sites will likely increase the number of transcription factors bound in the promoter region,

thereby synergistically increasing the activation potency of the reporter gene. As a matter of fact,

it is not uncommon in nature for multiple DNA response elements to exist in a given gene's

promoter region. For instance, yeast transcriptional activator GAL4 binds to multiple sites on

DNA to activate transcription synergistically; the presence of two such sites can more than

double the level of transcription from a single site (136). In the case of XRE, at least six XRE

sites have been identified in the upstream region of both rat and human CYP1A1 genes (137),

and eight XREs are located within 2.3 kb of the 5'-flanking region of human CYP1B1 gene (138).

Although it is unclear as to the synergistic activation roles of these different XREs on

gene regulation, in vitro experiments revealed that the AhR/Arnt heterodimer has different

affinities for these XRE variants sharing the 5'-GCGTG core sequence (137,138). These different

XRE sites in the same promoter may be used to perform complicated regulation of downstream

gene expression, as in the case of cI repressor and Cro proteins in phage λ or GAL4 in yeast

(136,139). Alternatively, it is also possible that these XRE sites are just evolutionary footprints

from the different positions where XRE sites were placed to find a favorable spatial

conformation for optimal, synergistic gene regulation. These trials eventually led to a fraction of

53

the total sites being physiologically relevant. Therefore, the presence of more XRE sites may

simply enable transcription factors to perform their regulatory functions more advantageously.

However, the presence of more target elements in the promoter region also risks

increased nonspecific interactions from endogenous biomolecules. Background expression will

be more “leaky” as the number of DNA targets increases. Indeed, background expression

increases by 3.6-fold in our studies when the number of XRE sites integrated into the yeast

genome increases from three to six. The increase in background expression can become

overwhelming when too many binding sites are present in the promoter. We constructed a yeast

strain with twenty-three integrated copies of XRE sites in a pilot experiment and found that

reporter gene activation from the negative control was so high that in the X-gal colony-lift filter

assay, the negative controls developed blue color within five minutes and intense blue after 15

minutes of incubation.

When we increased the number of XRE sites integrated into the yeast genome, we still

observed false negatives in our first trials with pCETT. Thus, we re-evaluated the role of

increased the number of target elements, which may be only one of many factors responsible for

false negatives. As insufficient transactivation potency from the single AD may also contribute

to the false negative (140), the double AD expression vector pCETT2 was additionally employed

to increase gene transcription levels.

3.5.3 Double AD system In addition to increasing the number of transcription factors bound to DNA to trigger

synergistic activation (136), oligomerization of the AD on a single transcriptional factor is

another strategy for enhancement of gene transcription. For example, a transcription factor

containing two copies of the VP16 AD upregulated transcription 5-fold over the single copy AD

activator (134).

54

Our strategy for AD oligomerization differs from that discussed above due to the

heterodimeric nature of the NAhR/NArnt complex. By use of the double AD coexpression

plasmid pCETT2, heterodimerization of GAL4AD-NAhR and GAL4AD-NArnt intrinsically

leads to doubling the local concentration of ADs present at the promoter. This double AD

heterodimer is somewhat different in structure from the multimerized AD on a single

transcriptional factor, but we expected our strategy to yield a similar effect.

When both NAhR and NArnt proteins were coexpressed as GAL4 AD fusion proteins in

strain YM4271[pLacZi/XRE-3], the sensitive HIS3 reporter selection assay showed positive

growth, demonstrating that doubling the AD concentration in the promoter region lead to

detectable transcriptional activation in the MY1H. However, the ONPG assay showed the

synergistic activation of this double AD in the three-copy strain to be insignificant (S/N 2.40).

Once the two fusion proteins were coexpressed in strain YM4271[pLacZi/XRE-6], the

synergistic activation of this double AD was so considerable that S/N increased over 4-fold

compared with the S/N ratios for the single AD heterodimer in the six-copy strain or the double

AD heterodimer in the three-copy stain. This result proves that both increasing the number of

DNA binding sites and increasing the number of activation domains contribute to the

considerable augmentation of transcription potency of the reporter gene through synergistic

activation, and that implementation of both strategies was critical for avoiding the false negative

result.

3.5.4 Synergistic activation is due to both increase of XRE target sites and doubling of AD

The data from trials with pCETT and pCETT2 reveal three important points. First,

increase of the number of XRE sites in the yeast genome from three to six raises background

expression. Thus, it is feasible to “amplify” the signal from a specific protein:DNA interaction

55

by increasing the copy number of DNA elements. However, the presence of too many DNA

target elements can adversely affect the differentiation of true positive and negative signals.

Second, only when both NAhR and NArnt are expressed as GAL4 AD fusion proteins can the

reporter be activated in the MY1H system. This result indicates that the double AD system leads

to more efficient reporter transcription. Third and most significantly, the considerable increase in

S/N requires both increase of the copy number of XRE sites and attachment of ADs to both

NAhR and NArnt. Overall, the data indicate that the combination of increasing target element

number and increasing the concentration of activation domains can make a false negative truly

positive.

Another potential bonus of introducing synergistic activation into our MY1H system is

that it increases the chances for detection of only weakly active proteins that would be

considered inactive, and therefore missed, during testing of large libraries by genetic assays.

In summary, by increasing the copy number of XRE sites integrated into the yeast

genome and doubling the GAL4 activation domains, we successfully turned a false negative

NAhR/NArnt:XRE interaction into a true positive control that paves the way to further study of

XRE-binding proteins in our MY1H system. Given that there is no general means for addressing

all possible false positives and false negatives, the methodology we present here provides an

effective, productive way to pinpoint false negatives that arise from unproductive transcription.

3.6 Supplemental Information Supplemental Information associated with this manuscript is provided in Appendix B.

56

Chap 4ter

Forced protein heterodimerization and specific DNA binding to a nonpalindromic DNA sequence in vivo and in vitro: bHLHZ-like hybrid heterodimers of bHLH/PAS proteins AhR and Arnt and

bZIP proteins JunD and Fos as a model Gang Chen, Antonia T. De Jong, S. Hesam Shahravan, and Jumi A. Shin

(Short running title: DNA binding forces heterodimerization of hybrids of bHLH/PAS and bZIP)

Contributions:

I initiated this project and performed all the in vivo experiments. Antonia De Jong and

Hesam Shahravan are now working on protein expression and purification and the in vitro

quantitative fluorescent anisotropy analysis. I completed the in vivo data interpretation and wrote

the manuscript. Jumi Shin provided supervision and intellectual input to the project and

manuscript.

57

4.1 Abstract The molecular basis of specific partner selection and DNA binding of the basic helix-

loop-helix (bHLH) superfamily of dimeric transcription factors is of great interest to both

biologists and chemists. The bHLH proteins use the helix-loop-helix dimerization domain to

form a four-helix bundle, which positions the contiguous basic regions for targeting the DNA

major groove. In bHLHZ and bHLH/PAS proteins, dimerization is further controlled by an

adjacent leucine zipper (LZ) or Per-Arnt-Sim homology (PAS) domain, respectively. Domain

swapping was employed to probe the relationship between the bHLH and PAS domains of the

bHLH/PAS proteins AhR and Arnt as well as their roles in specific DNA binding. Two hybrid

proteins, AhRJunD and ArntFos, were designed wherein the leucine zippers of JunD and Fos

were fused to the bHLH domains of AhR and Arnt, respectively. In the modified yeast one-

hybrid (MY1H) system, the HIS3 assay and two lacZ-based assays, X-gal colony-lift assay and

quantitative ONPG liquid assay, demonstrate that these two bHLH•LZ hybrids specifically

bound to the XRE cognate sequence with enhanced DNA-binding affinity relative to the

heterodimer of the truncated AhR and Arnt bHLH domains, with no PAS or LZ domains. The

JunD or Fos LZ domains may serve to induce properly folded, α-helical structure that stabilizes

the hydrophobic HLH dimerization interface as a bundle of four parallel α-helices.

Although each monomer is able to homodimerize, as demonstrated in the Y2H, we

demonstrate that the presence of the nonpalindromic XRE cognate sequence forces

heterodimerization of the two hybrid monomers for specific DNA binding in vivo. These hybrids

provide a scaffold for design of dimeric helical proteins forced to heterodimerize by a specific

DNA ligand and might be informative for further exploration of the relationship between protein

structure and DNA-binding function. Moreover, they provide additional evidence that supports

the hypothesis of domain shuffling in the evolutionary pathway of the bHLH superfamily.

58

4.2 Introduction The basic helix-loop-helix (bHLH) proteins are a large and diverse class of DNA-binding

proteins that target specific DNA sites for transcriptional regulation of genes involved in

fundamental biological processes, such as cell cycle and developmental regulation, apoptosis and

homeostasis, and stress response pathways (57-60). The bHLH proteins typically associate in

homo- or heterodimeric complexes that recognize the hexameric E-box DNA site, 5'-CANNTG

(58,65). Members of the bHLH superfamily share a highly conserved structural motif comprising

a DNA-binding basic region and a dimerization domain. Similar to that in bZIP proteins, the

basic region in bHLH proteins targets the DNA major groove. The dimerization domain

comprises two amphipathic helices separated by a nonconserved loop typically 5-12 residues in

length; upon dimerization, a compact hydrophobic four-helix bundle forms that positions the

contiguous basic regions for DNA binding (61,70, Kewley, 2004 #206,141).

Two subclasses of bHLH proteins, namely the bHLHZ and bHLH/PAS families, contain

an additional leucine zipper (LZ) or Per-Arnt-Sim (PAS) domain contiguous with the bHLH

domain, respectively, which further regulates protein dimerization (72,142). The LZ region in

bHLHZ proteins is believed to contribute to the overall stability of the proteins as well as to

determine partner selection (69,70).

In comparison, the PAS domain in bHLH/PAS proteins functions as a conserved

signaling module in response to environmental stimulus (128,143-146). Structurally, the PAS

domain is known to be involved in protein dimerization and specification of heterodimerization

partner and may also directly interact with the DNA target element as well as the adjacent bHLH

domain (72,74). However, the function of the PAS domain in protein dimerization and DNA

binding is far from being well understood. Although high-resolution structural data of the bHLH

domain of bHLH/PAS proteins is not available, the bHLH domains of bHLH/PAS proteins are

59

predicted to adopt a similar DNA-binding mode and to be structurally similar to the closely

related bHLH and bHLHZ proteins, based on their homologous primary sequences and DNA-

binding specificities (59).

AhR and Arnt are bHLH/PAS proteins that heterodimerize in the presence of

environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and

polychlorinated biphenyls (PCBs). The activated complex binds specific xenobiotic response

elements (XREs, also known as the dioxin response element, DRE), 5'-TNGCGTG, and activates

genes encoding xenobiotic metabolizing enzymes (128,143-145). Biochemical and genetics

studies have concluded that the AhR/Arnt heterodimer functions by binding the atypical XRE

cognate sequence (79,80): AhR targets the 5'-TNGC half site, and Arnt the 5'-GTG half site.

Arnt is able to homodimerize and target the E-box site (124,126,131,147). However, AhR is

unable to form homodimers and can only function through heterodimerization with Arnt.

In order to gain more insight into the role the PAS domains of AhR and Arnt play in

protein dimerization and DNA binding, and to further understand DNA recognition by

heterodimeric proteins, we constructed a pair of bHLHZ-like hybrid proteins, in which the bHLH

domains of AhR and Arnt are fused to the LZ regions of bZIP proteins JunD and Fos,

respectively (AhRJunD and ArntFos). We used in vivo yeast genetic assays and are currently

conducting in vitro quantitative fluorescence anisotropy titrations to explore their dimerization

and specific DNA-binding activities. In vivo results reveal that the presence of the

nonpalindromic XRE cognate sequence forces heterodimerization of the two hybrids for specific

DNA binding.

This chapter describes the results of the dimerization and specific DNA binding of

AhRJunD and ArntFos obtained from MY1H and Y2H assays. The in vitro quantitative

fluorescence anisotropy assays to assess the free energies of protein:DNA binding are ongoing,

60

as well as western blotting assays to examine the expression of the hybrid proteins examined in

the Y2H. The data presented here is not complete, and work remains to confirm these

interactions observed in vivo and accordingly determine the affinities of the corresponding

interactions in vitro.

4.3 Materials and methods Reagents were purchased from BioShop Canada (Burlington, ON), enzymes were


by Operon Biotechnologies (Huntsville, AL) or Integrated DNA Technologies (Coralville, IA)

unless otherwise stated.

4.3.1 Bacterial and yeast strains Escherichia coli DH5α (Stratagene, La Jolla, CA), SURE® strain (Stop Unwanted

Rearrangement Events, Stratagene), or dam-/dcm- C2925 (New England Biolabs) was used for

standard cloning and rescue of plasmids from yeast cells. SURE cells were used for routine

cloning of DNA with secondary structures likely to be rearranged or deleted in conventional

strains. C2925 is a methyltransferase-deficient E. coli strain used for growth and purification of

plasmids free of dam and dcm methylation; this allows cloning to be performed with dam- or

dcm-sensitive restriction sites.

Saccharomyces cerevisiae YM4271 [MATa, ura3-52, his3-200, ade2-101, lys2-801, leu2-

3, 112, trp1-901, tyr1-501, gal4-∆512, gal80-∆538, ade5::hisG] was used for reporter strain

construction. Two yeast reporter strains, YM4271[pHISi-1/XRE-6] and YM4271[pLacZi/XRE-

6], for reporter assay analysis in the MY1H were created as reported previously (148). These two

strains contain six tandem copies of the consensus xenobiotic response element (XRE), 5'-

TTGCGTG, upstream of the HIS3 and lacZ reporter genes, respectively. 3-amino-1,2,4-triazole

(3-AT) titration assay revealed that 30 mM 3-AT, a competitive inhibitor of the HIS3 gene

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product leading to background expression, was sufficient to suppress YM4271[pHISi-1/XRE-6]

background growth on SD/-H medium. AH109 [MATa, trp1-901, leu2-3, 112, ura3-52, his3-200,

gal4∆, gal80∆, LYS2 : : GAL1UAS-GAL1TATA-HIS3, GAL2UAS-GAL2TATA-ADE2, URA3 : :

MEL1UAS-MEL1 TATA-lacZ, MEL1] was used for assessing protein-protein interactions in the Y2H

system. Both YM4271 and AH109 were purchased from Clontech (Palo Alto, CA).

4.3.2 Transformation, DNA preparation, and plasmid rescue The procedures for molecular cloning, plasmid harvesting, and yeast transformation were

performed as described previously (127,148). The following is a brief summary. PCR reactions

were performed with Phusion™ high-fidelity DNA polymerase (New England Biolabs). PCR

products and DNA fragments for cloning were purified using the corresponding Qiagen DNA

purification kits (Qiagen, Mississauga, ON). Recombinant plasmids were transformed into E.

coli by the standard TSS procedure (108) and harvested from bacteria using the Wizard® Plus

SV Minipreps DNA Purification System (Promega, Madison, WI). Yeast transformations were

performed using either the standard lithium acetate method (Yeast Protocols Handbook,

Clontech) or the transformation procedure developed by Dohmen et al. (129).

4.3.3 Plasmid Construction Detailed information of plasmid construction is provided in the Supplemental

Information. All new constructs were confirmed by dideoxynucleotide DNA sequencing

performed at The Centre for Applied Genomics, The Hospital for Sick Children (Toronto, ON).

4.3.3.1 pCETT2/AhRbHLH/ArntbHLH All AhR and Arnt fragments used in this study were based on the human AhR and Arnt

cDNA (130) generously provided by Patricia Harper and Allan Okey (Department of

Pharmacology and Toxicology, University of Toronto). The AhRbHLH sequence encoding

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amino acids 20-90 of human AhR was amplified from AhR cDNA and cloned into MCS I of

pCETT2 to generate pCETT2/AhRbHLH. The ArntbHLH sequence encoding amino acids 82-

149 of human Arnt isoform 1 was amplified from the Arnt82-464 cDNA fragment (148) and

cloned into MCS II of pCETT2 and pCETT2/AhRbHLH to generate pCETT2//ArntbHLH and

pCETT2/AhRbHLH/ArntbHLH, respectively.

4.3.3.2 pCETT/AhRJunD/ArntFos and pCETT2/AhRJunD/ArntFos The AhRJunD fragment encoding a hybrid protein of human AhR20-86 fused to human

JunD296-332 was ligated into MCS I of both pCETT and pCETT2 to generate pCETT/AhRJunD

and pCETT2/AhRJunD, respectively. The ArntFos fragment encoding a hybrid protein of human

Arnt82-148 fused to human Fos165-201 was similarly ligated into MCS II of both

pCETT/AhRJunD and pCETT2/AhRJunD to generate pCETT/AhRJunD/ArntFos and

pCETT2/AhRJunD/ArntFos, respectively. The ArntFos fragment was also inserted into MCS II

of pCETT and pCETT2 to generate pCETT//ArntFos and pCETT2//ArntFos, respectively.

We note that AhRJunD contains AhR20-86 and ArntFos contains Arnt82-148, both of

which encompass the full-length bHLH domains of AhR and Arnt, respectively. This is in

comparison to AhR20-90 in the AhRbHLH construct and Arnt82-149 in the ArntbHLH construct

above that contain four and one additional amino acid(s) C-terminal to Helix 2 of AhR and Arnt,

respectively (Figure 4.1).

4.3.3.3 pCETT2/AhR(ΔL)JunD/ArntFos The AhR(ΔL)JunD fragment was constructed similarly to AhRJunD and ligated into

MCS I of both pCETT2 and pCETT2//ArntFos to generate pCETT2/AhR(ΔL)JunD and

pCETT2/AhR(ΔL)JunD/ArntFos, respectively. Compared with the AhRJunD fragment, the

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codon encoding Leu296 of JunD in AhR(ΔL)JunD was deleted. Therefore, this fragment encodes

a hybrid protein of human AhR20-86 fused to human JunD297-332 (Figure 4.1).

4.3.3.4 Plasmids constructed for the Y2H assay The aforementioned five fragments (AhRbHLH, ArntbHLH, AhRJunD, AhR(ΔL)JunD,

and ArntFos) were amplified with appropriate primers, and each was ligated into both GAL4

DNA-bidning domain (DBD) fusion vector pGBKT7 and GAL4 activation domain (AD) fusion

vector pGADT7 using the Matchmaker™ Two-hybrid System 3 (Clontech).

4.3.4 HIS3 reporter assay The HIS3 reporter assays were performed as described previously (148). A brief

summary of this procedure follows: fresh colonies of YM4271[pHISi-1/XRE-6] transformed

with an appropriate AD-fusion plasmid were resuspended in sterile water and then diluted in 10-

fold steps to ~0.01. 15 μL of each diluted resuspension were spotted on SD/-H/-L control plates

and on SD/-H/-L/ test plates containing 30 mM 3-AT. A positive interaction was indicated by the

growth of cultures on both types of plates.

4.3.4.1 Spot titration assay The HIS3 reporter gene was also used to compare the transactivation potency of different

GAL4 AD fusion proteins. Ten-fold serial dilutions of fresh colony resuspensions were

generated such that the diluted cultures of each transformant gave OD600 from 10-1 to 10-4. 15 µL

of each dilution were spotted on SD/-H/-L plates and SD/-H/-L test plates containing 30 mM 3-

AT. The relative strengths of reporter gene activation were estimated by comparing the growth

status of different transformants with the same cell densities on the test plates.

64

4.3.5 LacZ reporter assay Two commonly used β-galactosidase assays, qualitative X-gal colony-lift filter assay and

quantitative ortho-nitrophenyl-galactoside (ONPG) liquid assay, were performed as described

before (127). In the ONPG assay, results are presented as mean values ± standard error of the

mean (SEM) of 3-4 independent experiments, each performed in triplicate.

4.3.6 Protein-protein interactions in the Y2H system The Matchmaker™ GAL4 Two-hybrid System 3 (Clontech) was utilized to examine

homo- and heterodimerization of the proteins under investigation. The recombinant plasmids of

DBD fusion vector pGBKT7 and AD fusion vector pGADT7 (or their respective parental vectors

in the controls) were cotransformed into strain AH109. Transformed cells were selected on SD/-

L/-W plates at 30 °C, 4 days. Interactions between the two proteins under investigation were

determined by simultaneous activation of the HIS3, ADE2, and MEL1 reporter genes. Activation

of the three reporter genes was measured by spotting 15 μL diluted fresh cell resuspensions at

OD600 ~0.01 on SD/-L/-W plates and on SD/-A/-H/-L/-W/X-α-Gal plates. A positive interaction

was indicated by the growth of cultures on both types of plates, with blue colony color showing

on the latter plates. The X-α-Gal indicator plates were prepared as per the Yeast Protocol

Handbook, Clontech.

4.3.6.1 Spot titration assay Activation strength of two-hybrid activity was measured by spotting 15 μL of 10-fold

serial dilutions (OD600: 10-1-10-5) of fresh colony resuspensions at OD600 0.1 on both SD/-H/-L

control plates and SD/-A/-H/-L/X-α-Gal plates.

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4.4 Results The original goal of our design was to generate a protein heterodimer that targets a

specific cognate DNA element. Several considerations were made in the process of protein

design. First, we surmised that compared with palindromic or pseudopalindromic sites,

nonpalindromic sites are more suitable for heterodimer:DNA interactions in that these sites are

normally not targeted by homodimers, therefore minimizing the possibility of homodimeric

interference with DNA binding. So the atypical nonpalindromic XRE site targeted by the native

AhR/Arnt heterodimer became our ideal candidate; as a positive control, the interaction between

the bHLH/PAS domains of the AhR and Arnt proteins and XRE DNA site showed positive

signal in the MY1H (148). Second, in order for the hybrid proteins to target the specific DNA

site, the ideal scenario would be that neither of the two monomers be able to form homodimers,

and that heterodimerization of the two monomers must be of high affinity and stability, as it is

unlikely that monomeric proteins containing disordered basic regions strongly target the specific

DNA site without partner participation.

Arnt is able to homodimerize and bind to the 5'-CACGTG sequence (124,126,131,147),

and it serves as a common heterodimerization partner within the bHLH/PAS family to regulate a

number of proteins such as AhR, HIF-1α and SIM proteins (149). AhR alone, in contrast, is

unable to bind DNA. Jun and Fos, members of the bZIP superfamily, act in parallel fashion to

AhR and Arnt, in that Jun is able to form homodimers and is a common heterodimerization

partner within the Fos subfamily to regulate a number of proteins including c-Fos, whereas c-Fos

is restricted to pairing with Jun family members only (42). Additionally, Jun and Fos

preferentially heterodimerize rather than homodimerize, again similar to AhR and Arnt (150).

Members of the Jun subfamily have been shown to homodimerize with different affinities, and

JunD homodimerizes with lower affinity than does c-Jun (151).

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Our aim was to generate small bHLHZ-like hybrid protein structures that strongly

preferred to heterodimerize and bind a desired DNA site, in comparison with the much larger

native bHLH/PAS domains: the typical LZ is approximately 30 aa, whereas the PAS domain

exceeds 300 aa. These heterodimeric hybrid proteins of nonnative structure were also designed to

maintain the high DNA-binding affinity and sequence specificity of the full-length native

proteins.

MaxbHLHZ

basic region helix 1 loop helix 2 :

AhRJunD: AhR(ΔL)JunD

:

ArntFos:

MaxbHLHZ: AhRJunD:

AhR(ΔL)JunD

: ArntFos: AhR(20-90): Arnt(82-149):

ADKRAHHNALERKRR-DHIKDSFHSLRDSVP-SLQGEKAS -RAQILDKATEYIQYM- QKTVKPIPAEGIKSNPSKRHR-DRLNTELDRLASLLP-FPQDVINKLD-KLSVLRLSVSYLRAK- QKTVKPIPAEGIKSNPSKRHR-DRLNTELDRLASLLP-FPQDVINKLD-KLSVLRLSVSYLRAK- SSADKERLARENHSEIERRRR-NKMTAYITELSDMVP-TCSALARKPD-KLTILRMAVSHMKSL- ucine zipper leRRKNDT QQDIDD KRQNAL EQQVRA H L L LSFFDVA EEKVKT KSQNTE ASTASL REQVAQ KQKVLSHV L L L L LSFFDVA EEKVKT KSQNTE ASTASL REQVAQ KQKVLSHV- L L L L RGTGNTLQAETDQLEDEKSALQTEIANLLKEKEKLEFILAAHR basic region helix 1 loop helix 2

QKTVKPIPAEGIKSNPSKRHR-DRLNTELDRLASLLP-FPQDVINKLD-KLSVLRLSVSYLRAKSFF-DVALKSS SSADKERLARENHSEIERRRR-NKMTAYITELSDMVP-TCSALARKPD-KLTILRMAVSHMKSLRGT-GNTS

Figure 4.1 Primary sequence alignment of the native Max bHLHZ domain and bHLH•LZ hybrids. The sequence alignment is based on refs. (142) and (61). The bHLHZ region of reference sequence Max was aligned with the putative bHLH regions of AhR and Arnt, as well as the LZ of JunD and Fos. The bHLH domains of AhR or Arnt were fused to the LZ domains of JunD or Fos, respectively; the hydrophobic heptad repeat register of Helix 2 and the LZ is not maintained in AhR(ΔL)JunD, as the first leucine in the JunD LZ is deleted. The leucines/hydrophobic residues that define the leucine zipper motif are highlighted in bold, and the highly conserved residues in the basic regions of Max and Arnt are underlined.

We therefore decided to fuse the JunD LZ to the bHLH domain of the AhR protein, and

the c-Fos leucine zipper to the bHLH domain of the Arnt protein; the LZ is directly fused to

Helix 2 of the bHLH domain in order to maintain the register of the hydrophobic heptad repeat

that forms the protein-protein interface. Although the groups of Brennan and Whitelaw showed

that just the Arnt bHLH domain is sufficient for binding of the palindromic E-box DNA site (5'-

CACGTG) under certain conditions in in vitro studies (142,152), we found the Arnt bHLH

domain incapable of targeting the E-box in in vivo yeast studies (153). Therefore, we added the

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LZ not only to provide additional dimerization interface and promote heterodimerization, but

also to provide stability and nucleate the α-helical structure necessary for proper folding and

solubility. Although there are a few endogenous bZIP proteins present in yeas such as GCN4 and

Yap family proteins, it is unlikely that they interfere with the JunD/Fos LZ pair, as no interaction

between JunD or Fos zipper and either of the yeast bZIP proteins has been observed (151).

In order to better understand the role the leucine zipper plays in the dimerization structure

and DNA-binding function of the hybrid proteins, we also constructed the mutant AhR(ΔL)JunD,

in which the first leucine residue of the JunD LZ immediately after Helix 2 of AhR bHLH is

deleted—hence, disruption of the heptad repeat and hydrophobic interface (Figure 4.1).

4.4.1 The AhRJunD/ArntFos and AhR(ΔL)JunD/ArntFos heterodimers bind the XRE site in the MY1H

Two reporter assays, HIS3 and lacZ, were utilized to examine our protein design in the

MY1H system. These reporter assays provide indirect, qualititative (or semi-quantitative)

measurement of the DNA-binding affinities of the hybrid proteins under investigation. Because

transcriptional potency of reporter activation generally correlates with the strength of the

interaction under investigation (25), these in vivo measurements provide a reasonable guide

regarding the DNA-binding affinities of the hybrid proteins.

We first coexpressed both GAL4AD-AhRJunD and GAL4AD-ArntFos hybrid proteins,

which are fused to the GAL4 activation domain (GAL4AD), by use of pCETT2 in yeast strain

YM4271[pHISi-1/XRE-6] (Figure 4.2A), in which six tandem copies of the XRE site were

integrated into the yeast genome upstream of the HIS3 reporter gene. The transformant grew on

SD/-H/-L/30 mM 3-AT test plates, whereas its corresponding controls, in which only one of the

two hybrid proteins or none was expressed, did not grow on test plates; therefore, neither

AhRJunD nor ArntFos is capable of binding to the XRE site without a heterodimerization partner.

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Thus, the transcriptional activation of the HIS3 reporter gene in the XRE-integrated strain

requires the presence of both GAL4AD-AhRJunD and GAL4AD-ArntFos hybrid proteins. These

results indicate that in yeast, AhRJunD and ArntFos must heterodimerize and bind to the XRE

site, as the basic regions determine the DNA-binding specificity.

Figure 4.2 HIS3 reporter assay for detection of interactions between heterodimers and the XRE cognate sequence. The heterodimeric pairs of (A) AhRJunD/ArntFos, (B) AhR(ΔL)JunD/ArntFos, and (C) AhRbHLH/ArntbHLH were coexpressed by use of protein expression vector pCETT2 in strain YM4271[pHISi-1/XRE-6]. The transformants that coexpress both GAL4AD-fused monomers, as well as their corresponding controls, were grown as patches on SD/-H/-L control plates (left in each subfigure) that select only for the presence of the plasmid and replica-plated on SD/-H/-L/30mM 3-AT test plates (right in each subfigure) that select for HIS3 reporter activity (incubated at 30 °C, 5 days). The transformants on the same plate are identified according to the following order. (A) upper left: pCETT2/AhRJunD/ArntFos, upper right: pCETT2/AhRJunD, lower left: pCETT2//ArntFos, and lower right: pCETT2; (B) upper left: pCETT2/AhR(ΔL)JunD/ArntFos, upper right: pCETT2/AhR(ΔL)JunD, lower left: pCETT2//ArntFos, and lower right: pCETT2; (C) upper left: pCETT2/AhRbHLH/ArntbHLH, upper right: pCETT2/AhRbHLH, lower left: pCETT2//ArntbHLH, and lower right: pCETT2.

The coexpression of GAL4AD-AhR(ΔL)JunD and GAL4AD-ArntFos hybrids in the

same HIS3 reporter strain (Figure 4.2B) displayed similar results to that of GAL4AD-AhRJunD

and GAL4AD-ArntFos. Again, only the transformant that coexpresses both hybrid proteins was

69

able to grow on SD/-H/-L/30 mM 3-AT plates. The cells expressing only GAL4AD-

AhR(ΔL)JunD did not grow on the test plates. These data reveal that AhR(ΔL)JunD is also able

to heterodimerize with ArntFos, and this heterodimer targets the XRE as does the

AhRJunD/ArntFos heterodimer.

In contrast, the cells that coexpress GAL4AD-AhRbHLH and GAL4AD-ArntbHLH did

not grow on 3-AT containing test plates (Figure 4.2C), indicating that coexpression of just the

bHLH domains of AhR and Arnt is insufficient to trigger reporter gene activation.

Figure 4.3 HIS3 reporter spot titration assay for comparison of the relative strengths of AhRJunD/ArntFos:XRE and AhR(ΔL)JunD/ArntFos:XRE interactions. YM4271[pHISi-1/XRE-6] cells were transformed with either pCETT2/AhRJunD/ArntFos or pCETT2/AhR(ΔL)JunD/ArntFos and plated on SD/-H/-L plates. Fresh colonies were first resuspended in 1 mL H2O and diluted to OD600 0.1. 15 μL of 10-fold serial dilutions (OD600: 10-1 to 10-4) of the resuspensions at OD600 0.1 were spotted on SD/-H/-L plates and on SD/-H/-L plates containing 30 mM 3-AT.

Although the HIS3 reporter assay is qualititative and only shows growth or death, the

survival rate of a particular transformant in the presence of 3-AT generally correlates with

transcriptional activity (116). Therefore, lower survival of transformants on 3-AT-containing

plates indicates weaker transcriptional potency of the GAL4 AD fusion, probably caused by

weaker affinity of the protein of interest to its response element (25). As both AhRJunD and

AhR(ΔL)JunD are able to heterodimerize with ArntFos and target the XRE site, we performed a

spot titration assay to differentiate the transcriptional potencies of the transformants that

coexpress GAL4AD-ArntFos with either GAL4AD-AhRJunD or GAL4AD-AhR(ΔL)JunD

(Figure 4.3). Fresh colony dilutions of both transformants at a gradient of cell densities from

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OD600 10-1 to 10-4 were spotted on both SD/-H/-L control plates and on SD/-H/-L test plates

containing 30 mM 3-AT.

Figure 4.4 Colony-lift filter assay for the detection of interactions between heterodimers and the XRE cognate sequence. YM4271[pLacZi/XRE-6] cells were transformed with different pCETT2 recombinant plasmids for coexpression of heterodimeric pairs of (A) AhRJunD/ArntFos, (B) AhR(ΔL)JunD/ArntFos, (C) AhRbHLH/ArntbHLH, as well as their corresponding controls, and were plated on SD/-L/-U plates. X-gal colony-lift assay was performed after 4 days growth, 30 °C. Photos were taken after 60 min incubation at 30 °C. The transformants are identified from left to right: (A) pCETT2/AhRJunD/ArntFos, pCETT2/AhRJunD, pCETT2//ArntFos, and pCETT2; (B) pCETT2/AhR(ΔL)JunD/ArntFos, pCETT2/AhR(ΔL)JunD, pCETT2//ArntFos, and pCETT2; (C) pCETT2/AhRbHLH/ArntbHLH, pCETT2/AhRbHLH, pCETT2//ArntbHLH, and pCETT2.

71

As the hydrophobic heptad repeat continuing through AhR Helix 2 and the JunD leucine

zipper of AhR(ΔL)JunD is out of register due to the deletion of a leucine at the junction between

Helix 2 and the LZ, we expected that the AhR(ΔL)JunD/ArntFos heterodimer would display a

weaker signal from transcriptional activation as compared with AhRJunD/ArntFos in the MY1H.

Surprisingly, the growth of both transformants on the test plates was quite similar. Both were

capable of growth to OD600 10-4, indicating that the transcriptional potencies of both

heterodimers are comparable, thereby implying that both heterodimers are likely able to bind the

XRE site with comparable affinities.

Two lacZ-based β-galactosidase reporter assays, the X-gal colony-lift filter assay and

ONPG assay, further confirmed the results from the HIS3 reporter assays above. In the

qualititative colony-lift filter assay, the transformants coexpressing GAL4AD-ArntFos and either

GAL4AD-AhRJunD or GAL4AD-AhR(ΔL)JunD showed similar transcriptional activation of the

lacZ reporter gene. Both started to show blue color within 20 minutes and became bright blue

after 60 minutes (Figure 4.4A and B). None of their controls, which expressed only one of the

two proteins or neither, produced detectable blue color after 60 minutes. Similar to the negative

controls, the transformant expressing GAL4AD-AhRbHLH and GAL4AD-ArntbHLH (Figure

4.4C), as well as transformants expressing only one of the two proteins, showed color

development indiscernible from the background control transformed with pCETT2.

The ONPG assay further quantified these results. Expression of GAL4AD-ArntFos with

either GAL4AD-AhRJunD or GAL4AD-AhR(ΔL)JunD in the lacZ reporter strain led to the

same level of β-galactosidase activity, 95.7±3.1 and 95.1±9.0, respectively (Figure 4.5;

pCETT2/AhRJunD/ArntFos and pCETT2/AhR(ΔL)JunD/ArntFos), demonstrating that both

AhRJunD and AhR(ΔL)JunD confer similar XRE-binding capability upon heterodimerization

with ArntFos. These quantitative results corroborate those gained from the HIS3 reporter spot

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titration assay. As expected, expression of only one of the two proteins displayed β-galactosidase

activities comparable to background (Figure 4.5; pCETT2/AhRJunD, pCETT2/AhR(ΔL)JunD,

pCETT2//ArntFos, and pCETT2). Because expression of AhRbHLH and ArntbHLH displayed

negative signals in both the HIS3 reporter assay and colony-lift filter assay, the ONPG assay was

not performed.

Figure 4.5 ONPG measurements for detection of AhRJunD/ArntFos:XRE and AhR(ΔL)JunD/ArntFos:XRE interactions by use of protein expression vector pCETT2 in strain YM4271[pHISi-1/XRE-6]. Vertical axis indicates the mean values in β-galactosidase units. Error bars represent standard error from at least three independent trials conducted in triplicate.

4.4.2 Heterodimerization between two monomers confirmed in the Y2H assay

The homo- and heterodimerization activity of our hybrid proteins was examined in the

GAL4 yeast two-hybrid system. To exclude the possibility that the transformants show Ade+His+

and α-galactosidase-positive phenotypes simply because they encode a polypeptide that interacts

73

with either the GAL4 DBD of the fusion protein or the GAL4 AD of the fusion protein and not

the protein(s) under investigation, we cotransformed yeast strain AH109 with either the

corresponding GAL4 AD fusion plasmids (pGADT7-based) and the control containing only the

GAL4 DBD (pGBKT7), or the corresponding GAL4 DBD fusion plasmids (pGBKT7-based) and

the control containing only the GAL4 AD (pGADT7), and tested for adenine and histidine

prototrophy and α-galactosidase activity. Also, in order to exclude the possibility that some

proteins under investigation are transcriptionally active, a reverse test was performed that

exchanges the two proteins that are fused to the DBD and AD (17).

Figure 4.6 Y2H assay of homo- and heterodimerization of AhR and Arnt hybrid proteins. Each transformant is identified by a single letter and number, indicated in the figure and described in Table 4.1. The transformants were grown as patches on SD/-L/-W medium (A) that selects only for the presence of the plasmids and replica-plated on SD/-A/-H/-L/-W/X-α-Gal medium (B) that selects for two-hybrid activity.

None of these controls exhibited two-hybrid activity in the test except one (Figure 4.6,

Table 4.1). The GAL4AD-ArntFos fusion gave a weak positive signal when expressed with the

GAL4 DBD, indicating that there is no dependence on any hybrid proteins, i.e. ArntFos,

AhRJunD, or AhR(ΔL)JunD, that are fused to the GAL4 DBD to activate reporter transcription.

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Although this transformant is able to grow on the test plate, its blue color is much less intense

than the other true positives, which indicates this transactivation is relatively weak.

Table 4.1 Dimerization of AhR- and Arnt-hybrid proteins.1 pGBKT7/DBD fusion pGADT7/AD fusion X-α-Gal plate

A1 AhRJunD ArntFos + A2 AhRJunD vector only − A3 vector only ArntFos −2 A4 ArntFos AhRJunD + A5 ArntFos vector only − A6 vector only AhRJunD − B1 AhRbHLH ArntbHLH − B2 AhRbHLH vector only − B3 vector only ArntbHLH − B4 ArntbHLH AhRbHLH − B5 ArntbHLH vector only − B6 vector only AhRbHLH − C1 AhR(ΔL)JunD ArntFos + C2 AhR(ΔL)JunD vector only − C3 ArntFos AhR(ΔL)JunD + C4 vector only AhR(ΔL)JunD − C5 AhRJunD AhRJunD + C6 ArntFos ArntFos + D1 AhRbHLH AhRbHLH − D2 ArntbHLH ArntbHLH − D3 AhR(ΔL)JunD AhR(ΔL)JunD + D4 vector only vector only −

1 The pGBKT7- and pGADT7-encoded fusions were coexpressed in yeast AH109 cells and tested for adenine and histidine prototrophy, as well as expression of X-α-galactosidase; + : blue color detected; − : no detectable growth and/or no color development. 2 False positive.

The most straightforward interpretation of this result is that ArntFos binds to the GAL4

DBD or a DNA sequence in the promoter of the reporter gene. According to the intensity of the

blue color, the GAL4AD-ArntFos fusion displayed measurably stronger transcriptional activity

in the presence of the GAL4 DBD fusions with AhRJunD, AhR(ΔL)JunD, or ArntFos than when

expressed with the GAL4DBD alone; thus, the ArntFos protein interacts with AhRJunD,

AhR(ΔL)JunD, and ArntFos. In addition, since the GAL4DBD-ArntFos fusion gave a negative

signal when expressed with the GAL4AD, but displayed a positive signal when expressed with

the GAL4AD fusions with AhRJunD, AhR(ΔL)JunD, or ArntFos, a conclusion can be drawn

with confidence that ArntFos can homodimerize and also heterodimerize with AhRJunD or

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AhR(ΔL)JunD. Additionally, both AhRJunD and AhR(ΔL)JunD can homodimerize, while

neither AhRbHLH nor ArntbHLH can homodimerize or heterodimerize with each other in yeast.

As transcriptional potency of reporter activation usually reflects the strength of the

assayed interaction in the Y2H (25), a similar spot assay to that in the MY1H assay was

performed in order to further examine the relative strengths of homo- or heterodimerization of

the hybrid proteins, as they possess relatively similar primary sequences and structures. Fresh

colony dilutions of transformants at a gradient of cell densities from OD600 10-1 to 10-5 were

spotted on both SD/-L/-W control plates and SD/-A/-H/-L/-W/X-α-Gal test plates. The survival

status and color development are dependent on the relative strength of the interaction between

the two proteins coexpressed in the transformant: the stronger the interaction, the lower the

density of the spot at which the transformant is able to survive and the more blue color the

transformant develops on the test plate.

All five transformants showed different dimerization affinities, as measured by

transcription potencies in the Y2H. The transformant expressing GAL4DBD-ArntFos and

GAL4AD-AhRJunD showed similar dimerization strength to the transformant expressing

GAL4DBD-AhRJunD and GAL4AD-AhRJunD. These two transformants displayed the

strongest transcriptional potencies among the five. The transformant expressing GAL4DBD-

ArntFos and GAL4AD-AhR(ΔL)JunD displayed similar dimerization affinity to the transformant

expressing GAL4DBD-AhR(ΔL)JunD and GAL4AD-AhR(ΔL)JunD.

Therefore, both AhRJunD and AhR(ΔL)JunD are able to homodimerize and

heterodimerize with ArntFos, and the homo- and heterodimers that contain the AhRJunD

monomer are of stronger dimerization affinity than those containing AhR(ΔL)JunD. In addition,

ArntFos was also titrated to test the strength of dimerization. As GAL4AD-ArntFos gave false

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positive results in the yeast reporter assays, no reliable conclusion can be made as to the relative

strength of the ArntFos homodimer without in vitro measurement.

4.5 Discussion

4.5.1 Fusion of the JunD and Fos LZ domains to the Arnt and AhR bHLH domains reconstitutes the heterodimeric structure and specific DNA-binding function of the bHLH/PAS domains of AhR and Arnt

Our results clearly reveal that in yeast, the GAL4 AD fusions of the bHLH domains of

AhR and Arnt are unable to activate the transcription of reporter genes. However, when the

leucine zippers of bZIP proteins JunD and Fos are fused to the C-termini of the bHLH domains

of AhR and Arnt, respectively, these two hybrid proteins are able to heterodimerize and

specifically target the cognate XRE sequence in yeast, with concomitant reporter gene activation.

The results suggest two points: first, although the bHLH domains of the bHLH, bHLHZ,

and bHLH/PAS proteins are likely to be structurally similar and are proposed to have a similar

mode of DNA binding (59), there are still fine structural differences among these bHLH domains.

Structural analysis of bHLH and bHLHZ proteins demonstrates that the HLH domains from the

two monomers dimerize and form a compact four-helix bundle, which positions the basic regions

for specific DNA binding. For bHLH proteins, such as E-proteins, the HLH domains intrinsically

possess the capability of forming stable homo- or heterodimers with partner bHLH proteins

without the aid of a secondary dimerization region. In contrast, for bHLHZ proteins, the leucine

zipper domain, which is fused to the C- terminus of Helix 2 of the bHLH domain, is necessary

for achieving stable dimeric structure.

The LZ enhances DNA binding by elongating the dimerization interface in a long rigid α-

helical coiled coil (61,70,76,141). We observed that in the Y1H system, deletion of the LZ from

the Max bHLHZ abolished homodimerization and DNA binding of Max, indicating that the LZ

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is critical for Max dimerization in addition to determination of partner specificity (J. Xu, J. A.

Shin, unpublished results). These observations show that unlike bHLH proteins, the isolated

bHLH domains of bHLHZ proteins are incapable of forming stable DNA-binding dimers without

the additional "inducer" element LZ necessary for generating the dimeric structure capable of

specific DNA binding. In addition, analysis of HLH sequences from bHLH and bHLHZ proteins

indicates that more potential attractive pairs of residues exist between the monomers of bHLH

proteins than bHLHZ proteins (71), further proving the requirement of the additional LZ in

bHLHZ proteins for efficient dimerization and DNA-binding function.

Similar to the LZ in the bHLHZ family, the PAS domain in bHLH/PAS proteins also

serves as a secondary dimerization interface to initiate and/or stabilize folding and dimerization

of the bHLH domains (72,142,152). Although the isolated bHLH domains of AhR and Arnt

proteins can homo- and heterodimerize and weakly bind to specific DNA sites in vitro, the

inclusion of the PAS A domains considerably increases the binding affinity and specificity of the

heterodimeric AhR/Arnt:DNA interaction (152,154), indicating that PAS domains serve as the

additional inducer for high affinity DNA binding of the bHLH domains of AhR and Arnt. In this

regard, LZ regions in bHLHZ proteins and PAS domains in bHLH/PAS proteins are the primary

driving force dictating partner specificity leading to heterodimerization and DNA-binding

activity.

The second point suggested by our results is that the leucine zippers of JunD and Fos may

replace the dimerization and protein stabilization function of the PAS domains in AhR and Arnt.

Attachment of the JunD or Fos LZ to the bHLH domains of AhR or Arnt restored protein

heterodimerization and DNA binding at the XRE in vivo; similar chimeric proteins in which the

LZ of Myc and Max were fused to bHLH domains of AhR and Arnt, respectively, were reported

to be capable of heterodimerization and binding to the XRE in vitro (72). We emphasize that we

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do not directly measure protein dimerization in the MY1H, but that we infer dimerization with

positive DNA-binding signal, as we have shown above that protein constructs capable of only

monomeric or homodimeric DNA binding showed no detectable binding to the XRE in in vivo

yeast assays.

Compared with the LZ domains in bHLHZ proteins, the function of the PAS domain in

protein dimerization and DNA binding is quite diverse and not fully understood. The PAS

domain clearly forms a secondary dimerization interface and stabilizes dimer formation (74). In

the absence of the bHLH domain, the interaction between the isolated PAS domains of the AhR

and Arnt can be detected using the mammalian two-hybrid assay (155), and deletion of the entire

PAS region of Arnt markedly reduces dimerization with the AhR (156). The PAS domain of

Arnt may have a role in stabilizing the bHLH domain in the absence of DNA (152). In addition

to providing additional protein dimerization interface and contributing to dimerization strength,

the PAS domains also confer the specificity of partner choice within bHLH/PAS family. The

PAS A domain restricts AhR to heterodimerization with Arnt, as the isolated AhR bHLH domain

is capable of homodimerization (154). Moreover, the PAS domains of bHLH/PAS proteins can

contribute directly to DNA-binding activity, and interdomain communication between the bHLH

and PAS domains of bHLH/PAS proteins probably exists (72,74).

For hybrid proteins AhRJunD and ArntFos, we believe the LZ pair mimics the

dimerization and stabilization function of the PAS domains in bHLH/PAS proteins. Although it

is unlikely that the LZ coiled coil makes direct contact with the XRE DNA site, we suspect the

attachment of the JunD or Fos leucine zippers to the AhR or Arnt bHLH domains likely

increases the stability and/or helicity of the AhR and Arnt bHLH domains, with attendant

increase in DNA-binding affinity. For example, the DNA binding affinity of a MyoD hybrid was

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increased ~4-fold by introducing a disulfide-stabilized helix at the N-terminus of the basic

domain of MyoD (157).

Our results show that the effect of the PAS domains on specific DNA binding by

bHLH/PAS proteins is not unique, as the replacement of PAS domains with the LZ region can

also reconstitute the heterodimeric structure and DNA-binding function of the bHLH domains of

AhR and Arnt. Although our results do not conclusively prove that dimerization and DNA

binding by bHLH/PAS proteins, and similarly for bHLHZ proteins, occurs via an additive

process of dimerization between discrete protein modules, they do provide additional evidence of

the critical role that PAS domains play in dimerization of bHLH/PAS proteins.

4.5.2 The nonpalindromic XRE DNA target mediates the heterodimerized structure of AhRJunD and ArntFos, resulting in DNA-binding function

The LZ regions in bHLHZ proteins are believed to be responsible for dimerization

specificity, as well as providing an additional dimerization interface. The presence of two

distinct dimerization domains was proposed to ensure a high level of discrimination with respect

to dimer partner (82). Our original design also followed this plausible rule. However, our Y2H

results reveal that both AhRJunD and ArntFos are able to homodimerize, as well as

heterodimerize with each other; furthermore, spot assays in the Y2H indicate that AhRJunD

shows almost no preference for homodimerization over heterodimerization with ArntFos (Figure

4.6). Thus, the discrimination regarding dimerization partner selection is at least compromised, if

not totally lost, in our hybrid proteins.

As hypothesized, in the presence of the nonpalindromic XRE target site, neither the

AhRJunD homodimer nor ArntFos homodimer is able to bind the XRE site and activate reporter

gene expression in our MY1H system. Only when AhRJunD and ArntFos are coexpressed do

they heterodimerize and trigger reporter gene activation by specific XRE binding. Apparently,

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assembly of the AhRJunD/ArntFos:XRE complex is driven largely by the strength of each

monomer’s interaction with the DNA: i. e. the heterodimerization of bHLHZ-like AhRJunD and

ArntFos is mediated by the nonpalindromic XRE site and insignificantly, or not at all, by the

differential protein-partner interactions at the dimer interface. This result is in contrast to

dimerization-dependent DNA binding of AP-1 by bZIP proteins Jun and Fos. The Jun/Fos

heterodimer targets the AP-1 binding site in strong preference to the Jun/Jun homodimer.

However, this preference is due to increased dimer stability specified by several amino acids in

the LZ regions of both Jun and Fos, rather than any difference in how the two monomers contact

DNA (82). This mode of DNA-mediated heterodimerization was also found in the C/EBP-ATF

chimeric site targeted by ATF4/IGEBP1 and E-box site targeted by the MyoD-E47 heterodimer

(158,159). Thus, Nature may often utilize DNA-mediated heterodimerization as a common

strategy for specifying protein-protein partnering and protein:DNA recognition in the bZIP and

bHLH superfamilies, as well as using specific interactions at the protein dimer interface.

Therefore, DNA-binding specificity can serve as a feedback mechanism to ensure a

desired protein dimerization preference: a nonpalindromic DNA target site could force the

formation of heterodimer (kinetically) instead of formation of two homodimers that are also

stable in the absence of the DNA target site.

4.5.3 What causes the negative signal in XRE binding by the bHLH domains of AhR and Arnt in the MY1H?

There are several possibilities that may lead to the negative signal observed with

targeting the XRE site by the AhR bHLH and Arnt bHLH in yeast. First, the AhR bHLH and/or

Arnt bHLH may not be stably expressed or present in yeast. Under physiological conditions, the

full-length AhR either depends on chaperones, including Hsp90 and other proteins, or it

heterodimerizes with Arnt once it dissociates from chaperones (160); in both cases properly

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folded structure is most likely maintained. There is evidence that Hsp90 also interacts with the

bHLH domain of the AhR protein (161). In yeast cells, the lack of folding partner may lead to

protein degradation or aggregation. In addition, the expression of the AhR bHLH and Arnt

bHLH domains in E. coli has been shown to be problematic; even coexpression of the AhR

bHLH and Arnt bHLH domains in E. coli failed to recover any DNA binding activity, while

coexpression of the corresponding bHLH/PAS domains dramatically increased recovery of

functional proteins (152).

Second, the AhR bHLH may not be able to heterodimerize with the Arnt bHLH and

target XRE in yeast. The Arnt bHLH homodimer forms a less hydrophobic four-helix bundle

than other bHLH and bHLHZ proteins (142), such as E47 (75) and Max (61,141), both of which

form stable homodimers that target the E-box site. The AhR and Arnt bHLH domains may not be

stable or properly folded in yeast, as they lack the PAS domains to help stabilize the more

hydrophilic bHLH structure. PAS domains of bHLH/PAS proteins have been proposed to initiate

folding and/or stabilize dimerization of the bHLH region in a mode similar to that served by the

LZ in the bHLHZ Myc/Max heterodimer (152). Therefore, the LZ fused to the C-termini of the

AhR and Arnt bHLH domains serves in a manner similar to the LZ in bHLHZ proteins: a

nucleation device encouraging folding of the HLH domain from less ordered structure to

organized four-helix bundle. This may explain why we observed positive signals from the XRE

binding by bHLHZ-like AhRJunD and ArntFos in the MY1H, but not from just the bHLH

domains of AhR and Arnt.

4.5.4 Deletion of one leucine residue in the JunD LZ negatively affects heterodimerization but not DNA binding

The Y2H assay revealed that AhRJunD heterodimerizes with ArntFos more tightly than

does AhR(∆L)JunD. This must be due to the deletion of a Leu residue at the N-terminus of the

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JunD LZ in AhR(∆L)JunD; this deletion disrupts the hydrophobic heptad repeat register between

the AhR bHLH and JunD LZ. In the MY1H, however, the transformant coexpressing

AhR(∆L)JunD and ArntFos surprisingly displayed almost identical transcriptional activation of

the reporter gene to the one coexpressing AhRJunD and ArntFos.

Apparently, the register of the JunD LZ only affects the dimerization strength of the

heterodimer, not its DNA binding activity. This finding is probably due to the role that the LZ

plays in these hybrid proteins. The leucine zippers serve to stabilize the proper folding of the

contiguous bHLH domains of AhR and Arnt by nucleating α-helix formation and proper protein

folding; they do not contribute positively to the binding affinity between the heterodimer and the

target XRE site. Instead, there is evidence showing that the LZ can negatively affect the DNA-

binding affinity of Arnt bHLH in vitro. Quantitative fluorescence anisotropy analysis revealed

that fusion of the LZ from bZIP protein C/EBP with the bHLH of Arnt yields a protein hybrid

that binds the E-box DNA site with 4-fold reduced affinity compared with E-box binding by the

isolated Arnt bHLH domain (153). In the absence of DNA, however, the JunD/Fos LZ pair,

which is a secondary dimerization element, plays a more significant role with increased

contribution to the protein dimer interface.

In the presence of DNA, the four-helix bundle formed between the bHLH domains of

AhR and Arnt is the dominant interaction stabilizing the protein heterodimer, and ultimately,

contributes to the DNA-binding affinity. Furthermore, we note that the boundaries of each region

of these of bHLH domains were predicted based on sequence similarity with bHLHZ protein

Max; such alignment may not reflect the entire bHLH domain of bHLH/PAS proteins. The

fusion proteins were generated by direct attachment of the LZ to the C-termini of the bHLH

domains of bHLH/PAS proteins; no structural optimization was performed at the junction

between Helix 2 of the bHLH and the LZ, except that the hydrophobic heptad repeat was

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maintained in AhRJunD and ArntFos to give a structure hypothesized to mimic that of Max

bHLHZ. More definitive analysis of the structural differences between AhRJunD and

AhR(∆L)JunD, and conclusions about protein dimerization structure and DNA-binding function,

is possible once high-resolution structural information of the AhR bHLH domain is available and

in vitro measurements of the hybrid proteins are completed.

4.5.5 Our domain swapping experiments support the hypothesis of domain shuffling in the evolutionary pathway of the bHLH superfamily

In addition to the highly conserved HLH domain, members of the bHLH superfamily

may also contain LZ or PAS domains. In some family members, the basic region is even missing,

such as with Id, an HLH negative regulator of bHLH proteins (162). Moreover, the relative

position of the bHLH domain in different family members varies considerably (98). From an

evolutionary perspective, these conserved domains in bHLH proteins are usually independent

and separately evolved. Based on a study of the bHLH domains in 242 bHLH superfamily

members, Atchley and Fitch produced a phylogenetic tree to classify family members according

to evolutionary relationships, and found their analyses could not rigorously support the

hypothesis of a single evolutionary origin for the bHLH domain (98). However, further intensive

investigation of the non-bHLH components of 122 bHLH superfamily members provided strong

evidence that supports the hypothesis that bHLH proteins have undergone modular evolution by

domain shuffling, a process that involves domain insertion and rearrangement (163).

Our domain swapping experiment provides additional, complementary evidence for the

hypothesis of modular evolution by domain shuffling. We found that the PAS domains in the

bHLH/PAS AhR/Arnt heterodimer can be replaced by the LZ domain from the bZIP, a different

transcription factor family from the bHLH, while maintaining dimerized protein structure and

specific DNA-binding function. Taken together, our results provide further evidence that domain

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shuffling is likely the evolutionary pathway that Nature utilized to gain diverse structural

patterns and variable functions in the bHLH superfamily.

4.6 Supplemental Information Supplemental Information associated with this manuscript is provided in Appendix C.

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Chapter 5 Summary and future work

This chapter presents a summary and interpretation of this body of work and recommends

directions for future research.

5.1 Summary Due to the critical role that DNA-binding proteins play in the regulation of diverse

physiological functions including cell development, growth, and differentiation, quite a few

approaches have been employed to investigate the molecular basis of protein:DNA recognition

as well as the cooperative, enhancing (coactivating), and inhibitory effects of cofactors on

particular protein:DNA interactions. Owing to their unique physiologically relevant environment,

high efficiency and sensitivity, and relatively easy manipulation, yeast genetic systems have been

successfully applied to study these interactions.

This thesis is devoted to the development of a modified Y1H system useful for

examination of both protein-protein and protein:DNA interactions in vivo and to applying this

MY1H system to the study of heterodimeric protein:DNA interaction. Hence, we developed a

single plasmid-based MY1H capable of differential protein expression levels. In addition to a

GAL4 AD fusion protein, a second protein can be coexpressed at either comparable or higher

transcriptional levels from expression vectors pCETT or pCETF, respectively. This second

protein can play a structural, modifying, or inhibitory role that restores or blocks reporter gene

expression.

This system was validated by use of the well-characterized DNA-binding protein p53 and

its inhibitory partners, large T antigen (LTAg) and 53BP2. In the MY1H, the DNA binding of

p53 decreases by different extents dependent on different inhibitory partners and their expression

levels. As demonstrated, our system is particularly useful for examining the effects of a

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regulatory protein on the DNA-binding activity of a transcription factor. Moreover, in the study

of inhibitors of a DNA-binding protein or in DNA-binding competition assays, the regulatory

protein can be expressed at comparable or excess levels from MCS II compared with the

GAL4AD-fused protein from MCS I, therefore allowing qualitative assessment of the strength of

the protein:DNA or protein-protein interaction of interest by combined use of pCETT and

pCETF. This feature distinguishes our system from other reported systems and was fully

exemplified in the study of the repression of the Max:E-box DNA site interaction with several

competitor mutants of Max (J. Xu, J. A. Shin, unpublished results), as well as in the

p53/LTAg/53BP2 system (Chapter 2). In addition, this system can also be potentially applied to

investigate coregulatory or bridging proteins and to identify novel protein- or DNA-binding

partners through library screening under certain circumstances.

We then applied this MY1H system to investigate the DNA-binding activities of

heterodimeric proteins from the bHLH/PAS family. A positive control for the study of XRE-

binding proteins was first performed in the MY1H system in which the reporter genes were

driven by the XRE cognate sequence in the promoter. This positive control utilizes the

bHLH/PAS domains of AhR and Arnt (NAhR and NArnt) that are known to form heterodimers

and bind the cognate XRE sequence both in vitro and in vivo. However, negative signals were

observed when these two proteins were coexpressed from pCETT in the MY1H. Several

strategies were implemented together to turn this false negative into a true positive. By

increasing the copy number of XRE sites integrated into the yeast genome from three to six, and

at the same time fusing to each monomer a GAL4 activation domain, the heterodimer formed by

NAhR and NArnt specifically bound the cognate XRE sequence, which displays clear positive

binding signals with high signal-to-noise ratios in the MY1H system. Turning this false negative

interaction into a true positive control made possible the further study of XRE-binding proteins

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in the MY1H system. Moreover, this methodology provides an effective way for trouble-

shooting the false negatives that arise from unproductive transcription in yeast genetic assays.

In order to gain more insight into the role the PAS domains of AhR and Arnt play in

protein dimerization and DNA binding, and to further understand DNA recognition by

heterodimeric proteins, a pair of bHLHZ-like proteins, AhRJunD and ArntFos, were designed

and their dimerization and specific DNA-binding activities were explored in the MY1H and Y2H

systems and are now undergoing in vitro analysis. In vivo results reveal that both AhRJunD and

ArntFos are able to form homodimers, and that the presence of the nonpalindromic XRE cognate

sequence forces heterodimerization of the two hybrids for specific DNA binding.

Therefore, of the five scenarios that can be examined by use of the MY1H system (Figure

2.1), three have been successfully validated: the system investigating the inhibition of

LTAg/53BP2 on DNA binding of p53, where a protein-protein interaction was assayed and the

second protein serves as a blocker; the system investigating the repression of Max mutants on

DNA binding of Max bHLHZ protein, where a protein-DNA interaction was assayed and the

second protein functions as a repressor of DNA binding; the NAhR/NArnt:XRE or

AhRJunD/ArntFos:XRE system where protein heterodimer-DNA interaction was assayed. These

systems clearly demonstrate the wide applicability of this MY1H system.

5.2 Future work In this thesis, emphasis has been placed on validation of the MY1H system useful for

examination of simultaneous protein-protein and protein:DNA interactions and application of the

MY1H system to DNA-binding studies of the bHLH/PAS family of proteins. The following are

some suggestions for future improvements and new research directions.

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5.2.1 Downstream work on the AhRJunD/ArntFos:XRE interaction In addition to in vivo examination, in vitro quantitative assessment of the interaction of

AhRJunD/ArntFos heterodimer with its XRE target element by fluorescence anisotropy titration

is to be performed. The bacterially expressed proteins are currently being expressed and purified

by Antonia De Jong. Circular dichroism (CD) titration assays will also be applied to measure the

dissociation constants of the homodimers formed by these hybrid proteins. As the four-helix

bundle forms upon dimerization in bHLH proteins, an increase in helicity should be detected

with CD spectroscopy. Moreover, western blotting assays are also currently being utilized to

examine the expression of the hybrid proteins examined in the Y2H, for the Y2H is needed to

confirm that all proteins are present in the system as expected and any negative signals are not

due to false negatives.

5.2.2 Improvement on the MY1H system Although great success was achieved in the DNA binding studies of the heterodimer

between bHLHZ-like hybrids AhRJunD and ArntFos by fusing an activation domain to each

hybrid protein, we observed reduced signals when the single AD expression vector pCETT was

utilized to coexpress the two proteins in the MY1H system. For example, the NAhR/NArnt:XRE

interaction is positive only in double AD system, whereas the AhRJunD/ArntFos:XRE

interaction is positive in both the double AD system (ONPG: ~90) and single AD system (ONPG:

~40). Such observations may reflect the differences between strong and weak function; more

activation domains may amplify a weak signal, for instance. However, in some cases, the single

AD system is preferred, as heterodimeric proteins in the same family, usually containing a

monomer capable of both homo- and heterodimerization with a partner incapable of

homodimerization, are able to bind the same or similar DNA target sequences, commonly

palindromic or pseduopalindromic sequences. The double AD system will complicate the

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interactions under question as both homodimer and heterodimer occupy the same DNA binding

site, thereby activating the reporter gene. In contrast, in the single AD system, the problem can

be avoided by fusing the AD to the monomer that is incapable of forming homodimers. Although

the GAL4 AD possesses reasonable activation strength, we may utilize a stronger AD, such as

VP16, in order to facilitate the examination of the aforementioned heterodimeric protein:DNA

interactions in the MY1H system.

A second straightforward experiment would be to introduce a third protein into the

system to study the effects of this protein on the DNA-binding activity of heterodimeric proteins.

Given the fact that unregulated expression of quite a few transcription factors, for instance, the

oncogenic Myc/Max heterodimer, often leads to human diseases such as cancer, dominant

negative inhibitors could be a strategy for suppression of the aberrant function of such

transcription factors (164,165). If a third protein can be expressed in the MY1H system, the

dominant negative function of a protein on a heterodimeric transcription factor can be examined

in the MY1H system. This can be achieved by introducing a protein expression vector similar to

pGAD424-MCS IIΔAD (127) with a different nutritional marker other than LEU2.

5.2.3 Other DNA-binding hybrid proteins of interest As the basic region dictates specific DNA binding, we are now trying to evolve a

nonnative Myc/Max monomer to target the nonnative site 5'-CACCAC. This Myc-Max

monomer contains the basic regions of Myc and Max connected by a short linker. The basic idea

is that rather than using a dimer to target DNA, we fuse the basic regions of the two proteins,

each of which specifically targets a 5'-CAC half site; such covalent fusion of both basic regions

eliminates any need for dimerization and effectively increases the concentration of functional

DNA-binding structural modules at the protein:DNA interface; i.e. analogous to enzyme

catalysis, a similar concept of bringing reactants into close proximity to increase the rate of

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reaction. We anticipate that a protein conformation that optimally positions the two basic regions

of Myc and Max into the major groove of the target element can be found by mutagenizing the

short linker and performing library screening in the Y1H system. Furthermore, once the linker of

the Myc/Max monomer is optimized, one of the monomers can also be mutated to target another

DNA site as in 5'-CACNNN or 5'-NNNCAC; a library can be generated in which one of the two

basic regions is mutated and the other acts as the docking region. Eventually, this monomer

system, similar to zinc finger proteins, can potentially be used to evolve proteins that bind to any

hexameric DNA sequences.

Another hybrid protein of interest is a Max monomer, similar to the preceding concept,

that contains the basic region contiguous with a degenerate short peptide intended to target the

other half site of the E-box. We are trying to evolve a Max monomer to target the E-box cognate

sequence, which is naturally targeted by the Max bHLHZ dimer. As discussed above, this Max

monomer can protentially serve as a dominant negative inhibitor to suppress the expression of E-

box responsive genes.

In the present GAL4-based Y1H system, numerous false positives can appear during

library screening, which limits its use for effectively selecting nonnative DNA-binding protein

precursors that may bind to a DNA target with low affinity. In addition, multiple plasmids can be

transformed into a single yeast cell during library transformation, as high concentrations of DNA

is present, and such aberrant transformation of multiple plasmids makes any positive results

difficult to characterize: which gene is responsible for the protein that produced the positive

signal? We are now introducing the concept of directed evolution into the screening of the Max

monomer library in the Y1H system in an attempt to uncover any weak DNA-binding proteins

that may serve as progenitors of more active progeny and segregation of the multiple plasmids

that are often transformed into a single cell during library transformation.

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Chap 6ter Materials and Methods

6.1 General Details about reagent sources, bacterial and yeast strains, transformation, DNA

preparation, plasmid rescue, and plasmid sequencing are detailed in the Materials and Methods

sections of Chapters 2-4.

6.1.1 Two reporter assays used in the MY1H In the MY1H systems, HIS3 and lacZ are used as reporter genes. The HIS3 reporter gene

is used to test whether the transformants are auxotrophic for histidine. The lacZ reporter gene is

used to perform the β-galactosidase assay that enable colorimetric visualization of protein:DNA

binding. Two commonly used assays based on the lacZ reporter are the qualitative X-gal colony-

lift filter assay and quantitative ONPG liquid assay.

6.1.1.1 HIS3 reporter assay The detailed procedure was described in Chapter 3.

6.1.1.1.1 Spot titration assay The detailed procedure was described in Chapter 4.

6.1.1.2 X-gal colony-lift filter assay The detailed procedure was described in Chapter 2.

6.1.1.3 ONPG liquid assay The ONPG liquid assay was performed according to the protocol provided in the Yeast

Protocols Handbook (Clontech) with the following modifications. In the ONPG liquid assay,

yeast cells were grown as described below before harvesting for lysis. Transformed yeast cells

were initially grown at 30 °C with shaking in SD/-L media for 2 days or until OD600 >1.5 was

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reached, and then used to inoculate a fresh culture of SD/-L media. This secondary culture was

grown overnight until OD600 1.0-1.3 was reached. An aliquot of the secondary culture was

resuspended in YPDA to give starting OD600 ~0.2. The YPDA culture was then grown for 3-5

hrs until OD600 0.60-0.65 was reached. The units of β-galactosidase were calculated according to

β-galactosidase units = 1000 × A420 / (t × v × A600), where t = time (min) required for the reaction

and v = 0.1 × concentration factor. One unit of β-galactosidase is defined as the amount which

hydrolyzes 1 μmol of ONPG to o-nitrophenol and D-galactose per min per cell (114). Results are

presented as mean values ± S.E.M. of 3-4 independent experiments, each performed in triplicate.

6.1.2 Examination of protein-protein interactions in the Y2H system The Matchmaker™ GAL4 Two-hybrid System 3 (Clontech) was utilized to examine

homo- and heterodimerization of the proteins under investigation. The detailed procedure was

described in Chapter 4.

6.2 Modified Y1H for identification of protein-protein/protein:DNA interactions

6.2.1 Construction of p53 target-reporter strains Two yeast reporter strains, YM4271[p53HIS] and YM4271[p53BLUE], were created for

expression analysis in the MY1H from the integrating reporter vectors, p53HIS and p53BLUE

(Clontech), respectively. The p53HIS plasmid contains the minimal promoter of the HIS3 locus

(PminHIS), in which three tandem copies of the consensus p53 binding site, 5'-

AGGCATGCCTAGGCATGCCT, were inserted upstream of the HIS3 reporter gene. Likewise,

p53BLUE contains the minimal promoter of the yeast iso-1-cytochrome C gene (PCYC1), in

which three tandem copies of the consensus p53 binding site were inserted upstream of the lacZ

reporter gene. The p53HIS plasmid was linearized at the Xho I site and integrated into YM4271

93

by recombination to create YM4271[p53HIS]. Similarly, p53BLUE was linearized at the Nco I

site and integrated to generate YM4271[p53BLUE].

To assess background HIS3 expression, 3-AT was used as a competitive inhibitor of the

yeast HIS3 protein. 45 mM 3-AT is sufficient to completely suppress background growth due to

leaky HIS3 expression in the YM4271[p53HIS] reporter strain as per the Matchmaker One-

Hybrid System User Manual (Clontech).

6.2.2 Plasmid construction Detailed information was provided in Chapter 2.

6.2.3 3-AT titration analysis The detailed procedure of the 3-AT titration analysis was described in Chapter 2.

6.3 Turning false negatives into true positives in the MY1H

6.3.1 Construction of XRE target-reporter strains The details of reporter strain construction were described in Appendix B.

6.3.2 Plasmid construction The details of plasmid construction were described in Chapter 3 as well as Appendix B.

6.4 DNA binding forces heterodimerization of hybrids of bHLH/PAS and bZIP

6.4.1 Plasmid construction The details of plasmid construction were described in Chapter 4 as well as Appendix C.

94

References 1. Fields, S. and Song, O.K. (1989) A novel genetic system to detect protein-protein

interactions. Nature, 340, 245-246.

2. Wang, M.M. and Reed, R.R. (1993) Molecular cloning of the olfactory neuronal transcription factor Olf-1 by genetic selection in yeast. Nature, 364, 121-126.

3. Luo, Y., Vijaychander, S., Stile, J. and Zhu, L. (1996) Cloning and analysis of DNA-binding proteins by yeast one-hybrid and one-two-hybrid systems. Biotechniques, 20, 564-568.

4. Li, J.J. and Herskowitz, I. (1993) Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system. Science, 262, 1870-1874.

5. Ahn, J.H., Chiou, C.J. and Hayward, G.S. (1998) Evaluation and mapping of the DNA binding and oligomerization domains of the IE2 regulatory protein of human cytomegalovirus using yeast one and two hybrid interaction assays. Gene, 210, 25-36.

6. West, M., Flanery, D., Woytek, K., Rangasamy, D. and Wilson, V.G. (2001) Functional mapping of the DNA binding domain of bovine papillomavirus E1 protein. J. Virol., 75, 11948-11960.

7. James, P., Halladay, J. and Craig, E.A. (1996) Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics, 144, 1425-1436.

8. Vidal, M., Brachmann, R.K., Fattaey, A., Harlow, E. and Boeke, J.D. (1996) Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions. Proc. Natl. Acad. Sci. U. S. A., 93, 10315-10320.

9. Vidal, M. and Legrain, P. (1999) Yeast forward and reverse 'n'-hybrid systems. Nucleic Acids Res., 27, 919-929.

10. Vidalain, P.O., Boxem, M., Ge, H., Li, S.M. and Vidal, M. (2004) Increasing specificity in high-throughput yeast two-hybrid experiments. Methods, 32, 363-370.

11. Cormack, R.S., Hahlbrock, K. and Somssich, I.E. (1998) Isolation of putative plant transcriptional coactivators using a modified two-hybrid system incorporating a GFP reporter gene. Plant J., 14, 685-692.

12. Endoh, H., Walhout, A.J.M. and Vidal, M. (2000) A green fluorescent protein-based reverse two-hybrid system: Application to the characterization of large numbers of potential protein-protein interactions. Methods Enzymol., 328, 74-88.

13. Barz, T., Ackermann, K. and Pyerin, W. (2002) A positive control for the green fluorescent protein-based one-hybrid system. Analytical Biochemistry, 304, 117-121.

14. Barnard, E., McFerran, N.V., Trudgett, A., Nelson, J. and Timson, D.J. (2008) Detection and localisation of protein-protein interactions in Saccharomyces cerevisiae using a split-GFP method. Fungal Genetics and Biology, 45, 597-604.

15. Miller, J.P., Lo, R.S., Ben-Hur, A., Desmarais, C., Stagljar, I., Noble, W.S. and Fields, S. (2005) Large-scale identification of yeast integral membrane protein interactions. Proc. Natl. Acad. Sci. U. S. A., 102, 12123-12128.

95

16. Obrdlik, P., El-Bakkoury, M., Hamacher, T., Cappellaro, C., Vilarino, C., Fleischer, C., Ellerbrok, H., Kamuzinzi, R., Ledent, V., Blaudez, D. et al. (2004) K+ channel interactions detected by a genetic system optimized for systematic studies of membrane protein interactions. Proc. Natl. Acad. Sci. U. S. A., 101, 12242-12247.

17. Izumchenko, E., Wolfson, M., Golemis, E.A. and Serebriiskii, I.G. (2007), Yeast Gene Analysis, 2nd Edition. Elsevier Academic Press Inc. , San Diego, Vol. 36, pp. 103-137.

18. Dalton, S. and Treisman, R. (1992) Characterization of SAP-1, a protein recruited by serum response factor to the c-fos serum response element. Cell, 68, 597-612.

19. Naya, F.J., Stellrecht, C.M.M. and Tsai, M.J. (1995) Tissue-specific regulation of the insulin gene by a novel basic helix-loop-helix transcription factor. Genes Dev., 9, 1009-1019.

20. Peterson, B.R., Sun, L.J. and Verdine, G.L. (1996) A critical arginine residue mediates cooperativity in the contact interface between transcription factors NFAT and AP-1. Proc. Natl. Acad. Sci. U. S. A., 93, 13671-13676.

21. Sun, L.J., Peterson, B.R. and Verdine, G.L. (1997) Dual role of the nuclear factor of activated T cells insert region in DNA recognition and cooperative contacts to activator protein 1. Proc. Natl. Acad. Sci. U. S. A., 94, 4919-4924.

22. Yu, Y., Yussa, M., Song, J.B., Hirsch, J. and Pick, L. (1999) A Double Interaction Screen identifies positive and negative ftz gene regulators and Ftz-interacting proteins. Mech. Dev., 83, 95-105.

23. Koszewski, N.J., Henry, K.W., Lubert, E.J., Gravatte, H. and Noonan, D.J. (2003) Use of a modified yeast one-hybrid screen to identify BAF60a interactions with the Vitamin D receptor heterodimer. J. Steroid Biochem. Mol. Biol., 87, 223-231.

24. Li, B. and Fields, S. (1993) Identification of mutations in p53 that affect its binding to SV40 large T antigen by using the yeast two-hybrid system. Faseb J., 7, 957-963.

25. Estojak, J., Brent, R. and Golemis, E.A. (1995) Correlation of two-hybrid affinity data with in vitro measurements. Mol. Cell. Biol., 15, 5820-5829.

26. Brent, R. and Finley, R.L. (1997) Understanding gene and allele function with two-hybrid methods. Annual Review of Genetics, 31, 663-704.

27. Walhout, A.J.M., Sordella, R., Lu, X.W., Hartley, J.L., Temple, G.F., Brasch, M.A., Thierry-Mieg, N. and Vidal, M. (2000) Protein interaction mapping in C-elegans using proteins involved in vulval development. Science, 287, 116-122.

28. Walhout, A.J.M., Boulton, S.J. and Vidal, M. (2000) Yeast two-hybrid systems and protein interaction mapping projects for yeast and worm. Yeast, 17, 88-94.

29. Bartel, P., Chien, C.-T., Sternglantz, R. and Fields, S. (1993) Elimination of false positives that arise in using the two-hybrid system. Biotechniques, 14, 920-924.

30. Golemis, E.A. and Brent, R. (1992) Fused protein domains inhibit DNA binding by LexA. Mol. Cell. Biol., 12, 3006-3014.

31. Blancafort, P., Segal, D.J. and Barbas, C.F. (2004) Designing transcription factor architectures for drug discovery. Molecular Pharmacology, 66, 1361-1371.

96

32. Marmorstein, R. and Fitzgerald, M.X. (2003) Modulation of DNA-binding domains for sequence-specific DNA recognition. Gene, 304, 1-12.

33. Matthews, B.W. (1988) No code for recognition. Nature, 335, 294-295.

34. Benos, P.V., Lapedes, A.S. and Stormo, G.D. (2002) Is there a code for protein-DNA recognition? Probab(ilistical)ly. Bioessays, 24, 466-475.

35. Sarai, A. and Kono, H. (2005) Protein-DNA recognition patterns and predictions. Annual Review of Biophysics and Biomolecular Structure, 34, 379-398.

36. Pabo, C.O. and Sauer, R.T. (1992) TRANSCRIPTION FACTORS: Structural families and principles of DNA recognition. Annual Review of Biochemistry, 61, 1053-1095.

37. Luscombe, N.M., Austin, S.E., Berman, H.M. and Thornton, J.M. (2000) An overview of the structures of protein-DNA complexes. Genome Biol., 1, reviews001.001-reviews001.037.

38. Garvie, C.W. and Wolberger, C. (2001) Recognition of specific DNA sequences. Molecular Cell, 8, 937-946.

39. Nelson, H.C.M. (1995) Structure and function of DNA-binding proteins. Current Opinion in Genetics and Development, 5, 180-189.

40. Vinson, C., Myakishev, M., Acharya, A., Mir, A.A., Moll, J.R. and Bonovich, M. (2002) Classification of human B-ZIP proteins based on dimerization properties. Mol. Cell. Biol., 22, 6321-6335.

41. Deppmann, C.D., Acharya, A., Rishi, V., Wobbes, B., Smeekens, S., Taparowsky, E.J. and Vinson, C. (2004) Dimerization specificity of all 67 B-ZIP motifs in Arabidopsis thaliana: A comparison to Homo sapiens B-ZIP motifs. Nucleic Acids Res., 32, 3435-3445.

42. Hurst, H.C. (1994) Transcription factors 1: bZIP proteins. Protein Profile, 1, 123-168.

43. Ellenberger, T.E., Brandl, C.J., Struhl, K. and Harrison, S.C. (1992) The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted α Helices: Crystal structure of the protein-DNA complex Cell, 71, 1223-1237.

44. Struhl, K. (1989) Helix-turn-helix, zinc-finger, and leucine-zipper motifs for eukaryotic transcriptional regulatory proteins. Trends in Biochemical Sciences, 14, 137-140.

45. Landschulz, W.H., Johnson, P.F. and McKnight, S.L. (1988) The leucine zipper: A hypothetical structure common to a new class of DNA binding proteins. Science, 240, 1759-1764.

46. Agre, P., Johnson, P.F. and McKnight, S.L. (1989) Cognate DNA-binding specificity retained after leucine zipper exchange between GCN4 and C/EBP. Science, 246, 922-926.

47. Konig, P. and Richmond, T.J. (1993) The X-ray structure of the GCN4-bZIP bound to ATF/CREB site DNA shows the complex depends on DNA flexibility. J. Mol. Biol., 233, 139-154.

48. Keller, W., Konig, P. and Richmond, T.J. (1995) Crystal structure of a bZIP/DNA complex at 2.2 Å: Determinants of DNA specific recognition. J. Mol. Biol., 254, 657-667.

97

49. Glover, J.N.M. and Harrison, S.C. (1995) Crystal structure of the heterodimeric bZIP transcription factor c-Fos-c-Jun bound to DNA. Nature, 373, 257-261.

50. Schumacher, M.A., Goodman, R.H. and Brennan, R.G. (2000) The structure of a CREB bZIP·somatostatin CRE complex reveals the basis for selective dimerization and divalent cation-enhanced DNA binding. J. Biol. Chem., 275, 35242-35247.

51. Fujii, Y., Shimizu, T., Toda, T., Yanagida, M. and Hakoshima, T. (2000) Structural basis for the diversity of DNA recognition by bZIP transcription factors. Nat. Struct. Biol., 7, 889-893.

52. Vinson, C.R., Sigler, P.B. and McKnight, S.L. (1989) Scissors-grip model for DNA recognition by a family of leucine zipper proteins. Science, 246, 911-916.

53. O'Neil, K.T., Shuman, J.D., Ampe, C. and Degrado, W.F. (1991) DNA-induced increase in the α-helical content of C/EBP and GCN4. Biochemistry, 30, 9030-9034.

54. Saudek, V., Pasley, H.S., Gibson, T., Gausepohl, H., Frank, R. and Pastore, A. (1991) Solution structure of the basic region from the transcriptional activator GCN4. Biochemistry, 30, 1310-1317.

55. Weiss, M.A., Ellenberger, T., Wobbe, C.R., Lee, J.P., Harrison, S.C. and Struhl, K. (1990) Folding transition in the DNA-binding domain of GCN4 on specific binding to DNA. Nature, 347, 575-578.

56. Vinson, C., Acharya, A. and Taparowsky, E.J. (2006) Deciphering B-ZIP transcription factor interactions in vitro and in vivo. Biochim. Biophys. Acta-Gene Struct. Expression, 1759, 4-12.

57. Littlewood, T.D. and Evan, G.I. (1994) Transcription factors 2: Helix-loop-helix. Protein Profile, 1, 639-709.

58. Jones, S. (2004) An overview of the basic helix-loop-helix proteins. Genome Biol., 5, 226.001-226.006.

59. Kewley, R.J., Whitelaw, M.L. and Chapman-Smith, A. (2004) The mammalian basic helix-loop-helix/PAS family of transcriptional regulators. International Journal of Biochemistry & Cell Biology, 36, 189-204.

60. Murre, C., Bain, G., Vandijk, M.A., Engel, I., Furnari, B.A., Massari, M.E., Matthews, J.R., Quong, M.W., Rivera, R.R. and Stuiver, M.H. (1994) Structure and function of helix-loop-helix proteins. Biochim. Biophys. Acta-Gene Struct. Expression, 1218, 129-135.

61. Ferré-D'Amaré, A.R., Prendergast, G.C., Ziff, E.B. and Burley, S.K. (1993) Recognition by Max of its cognate DNA through a dimeric b/HLH/Z domain. Nature, 363, 38-45.

62. Phillips, S.E.V. (1994) Built by association: Structure and function of helix-loop-helix DNA-binding proteins. Structure, 2, 1-4.

63. Swanson, H.I. and Yang, J.H. (1996) Mapping the protein/DNA contact sites of the Ah receptor and Ah receptor nuclear translocator. J. Biol. Chem., 271, 31657-31665.

64. Ma, P.C.M., Rould, M.A., Weintraub, H. and Pabot, C.O. (1994) Crystal structure of MyoD bHLH domain-DNA complex: Perspectives on DNA recognition and implications for transcriptional activation. Cell, 77, 451-459.

98

65. Massari, M.E. and Murre, C. (2000) Helix-loop-helix proteins: Regulators of transcription in eucaryotic organisms. Mol. Cell. Biol., 20, 429-440.

66. Taylor, B.L. and Zhulin, I.B. (1999) PAS domains: Internal sensors of oxygen, redox potential, and light. Microbiology and Molecular Biology Reviews, 63, 479-506.

67. Berkes, C.A. and Tapscott, S.J. (2005) MyoD and the transcriptional control of myogenesis. Seminars in Cell & Developmental Biology, 16, 585-595.

68. Baudino, T.A. and Cleveland, J.L. (2001) The Max network gone mad. Mol. Cell. Biol., 21, 691-702.

69. Ellenberger, T. (1994) Getting a grip on DNA recognition: Structures of the basic region leucine zipper, and the basic region helix-loop-helix DNA-binding domains. Curr. Opin. Struct. Biol., 4, 12-21.

70. Nair, S.K. and Burley, S.K. (2003) X-ray structures of Myc-Max and Mad-Max recognizing DNA: Molecular bases of regulation by proto-oncogenic transcription factors. Cell, 112, 193-205.

71. Shirakata, M., Friedman, F.K., Wei, Q. and Paterson, B.M. (1993) Dimerization specificity of myogenic helix-loop-helix DNA-binding factors directed by nonconserved hydrophilic residues. Genes Dev., 7, 2456-2470.

72. Chapman-Smith, A. and Whitelaw, M.L. (2006) Novel DNA binding by a basic helix-loop-helix protein: The role of the dioxin receptor PAS domain. J. Biol. Chem., 281, 12535-12545.

73. Card, P.B., Erbel, P.J.A. and Gardner, K.H. (2005) Structural basis of ARNT PAS-B dimerization: Use of a common beta-sheet interface for hetero- and homodimerization. J. Mol. Biol., 353, 664-677.

74. Yildiz, O., Doi, M., Yujnovsky, I., Cardone, L., Berndt, A., Hennig, S., Schulze, S., Urbanke, C., Sassone-Corsi, P. and Wolf, E. (2005) Crystal structure and interactions of the PAS repeat region of the Drosophila clock protein PERIOD. Molecular Cell, 17, 69-82.

75. Ellenberger, T., Fass, D., Arnaud, M. and Harrison, S.C. (1994) Crystal structure of transcription factor E47: E-box recognition by a basic region helix-loop-helix dimer. Genes Dev., 8, 970-980.

76. Ferré-D'Amaré, A.R., Pognonec, P., Roeder, R.G. and Burley, S.K. (1994) Structure and function of the b/HLH/Z domain of USF. Embo Journal, 13, 180-189.

77. Sauve, S., Tremblay, L. and Lavigne, P. (2004) The NMR solution structure of a mutant of the max b/HLH/LZ free of DNA: Insights into the specific and reversible DNA binding mechanism of dimeric transcription factors. J. Mol. Biol., 342, 813-832.

78. Lavigne, P., Crump, M.P., Gagne, S.M., Hodges, R.S., Kay, C.M. and Sykes, B.D. (1998) Insights into the mechanism of heterodimerization from the 1H-NMR solution structure of the c-Myc-Max heterodimeric leucine zipper. J. Mol. Biol., 281, 165-181.

79. Bacsi, S.G., Reiszporszasz, S. and Hankinson, O. (1995) Orientation of the heterodimeric aryl-hydrocarbon (Dioxin) receptor complex on its asymmetric DNA recognition sequence. Molecular Pharmacology, 47, 432-438.

99

80. Rowlands, J.C. and Gustafsson, J.A. (1997) Aryl hydrocarbon receptor-mediated signal transduction. Critical Reviews in Toxicology, 27, 109-134.

81. Antonsson, C., Arulampalam, V., Whitelaw, M.L., Pettersson, S. and Poellinger, L. (1995) Constitutive function of the basic helix-loop-helix/PAS factor Arnt: Regulation of target promoters via the E box motif. J. Biol. Chem., 270, 13968-13972.

82. Lamb, P. and McKnight, S.L. (1991) Diversity and specificity in transcriptional regulation: The benefits of heterotypic dimerization Trends in Biochemical Sciences, 16, 417-422.

83. White, C.L., Suto, R.K. and Luger, K. (2001) Structure of the yeast nucleosome core particle reveals fundamental changes in internucleosome interactions. Embo Journal, 20, 5207-5218.

84. Cremer, T. and Cremer, C. (2001) Chromosome territories, nuclear architecture and gene regulation in mammalian cells. Nature Reviews Genetics, 2, 292-301.

85. Larschan, E. and Winston, F. (2001) The S. cerevisiae SAGA complex functions in vivo as a coactivator for transcriptional activation by Gal4. Genes Dev., 15, 1946-1956.

86. Mak, K.L., Longcor, L.C., Johnson, S.E., Lemercier, C., To, R.Q. and Konieczny, S.F. (1996) Examination of mammalian basic helix-loop-helix transcription factors using a yeast one-hybrid system. DNA and Cell Biology, 15, 1-8.

87. Hanel, F., Peukert, K. and Munder, T. (1997) Control of the Myc-Max mediated transactivation in yeast by natural promoter elements. Journal of Basic Microbiology, 37, 23-28.

88. Kasten, M.M., Ayer, D.E. and Stillman, D.J. (1996) SIN3-dependent transcriptional repression by interaction with the Mad1 DNA-binding protein. Mol. Cell. Biol., 16, 4215-4221.

89. Carver, L.A., Jackiw, V. and Bradfield, C.A. (1994) The 90-kDa heat shock protein is essential for Ah receptor signaling in a yeast expression system. J. Biol. Chem., 269, 30109-30112.

90. Whitelaw, M.L., McGuire, J., Picard, D., Gustafsson, J.A. and Poellinger, L. (1995) Heat shock protein hsp90 regulates dioxin receptor function in vivo. Proc. Natl. Acad. Sci. U. S. A., 92, 4437-4441.

91. Yamaguchi, Y. and Kuo, M.T. (1995) Functional analysis of aryl hydrocarbon receptor nuclear translocator interactions with aryl hydrocarbon receptor in the yeast two-hybrid system. Biochemical Pharmacology, 50, 1295-1302.

92. Miller, C.A. (1999) A human aryl hydrocarbon receptor signaling pathway constructed in yeast displays additive responses to ligand mixtures. Toxicology and Applied Pharmacology, 160, 297-303.

93. Sugawara, T. and Nomura, E. (2006) Development of a recombinant yeast assay to detect Ah-receptor ligands. Toxicology Mechanisms and Methods, 16, 287-294.

94. Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

100

95. Ptashne, M. and Gann, A. (1997) Transcriptional activation by recruitment. Nature, 386, 569-577.

96. Wolberger, C. (1998) Combinatorial transcription factors. Curr. Opin. Genet. Dev., 8, 552-559.

97. Ransone, L.J. and Verma, I.M. (1990) Nuclear proto-oncogenes Fos and Jun. Annual Review of Cell Biology, 6, 539-557.

98. Atchley, W.R. and Fitch, W.M. (1997) A natural classification of the basic helix-loop-helix class of transcription factors. Proc. Natl. Acad. Sci. U. S. A., 94, 5172-5176.

99. Wang, Y., Schwedes, J.F., Parks, D., Mann, K. and Tegtmeyer, P. (1995) Interaction of p53 with its consensus DNA-binding site. Mol. Cell. Biol., 15, 2157-2165.

100. Zhao, K.H., Chai, X.M., Johnston, K., Clements, A. and Marmorstein, R. (2001) Crystal structure of the mouse p53 core DNA-binding domain at 2.7 Å resolution. J. Biol. Chem., 276, 12120-12127.

101. Farmer, G., Bargonetti, J., Zhu, H., Friedman, P., Prywes, R. and Prives, C. (1992) Wild-type p53 activates transcription in vitro. Nature, 358, 83-86.

102. Lilyestrom, W., Klein, M.G., Zhang, R.G., Joachimiak, A. and Chen, X.J.S. (2006) Crystal structure of SV40 large T-antigen bound to p53: Interplay between a viral oncoprotein and a cellular tumor suppressor. Genes Dev., 20, 2373-2382.

103. Iwabuchi, K., Bartel, P.L., Li, B., Marraccino, R. and Fields, S. (1994) Two cellular proteins that bind to wild-type but not mutant p53. Proc. Natl. Acad. Sci. U. S. A., 91, 6098-6102.

104. Bargonetti, J., Reynisdottir, I., Friedman, P.N. and Prives, C. (1992) Site-specific binding of wild-type p53 to cellular DNA is inhibited by SV40 T antigen and mutant p53. Genes Dev., 6, 1886-1898.

105. Segawa, K., Minowa, A., Sugasawa, K., Takano, T. and Hanaoka, F. (1993) Abrogation of p53-mediated transactivation by SV40 large T antigen. Oncogene, 8, 543-548.

106. Gorina, S. and Pavletich, N.P. (1996) Structure of the p53 tumor suppressor bound to the ankyrin and SH3 domains of 53BP2. Science, 274, 1001-1005.

107. Tidow, H., Veprintsev, D.B., Freund, S.M.V. and Fersht, A.R. (2006) Effects of oncogenic mutations and DNA response elements on the binding of p53 to p53-binding protein 2 (53BP2). J. Biol. Chem., 281, 32526-32533.

108. Chung, C.T., Niemela, S.L. and Miller, R.H. (1989) One-step preparation of competent Escherichia coli: Transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sci. U. S. A., 86, 2172-2175.

109. Orrweaver, T.L., Szostak, J.W. and Rothstein, R.J. (1981) Yeast transformation: A model system for the study of recombination. Proc. Natl. Acad. Sci. U. S. A., 78, 6354-6358.

110. Bartel, P.L., Chien, C.-T., Sternglanz, R. and Fields, S. (1993) In Hartley, D. A. (ed.), Cellular interactions in development: A practical approach. Oxford University Press, Oxford, England, pp. 153-179.

111. Dillon, P.J. and Rosen, C.A. (1990) A rapid method for the construction of synthetic genes using the polymerase chain reaction. Biotechniques, 9, 298-300.

101

112. Oliphant, A.R., Nussbaum, A.L. and Struhl, K. (1986) Cloning of random-sequence oligodeoxynucleotides. Gene, 44, 177-183.

113. Tornow, J. and Santangelo, G.M. (1990) Efficient expression of the Saccharomyces cerevisiae glycolytic gene ADH1 is dependent upon a cis-acting regulatory element (UASRPG) found initially in genes encoding ribosomal proteins. Gene, 90, 79-85.

114. Clontech. (2001) Yeast Protocols Handbook, Palo Alto, CA.

115. Ammerer, G. (1983) Expression of genes in yeast using the ADCI promoter. Methods Enzymol., 101, 192-201.

116. Titz, B., Thomas, S., Rajagopala, S.V., Chiba, T., Ito, T. and Uetz, P. (2006) Transcriptional activators in yeast. Nucleic Acids Res., 34, 955-967.

117. Thukral, S.K., Blain, G.C., Chang, K.K.H. and Fields, S. (1994) Distinct residues of human p53 implicated in binding to DNA, simian virus 40 large T antigen, 53BP1, and 53BP2. Mol. Cell. Biol., 14, 8315-8321.

118. Luo, Y., Batalao, A., Zhou, H. and Zhu, L. (1997) Mammalian two-hybrid system: A complementary approach to the yeast two-hybrid system. Biotechniques, 22, 350-352.

119. Hu, J.C. (2001) Model systems: Studying molecular recognition using bacterial n-hybrid systems. Trends in Microbiology, 9, 219-222.

120. Walhout, A.J.M. (2006) Unraveling transcription regulatory networks by protein-DNA and protein-protein interaction mapping. Genome Res., 16, 1445-1454.

121. Legrain, P. and Selig, L. (2000) Genome-wide protein interaction maps using two-hybrid systems. FEBS Letters, 480, 32-36.

122. Serebriiskii, I.G. and Golemis, E.A. (2001) Two-hybrid system and false positives. Approaches to detection and elimination. Methods in molecular biology, 177, 123-134.

123. Hanes, S.D., Riddihough, G., Ish-Horowicz, D. and Brent, R. (1994) Specific DNA recognition and intersite spacing are critical for action of the bicoid morphogen. Mol. Cell. Biol., 14, 3364-3375.

124. Swanson, H.I., Chan, W.K. and Bradfield, C.A. (1995) DNA binding specificities and pairing rules of the Ah receptor, ARNT, and SIM proteins. J. Biol. Chem., 270, 26292-26302.

125. Dong, L.Q., Ma, Q. and Whitlock, J.P., Jr. (1996) DNA binding by the heterodimeric Ah receptor: Relationship to dioxin-induced CYP1A1 transcription in vivo. J. Biol. Chem., 271, 7942-7948.

126. Kikuchi, Y., Ohsawa, S., Mimura, J., Ema, M., Takasaki, C., Sogawa, K. and Fujii-Kuriyama, Y. (2003) Heterodimers of bHLH-PAS protein fragments derived from AhR, AhRR, and Arnt prepared by co-expression in Escherichia coli: Characterization of their DNA binding activity and preparation of a DNA complex. J. Biochem., 134, 83-90.

127. Chen, G., DenBoer, L.M. and Shin, J.A. (2008) Design of a single plasmid-based modified yeast one-hybrid system for investigation of in vivo protein-protein and protein-DNA interactions. Biotechniques, in press.

128. Fujisawa-Sehara, A., Sogawa, K., Yamane, M. and Fujii-Kuriyama, Y. (1987) Characterization of xenobiotic responsive elements upstream from the drug-metabolizing

102

cytochrome P-450c gene: A similarity to glucocorticoid regulatory elements. Nucleic Acids Res., 15, 4179-4191.

129. Dohmen, R.J., Strasser, A.W.M., Honer, C.B. and Hollenberg, C.P. (1991) An efficient transformation procedure enabling long-term storage of competent cells of various yeast genera. Yeast, 7, 691-692.

130. Wong, J.M.Y., Harper, P.A., Meyer, U.A., Bock, K.W., Morike, K., Lagueux, J., Ayotte, P., Tyndale, R.F., Sellers, E.M., Manchester, D.K. et al. (2001) Ethnic variability in the allelic distribution of human aryl hydrocarbon receptor codon 554 and assessment of variant receptor function in vitro. Pharmacogenetics, 11, 85-94.

131. Swanson, H.I. and Yang, J.H. (1999) Specificity of DNA binding of the c-Myc/Max and ARNT/ARNT dimers at the CACGTG recognition site. Nucleic Acids Res., 27, 3205-3212.

132. Ghosh, S., Selby, M.J. and Peterlin, B.M. (1993) Synergism between Tat and VP16 in trans-activation of HIV-1 LTR. J. Mol. Biol., 234, 610-619.

133. Sathya, G., Li, W.Z., Klinge, C.M., Anolik, J.H., Hilf, R. and Bambara, R.A. (1997) Effects of multiple estrogen responsive elements, their spacing, and location on estrogen response of reporter genes. Molecular Endocrinology, 11, 1994-2003.

134. Ohashi, Y., Brickman, J.M., Furman, E., Middleton, B. and Carey, M. (1994) Modulating the potency of an activator in a yeast in vitro transcription system. Mol. Cell. Biol., 14, 2731-2739.

135. Tjian, R. and Maniatis, T. (1994) Transcriptional activation: A complex puzzle with few easy pieces. Cell, 77, 5-8.

136. Carey, M., Lin, Y.S., Green, M.R. and Ptashne, M. (1990) A mechanism for synergistic activation of a mammalian gene by GAL4 derivatives. Nature, 345, 361-364.

137. Denison, M.S., Fisher, J.M. and Whitlock, J.P., Jr. (1989) Protein-DNA interactions at recognition sites for the dioxin-Ah receptor complex. J. Biol. Chem., 264, 16478-16482.

138. Tsuchiya, Y., Nakajima, M. and Yokoi, T. (2003) Critical enhancer region to which AhR/ARNT and Sp1 bind in the human CYP1B1 gene. J. Biochem., 133, 583-592.

139. Ptashne, M. (1992) A genetic switch : Phage λ and higher organisms. 2nd ed. Cell Press & Blackwell Scientific Publications, Cambridge.

140. Majmudar, C.Y., Lum, J.K., Prasov, L. and Mapp, A.K. (2005) Functional specificity of artificial transcriptional activators. Chemistry & Biology, 12, 313-321.

141. Brownlie, P., Ceska, T.A., Lamers, M., Romier, C., Stier, G., Teo, H. and Suck, D. (1997) The crystal structure of an intact human Max-DNA complex: New insights into mechanisms of transcriptional control. Structure, 5, 509-520.

142. Huffman, J.L., Mokashi, A., Bachinger, H.P. and Brennan, R.G. (2001) The basic helix-loop-helix domain of the aryl hydrocarbon receptor nuclear transporter (ARNT) can oligomerize and bind E-box DNA specifically. J. Biol. Chem., 276, 40537-40544.

143. Cuthill, S., Wilhelmsson, A. and Poellinger, L. (1991) Role of the ligand in intracellular receptor function: Receptor affinity determines activation in vitro of the latent dioxin receptor to a DNA-binding form. Mol. Cell. Biol., 11, 401-411.

103

144. Whitelaw, M., Pongratz, I., Wilhelmsson, A., Gustafsson, J.A. and Poellinger, L. (1993) Ligand-dependent recruitment of the Arnt coregulator determines DNA recognition by the dioxin receptor. Mol. Cell. Biol., 13, 2504-2514.

145. Wu, L. and Whitlock, J.P., Jr. (1993) Mechanism of dioxin action: Receptor - enhancer interactions in intact cells. Nucleic Acids Res., 21, 119-125.

146. Schmid, T., Zhou, J. and Brüne, B. (2004) HIF-1 and p53: Communication of transcription factors under hypoxia. Journal of Cellular and Molecular Medicine, 8, 423-431.

147. Sogawa, K., Nakano, R., Kobayashi, A., Kikuchi, Y., Ohe, N., Matsushita, N. and Fujiikuriyama, Y. (1995) Possible function of Ah receptor nuclear translocator (Arnt) homodimer in transcriptional regulation. Proc. Natl. Acad. Sci. U. S. A., 92, 1936-1940.

148. Chen, G. and Shin, J.A. (2008) AhR/Arnt:XRE interaction: Turning false negatives into true positives in the modified yeast one-hybrid assay. Analytical Biochemistry, in press.

149. Swanson, H.I. (2002) DNA binding and protein interactions of the AHR/ARNT heterodimer that facilitate gene activation. Chemico-Biological Interactions, 141, 63-76.

150. O'shea, E.K., Rutkowski, R., Stafford, W.F. and Kim, P.S. (1989) Preferential heterodimer formation by isolated leucine zippers from Fos and Jun. Science, 245, 646-648.

151. Newman, J.R.S. and Keating, A.E. (2003) Comprehensive identification of human bZIP interactions with coiled-coil arrays. Science, 300, 2097-2101.

152. Chapman-Smith, A., Lutwyche, J.K. and Whitelaw, M.L. (2004) Contribution of the Per/Arnt/Sim (PAS) domains to DNA binding by the basic helix-loop-helix PAS transcriptional regulators. J. Biol. Chem., 279, 5353-5362.

153. Chow, H.-K., Xu, J., Shahravan, S.H., De Jong, A.T., Chen, G. and Shin, J.A. (2008) Minimalist protein design: A bHLHZ-like hybrid of the bHLH/PAS Arnt and C/EBP bZIP targets the E-box DNA site in vivo and in vitro. submitted to PLoS ONE.

154. Pongratz, I., Antonsson, C., Whitelaw, M.L. and Poellinger, L. (1998) Role of the PAS domain in regulation of dimerization and DNA binding specificity of the dioxin receptor. Mol. Cell. Biol., 18, 4079-4088.

155. Lindebro, M.C., Poellinger, L. and Whitelaw, M.L. (1995) Protein-protein interaction via PAS domains: Role of the PAS domain in positive and negative regulation of the bHLH/PAS dioxin receptor-Arnt transcription factor complex. EMBO Journal, 14, 3528-3539.

156. Reisz-Porszasz, S., Probst, M.R., Fukunaga, B.N. and Hankinson, O. (1994) Identification of functional domains of the aryl hydrocarbon receptor nuclear translocator protein (ARNT). Mol. Cell. Biol., 14, 6075-6086.

157. Turner, E.C., Cureton, C.H., Weston, C.J., Smart, O.S. and Allemann, R.K. (2004) Controlling the DNA binding specificity of bHLH proteins through intramolecular interactions. Chemistry & Biology, 11, 69-77.

158. Vinson, C.R., Hai, T.W. and Boyd, S.M. (1993) Dimerization specificity of the leucine zipper-containing bZIP motif on DNA binding: Prediction and rational design. Genes Dev., 7, 1047-1058.

104

159. Wendt, H., Thomas, R.M. and Ellenberger, T. (1998) DNA-mediated folding and assembly of MyoD-E47 heterodimers. J. Biol. Chem., 273, 5735-5743.

160. Gu, Y.Z., Hogenesch, J.B. and Bradfield, C.A. (2000) The PAS superfamily: Sensors of environmental and developmental signals. Annual Review of Pharmacology and Toxicology, 40, 519-561.

161. Antonsson, C., Whitelaw, M.L., McGuire, J., Gustafsson, J.A. and Poellinger, L. (1995) Distinct roles of the molecular chaperone hsp90 in modulating dioxin receptor function via the basic helix-loop-helix and PAS domains Mol. Cell. Biol., 15, 756-765.

162. Benezra, R., Davis, R.L., Lockshon, D., Turner, D.L. and Weintraub, H. (1990) The protein Id: A negative regulator of helix-loop-helix DNA binding proteins. Cell, 61, 49-59.

163. Morgenstern, B. and Atchley, W.R. (1999) Evolution of bHLH transcription factors: Modular evolution by domain shuffling? Molecular Biology and Evolution, 16, 1654-1663.

164. Jean-François, N., Frédéric, G., Raymund, W., Benoit, C. and Lavigne, P. (2003) Improving the thermodynamic stability of the leucine zipper of max increases the stability of its b-HLH-LZ:E-box complex. J. Mol. Biol., 326, 1577-1595.

165. Krylov, D., Kasai, K., Echlin, D.R., Taparowsky, E.J., Arnheiter, H. and Vinson, C. (1997) A general method to design dominant negatives to B-HLHZip proteins that abolish DNA binding. Proc. Natl. Acad. Sci. U. S. A., 94, 12274-12279.

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Appendix A

Design of a single plasmid-based modified yeast one-hybrid system for investigation of in vivo protein-protein and protein:DNA

interactions Gang Chen, Lisa M. DenBoer, and Jumi A. Shin

Appendix A contains the original Supplemental Information for the paper accepted by

BioTechniques (Chapter 2 in this thesis), with necessary changes for thesis organization purposes,

as well as western blot data confirming expression of the proteins examined in the MY1H system.

MCS I

CTA TTC GAT GAT GAA GAT ACC CCA CCA AAC CCA AAA AAA GAG ATC TTT AAT ACG ACT

CAC TAT AGG GCG AGC GCC GCC ATC ATG GAG GAG CAG AAG CTG ATC TCA GAG GAG GAC

CTG CAT ATG GCC ATG GAG GCC GCG GTC GAC AAG GTG CGC GCT CTA GAT GAT CAT GAA

TCG TAG ATA CTG AAA AAC CCC GCAAGTTCACTTCAACTGTGCATCGTGCAC

T7 Promoter

c-Myc Epitope Tag

STARTin vitro

GAL4 Activation Domain

BssH II

Xba I

STOP

STOP

Bcl I

STOP

Sac II

Sal I

STOP

STOP

MCS II

CCT CCA AAA AAG AAG AGA AAG GTC GAA TTG GGT ACC GCC GCC AAT AAA GAG ATC TTT

AAT ACG ACT CAC TAT AGG GCG AGC GCC GCC ATG GAG TAC CCA TAC GAC GTA CCA GAT

TAC GCT CAT ATG GCC ATG GAG GCC AGT GAA TTC CAC CCG GGT GGG CAT CGA TAC GGG

ATC CAT CGA GCT CGA GCT GCA GAT GAA TCG TAG ATA CTG AAA AAC CCC GCA AGT TCA

CTTCAACTGTGCATC

T7 Promoter

SV40 Nuclear Localization Sequence

STARTin vitro

HA Epitope Tag

Sma Xma I I/STOP

Nde I

Sfi I STOP

Xho I

Sac I

Pst I

EcoR I STOP

Cla I

BamH I

Figure A.1 Plasmids pCETT (truncated ADH1 promoter) and pCETF (full-length ADH1 promoter) were constructed for coexpression of two proteins in a yeast model system. The two vectors have unique restriction sites located in both the MCS I and MCS II regions. MCS I is at the 3'-end of the open reading frame for the GAL4 AD sequence allowing a fusion protein combining amino acids 768–881 of the GAL4 AD and the cloned protein of interest to be expressed at low levels from a truncated constitutive ADH1 promoter. The expression of genes inserted into MCS II is controlled by either the truncated ADH1 promoter (pCETT) or full length ADH1 promoter (pCETF). Therefore, a second protein can be coexpressed at either low levels (pCETT) or high levels (pCETF). Both vectors also contain a T7 promoter at both MCS regions, a c-Myc epitope tag at MCS I, and an HA epitope tag at MCS II.

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A.1 Western blot analysis Yeast cells were grown as described for the 3-AT titration assay. Cells were then

harvested by centrifugation for 5 min at 3300 rpm and resuspended in 100 µL cold lysis buffer

(50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1%

SDS, and 0.02% sodium azide) containing freshly added protease inhibitors [100 µg/ml

phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml

pepstatin]. The suspensions were incubated on ice for 5 min, and then subjected to 3 rounds of

sonication for 15 sec each. Lysates were again incubated on ice for 5 min, and then centrifuged

for 10 min at 4 °C. The pellet was resuspended in 100 µL 3M urea and incubated at room

temperature for 3 min. An equal volume of SDS-PAGE sample buffer (50 mM Tris-HCl, pH 6.8,

100 mM DTT, 2% SDS, 10% glycerol, and 0.1% bromophenol blue) was added and the samples

were boiled for 10 min. Samples were stored at -80 °C until use.

20 µL of samples were loaded on a 12% SDS-PAGE (Tris-Glycine) gel and run at 100V

for 90 min in SDS-PAGE Running Buffer (25 mM Tris-HCl, 192 mM glycine, 0.5% w/v SDS),

then transferred by tank electroblotting to a nitrocellulose membrane at 100V for 75 min in cold

transfer buffer (25 mM glycine, 192 mM Tris-HCl, 20% v/v methanol). The membrane was

washed in TBS (10 mM Tris-HCl, pH 7.5, 150 mM NaCl) for 10 min and washed twice in TBS-

T (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.05% v/v Tween-20) for 10 min each. The

membrane was blocked in TBS-T containing 5% non-fat skim milk powder at room temperature

with shaking overnight. The membrane was washed twice in TBS-T for 10 min each, then once

in TBS for 10 min. The primary antibody (HA.11 or 9E10(c-Myc), Covance Inc., Princeton, NJ)

was diluted 1000-fold in TBS and the membrane was incubated for 2 hrs at room temperature,

followed by washing as before, and incubation with the secondary antibody (goat anti-mouse

107

IgG-HRP conjugate, Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hr at room temperature.

The membrance was washed again, as before, and the protein bands were visualized using the

ECL Plus Western Blotting Detection System (Amersham Biosciences, Piscataway, NJ) on a

Molecular Dynamics Storm 840 PhosphorImager System.

The gel was washed in water, post-transfer, for 10 min, then stained with Coomassie

Brillant Blue (0.25% w/v in 50% v/v methanol) for 30 min, and de-stained in 10% v/v acetic acid

until the desired color was reached.

A.2 Expression levels of LTAg and 53BP2 in the MY1H system

Figure A.2 Promoter length dictates differential expression levels of inhibitory proteins when YM4271[p53HIS] cells were transformed with the indicated plasmids and grown to exponential phase in YPDA media. Cells were lysed by sonication and 20 µl aliquots were separated by SDS-PAGE and subjected to Western blotting. Proteins were immuno-detected using an anti-HA antibody and visualized by fluorescence detection on a Molecular Dynamics Storm 840 Phosphorimager.

Western blot analysis was performed to examine the expression levels of GAL4AD-p53

and the two inhibitory proteins LTAg and 53BP2 in the MY1H system. We obtained positive

signal from MCS II, but not from MCS I. As stated by Clontech, the truncated ADH1 promoter

in vector pGAD424, the protein expression plasmid provided in the Matchmaker™ One-Hybrid

System, leads to very low levels of fusion protein expression in yeast, such that the signal is not

108

detectable in western blot analysis (114). As our coexpression plasmids, pCETT and pCETF, are

based on pGAD424, they share the same expression cassette in MSC I. Although we also tried

different primary monoclonal antibodies (c-Myc mAb and p53 mAb), none displayed positive

signal.

In contrast, HA-tagged LTAg and 53BP2 fusion proteins expressed from both pCETT

and pCETF in the MY1H system are clearly detectable by using an anti-HA antibody in the

western blot analysis. The LTAg and 53BP2 fusion proteins are approximately 78 kDa and 33

kDa, respectively; the nonsense protein encoded by the cloning vector, which includes the HA

tag, is approximately 8.6 kDa. All HA-tagged proteins of expected molecular weights are

visualized in Figure A.2. Comparison of protein expression from pCETT and pCETF

demonstrates that genes inserted into MCS II of pCETF are expressed at higher levels than those

inserted into MCS II of pCETT, which is due to the different lengths of the ADH1 promoters that

control gene expression in MCS II: the full-length ADH1 promoter leads to higher expression

levels than the truncated promoter.

Although in pCETT, both MCS I and MCS II are under the control of the truncated

ADH1 promoter, western blot results revealed that only protein expressed from the MCS II, not

from the MCS I, is detectable. This observation may indicate the difference in levels of protein

expression between MCS I and MCS II in pCETT. An additional consideration that may affect

protein detection during western blot analysis is that different epitope tags (c-Myc vs. HA) are

used in MCS I and MCS II, with different positioning that may affect accessibility of the tags

during incubation with the respective mAb (c-Myc is in the middile of the fusion protein vs. HA

at the N-terminus)

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A.3 Expression of the AD-fusion protein from MCS I is not affected by expression from MCS II Although the expression of the GAL4AD-p53 fusion could not be detected in the western

blot analysis, we believe our conclusion that expression of the AD-fusion protein from MCS I is

not affected by expression from MCS II is still valid. The reasons are given briefly in the text, as

well as explicitly described below.

First, 3-AT titration analysis shows that expression of only GAL4AD-p53 from either

pCETT or pCETF leads to comparable reporter gene activation curves (Figure 2.3). The colony

lift assay shows no difference in blue color intensity for these two controls (Figure 2.4).

Additionally, in the ONPG assay, the β-galactosidase values are 492±11 and 465±34 when

GAL4AD-p53 only is expressed from vectors pCETT and pCETF, respectively (Figure 2.5 and

Table A.1). These statistically comparable data from ONPG analysis, together with 3-AT titation

and colony lift assay results, reveal that the different ADH1 promoters (truncated vs. full-length)

that control the transcription of genes cloned into MCS II do not affect the expression from MCS

I.

Table A.1 Reporter activation of GAL4AD fusions of p53 or Max expressed from different vectors Strain Plasmid β-galactosidase units

pGAD53m 457* pCETT/53 492±11 YM4271[p53BLUE] pCETF/53 465±34

pGAD424/Max 147±7** pCETT/Max 155±20** YM4271[pLacZi/E-box]pCETF/Max 137±13**

*: One trial performed in triplicate. **: J. Xu, J. A. Shin, unpublished results

Second, the expression of GAL4AD-p53 is not affected by the low or high levels of

nonsense protein (76 aa) expressed from cloning vectors pCETT and pCETF when no gene is

110

cloned into MCS II. We tested the effect of expression of this nonsense protein from MCS II on

transcription activation of GAL4AD-p53 expressed from MCS I by ONPG assay. We then

compared these results to those obtained when using singly transformed yeast expressing

GAL4AD-p53 from the original pGAD53m plasmid supplied by Clontech. The β-galactosidase

value from the tranformant with pGAD53m is 457 (Table A.1), which is comparable to 492±11

and 465±34, the values from transformants with pCETT/53 and pCETF/53, respectively, in

which p53 is expressed from MCS I described above. In addition, we did not observe any

retarding or enhancing growth in cells transformed with pCETT/53 or pCETF/53. These results

suggest that the nonsense protein expressed from MCS II in pCETT or pCETF, regardless of low

or high expression levels, is neutral to the expression of GAL4AD-p53 from MCS I.

A separate project supports the conclusion that the nonsense protein expressed from MCS

II in pCETT or pCETF does not affect the expression of the GAL4AD fusion from MCS I. In

this project, the E-box binding protein Max was expressed from MCS I from different protein

expression vectors including pGAD424, pCETT, and pCETF in the E-box integrated yeast strain

(J. Xu, J. A. Shin, unpublished results). The ONPG assay was performed to compare the reporter

gene activation of the GAL4AD-Max fusion expressed from these different vectors (Table A.1).

The β-galactosidase values are 147±7, 155±20, and 137±13 for pGAD424/Max, pCETT/Max,

and pCETF/Max, respectively: again, quantitative ONPG values that are virtually identical for

protein expressed from MCS I, regardless of no, low or high expression of nonsense protein from

MCS II. Therefore, these experiments further validate the conclusion that the nonsense protein

expressed from MCS II of pCETT or pCETF does not affect the expression of the GAL4AD

fusion expressed from MCS I.

111

Therefore, although we could not produce a western blot for the GAL4AD fusion protein

expressed from the MCS I, we believe that with the evidence from different systems described

both in the text and above, our MY1H system is validated.

112

Appendix B

AhR/Arnt:XRE interaction: turning false negatives into true positives in the modified yeast one-hybrid assay

Gang Chen and Jumi A. Shin

Appendix B is the Supplemental Information for the paper accepted by Analytical

Biochemistry (Chapter 3 in this thesis), with necessary changes for thesis organization purposes.

Table B.1 Oligonucleotides used in this study* No Sequence 3-1 AAAGAATTCTTGCGTGTTGCGTGTTGCGTG

3-2 AAATCTAGACACGCAACACGCAACACGCAA

3-3 AAAGTCGACCACGCAACACGCAACACGCAA

3-4 AAAGAATTCTTGCGTGTTGCGTGTTGCGTGTTGCGTGTTGCGTGTTGCGTG

3-5 AAATCTAGACACGCAACACGCAACACGCAACACGCAACACGCAACACGCAA

3-6 AAAGTCGACCACGCAACACGCAACACGCAACACGCAACACGCAACACGCAA

3-7 ATGCGTCGACGCCAACATCACCTACGCCAG

3-8 ATGCTCTAGATCAACTAGTGCCATTTTTAGTC

3-9 ATGCGGATCCATAGCTCTGCGGATAAAGAGAG

3-10 ATTTTCCCTGGCAAGTCTCTCTTTATCCGCAGAG

3-11 AGAGAGACTTGCCAGGGAAAATCACAGTGA

3-12 ATGCCTCGAGTCACACATTGGTGTTGGTACAGA

*Oligonucleotide sequences are shown in 5′ to 3′ direction. Restriction sites used for cloning are in bold.

B.1 Construction of reporter strains

B.1.1 Three-copy strains The integrated strains YM4271[pHISi-1/XRE-3] and YM4271[pLacZi/XRE-3] contain

three copies of the XRE site upstream of the HIS3 and lacZ reporter genes, respectively, in the

yeast genome.

113

A DNA fragment containing three tandem copies of the XRE target sequence (5'-

TTGCGTG-3') was assembled by mutually primed synthesis (112) using oligonucleotides 3-1

and 3-2 (Table B.1; all oligonucleotides discussed in Appendix B are listed in Table B.1 and also

described in Table D.1 of Appendix D). This fragment was inserted between EcoR I and Xba I

sites of the yeast integration and reporter vector pHISi-1 to create pHISi-1/XRE-3. pHISi-1

contains the yeast HIS3 gene downstream of the MCS and the minimal promoter of the his3

locus. The recombinant vector pHISi-1/XRE-3 was then linearized at the Xho I site in the 3’-

untranslated region immediately following the HIS3 marker and integrated into the mutated his3

locus of YM4271 to produce the reporter strain YM4271[pHISi-1/XRE-3]. The integration

confers a His+ phenotype on the transformants. The 3-AT titration assay was performed to

determine the concentration of 3-AT sufficient for suppression of background growth of

YM4271[pHISi-1/XRE-3]. It was found that 30 mM 3-AT was sufficient to suppress

YM4271[pHISi-1/XRE-3] background growth on SD/–His medium.

Similarly, another DNA fragment containing three tandem copies of the XRE target

sequence was assembled by mutually primed synthesis (112) using oligonucleotides 3-1 and 3-3.

This fragment was inserted between EcoR I and Sal I sites of the yeast integration and reporter

vector pLacZi to create pLacZi/XRE-3. This recombinant reporter plasmid was linearized at the

Nco I site and integrated into the mutated ura3 locus of YM4271. The integration confers a Ura+

phenotype on the transformants.

B.1.2 Six-copy strains YM4271[pHISi-1/XRE-6] and YM4271[pLacZi/XRE-6] contain six copies of the XRE

site upstream of the HIS3 and lacZ reporter genes, respectively, in the yeast genome.

The construction of YM4271[pHISi-1/XRE-6] and YM4271[pLacZi/XRE-6] strains was

similar to that of their corresponding counterparts described above, respectively. The

114

oligonucleotides used for construction of YM4271[pHISi-1/XRE-6] were oligos 3-4 and 3-5; the

oligonucleotides used for construction of YM4271[pLacZi/XRE-6] were oligos 3-4 and 3-6.

3-AT titration assay revealed that 30 mM 3-AT was also sufficient to suppress

YM4271[pHISi-1/XRE-6] background growth on SD/–His medium.

B.2 Construction of AhR6-436 (NAhR) fragment NAhR fragment was amplified from original human AhR cDNA (130) using

oligonucleotides 3-7 and 3-8 as the 5′ and 3′ primers, respectively.

B.3 Construction of Arnt82-464 (NArnt) fragment ArntP1 fragment was assembled by mutually primed synthesis (112) using

oligonucleotides 3-9 and 3-10; ArntP2 fragment was amplified from original human Arnt variant

3 cDNA (130) using oligonucleotides 3-11 and 3-12 as the 5′ and 3′ primers, respectively. The 3’

end of the ArntP1 fragment contains 22 bp homology with the 5′ end of the ArntP2 fragment.

The final NArnt fragment was then amplified with ArntP1 and ArntP2 as templates using

oligonucleotides 3-9 and 3-12 as the 5′ and 3′ primers, respectively.

115

Figure B.1 Colony-lift filter assay for detection of NAhR/NArnt:XRE interaction from protein expression vector pCETT or pCETT2 in YM4271[pHISi-1/XRE-3] (A and C) and YM4271[pHISi-1/XRE-6] (B and D) strains. The X-gal colony-lift assay was performed after 4 days growth of transformants on SD/-L/-U plates at 30 °C. Photos were taken after 60 min incubation at 30 °C. The transformants were identified according to the following order. A and B: upper left, pCETT/NAhR/NArnt; upper right, pCETT/NAhR; lower left, pCETT//NArnt; lower right, pCETT. C and D: upper left, pCETT2/NAhR/NArnt; upper right, pCETT2/NAhR; lower left, pCETT2//NArnt; lower right, pCETT2.

116

Appendix C

Forced protein heterodimerization and specific DNA binding to a nonpalindromic DNA sequence in vivo and in vitro: bHLHZ-like hybrid heterodimers of bHLH/PAS proteins AhR and Arnt and

bZIP proteins JunD and Fos as a model Gang Chen, Antonia T. De Jong, S. Hesam Shahravan, and Jumi A. Shin

Appendix C is the Supplemental Information for Chapter 4.

Table C.1 Oligonucleotides used in this study* No Sequence 4-1 TGCGTCGACCAGAAAACAGTAAAGCCAAT

4-2 TATTCTAGAGGAGGATTTTAATGCAACA

4-3 ACTGAATTCAGCTCTGCGGATAAAGAGAG

4-4 ATACTCGAGGGATGTGTTGCCAGTTCCCC

4-5 AGCTTCTTTGATGTTGCATTAGAAGAAAAGGTTAAGACC

4-6 CAAAGAAGCGGTAGAAGCCAATTCGGTGTTTTGAGACTTCAAGGTCTTAACCTTTTCTTC

4-7 GCTTCTACCGCTTCTTTGTTGAGAGAACAAGTTGCTCAATTGAAGCAAAAGGTTTTGTCT

4-8 GTCCTCTAGAAACGTGAGACAAAACCTTTTGCTT

4-9 TGCGGGGAACTGGCAACACACAAGCTGAAACTGACCAA

4-10 CAACAAGTTAGCAATTTCGGTTTGCAAAGCAGACTTTTCGTCTTCCAATTGGTCAGTTTC

4-11 GAAATTGCTAACTTGTTGAAGGAAAAGGAAAAGTTGGAGTTTATCTTGGCTGCTCACAGA

4-12 AAAGGATCCTGGTCTGTGAGCAGCCAAGAT

4-13 AGCTTCTTTGATGTTGCAGAAGAAAAGGTTAAGACC

4-14 TGCGAATTCCAGAAAACAGTAAAGCCAAT

4-15 TATGGATCCGGAGGATTTTAATGCAACA

4-16 ATACTGCAGGGATGTGTTGCCAGTTCCCC

4-17 GTCCGGATCCAACGTGAGACAAAACCTTTTGCTT

*Oligonucleotide sequences are shown in 5' to 3' direction. Restriction sites used for cloning are in bold.

117

C.1 Plasmid Construction

C.1.1 pCETT2/AhRbHLH/ArntbHLH The AhRbHLH sequence encoding amino acids 20-90 of human AhR (GeneID: 196) was

amplified from AhR cDNA (130) with oligonucleotides 4-1 and 4-2 (Table C.1; all

oligonucleotides discussed in Appendix C are listed in Table C.1 and also described in Table D.1

of Appendix D) and inserted between the Sal I and Xba I sites of pCETT2 to construct

pCETT2/AhRbHLH. The ArntbHLH sequence encoding amino acids 82-149 of human Arnt

(GeneID: 405) isoform 1 was amplified from the Arnt82-464 cDNA fragment (148) with

oligonucleotides 4-3 and 4-4 and inserted between the EcoR I and Xho I sites of pCETT2 and

pCETT2/AhRbHLH to generate pCETT2//ArntbHLH and pCETT2/AhRbHLH/ArntbHLH,

respectively.

C.1.2 pCETT/AhRJunD/ArntFos and pCETT2/AhRJunD/ArntFos The RJunD fragment containing the sequence encoding amino acids 81-86 of human

AhR followed by amino acids 296-332 of human JunD (GeneID: 3727) was assembled by self-

priming PCR (111) using oligonucleotides 4-5 to 4-8. The final AhRJunD fragment was then

amplified with RJunD and AhRbHLH described above as templates using oligonucleotides 4-1

and 4-8 as the 5' and 3' primers, respectively. This amplified fragment was digested with Sal I

and Xba I, and then ligated into these restriction sites in pCETT and pCETT2 to construct

pCETT//AhRJunD and pCETT2/AhRJunD, respectively. We note that the AhRJunD hybrid

contains amino acids 20-86 of human AhR, which is in contrast to the AhRbHLH fragment

constructed above that encodes amino acids 20-90 of human AhR.

The ArntFos fragment was constructed in a similar manner to that of AhRJunD: the TFos

fragment containing the sequence encoding amino acids 143-148 of human Arnt followed by

amino acids 165-201 of human c-Fos (GeneID: 2353) was assembled by self-priming PCR (111)

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using oligonucleotides 4-9 to 4-12; the final ArntFos fragment was then amplified with TFos and

ArntbHLH described above as templates using oligonucleotides 4-3 and 4-12 as the 5' and 3'

primers, respectively. This fragment was digested with EcoR I and BamH I and inserted into

these restriction sites in pCETT/AhRJunD and pCETT2/AhRJunD to generate

pCETT/AhRJunD/ArntFos and pCETT2/AhRJunD/ArntFos, respectively. The EcoR I/BamH I-

digested ArntFos fragment was also inserted between these restriction sites in pCETT and

pCETT2 to generate pCETT//ArntFos and pCETT2//ArntFos, respectively. We also note that the

ArntFos hybrid contains amino acids 82-148 of human Arnt, which is in contrast to the

ArntbHLH fragment constructed above that encodes amino acids 82-149 of human Arnt.

C.1.3 pCETT2/AhR(ΔL)JunD/ArntFos The AhR(ΔL)JunD fragment was constructed in a similar fashion to that of AhRJunD.

The R(ΔL)JunD fragment was assembled by self-priming PCR (111) using oligonucleotides 4-13

and 4-6 to 4-8. The final AhR(ΔL)JunD fragment was then amplified with R(ΔL)JunD and

AhRbHLH described above as templates using oligonucleotides 4-1 and 4-8 as the 5' and 3'

primers, respectively. This amplified fragment was digested with Sal I and Xba I, and then

ligated into these restriction sites of pCETT2 and pCETT2//ArntFos to generate

pCETT2/AhR(ΔL)JunD and pCETT2/AhR(ΔL)JunD/ArntFos, respectively.

C.1.4 pGBKT7/AhRbHLH and pGADT7/AhRbHLH The AhRbHLH fragment was amplified from AhR cDNA (130) with oligonucleotides 4-

14 and 4-15 and inserted between the EcoR I and BamH I sites of pGBKT7 and pGADT7 to

generate pGBKT7/AhRbHLH and pGADT7/AhRbHLH, respectively.

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C.1.5 pGBKT7/ArntbHLH and pGADT7/ArntbHLH The ArntbHLH fragment was amplified from NArnt cDNA (148) with oligonucleotides

4-3 and 4-16 and inserted between the EcoR I and Pst I sites of pGBKT7 to generate

pGBKT7/ArntbHLH; the EcoR I/Xho I-digested ArntbHLH fragment described above was

inserted into these restriction sites in pGADT7 to generate pGADT7/ArntbHLH.

C.1.6 pGBKT7/AhRJunD and pGADT7/AhRJunD The AhRJunD fragment was amplified from the AhRJunD fragment with

oligonucleotides 4-14 and 4-17 and inserted between the EcoR I and BamH I sites of pGBKT7

and pGADT7 to generate pGBKT7/AhRJunD and pGADT7/AhRJunD, respectively.

C.1.7 pGBKT7/AhR(ΔL)JunD and pGADT7/AhR(ΔL)JunD The AhR(ΔL)JunD fragment was amplified from the AhR(ΔL)JunD fragment with

oligonucleotides 4-14 and 4-17 and inserted between the EcoR I and BamH I sites of pGBKT7

and pGADT7 to generate pGBKT7/AhR(ΔL)JunD and pGADT7/AhR(ΔL)JunD, respectively.

C.1.8 pGBKT7/ArntFos and pGADT7/ArntFos The EcoR I/BamH I-digested ArntFos fragment was inserted into these restriction sites in

pGBKT7 and pGADT7 to generate pGBKT7/ArntFos and pGADT7/ArntFos, respectively.

C.2 Expression of GAL4AD-AhRJunD and ArntFos by use of pCETT in the MY1H We also tried to express the GAL4AD-AhRJunD and ArntFos proteins by use of pCETT

in both YM4271[pHISi-1/XRE-6] and YM4271[pLacZi/XRE-6]. As expected, both HIS3

(Figure C.1) and lacZ (Figure C.2 and C.3) reporter assays showed the transformant displayed

positive signals while its corresponding controls, in which only one of the two hybrid proteins or

none was expressed, displayed negative signals. In addition, when GAL4AD-AhRJunD and

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ArntFos were coexpressed in the lacZ reporter strain, the ONPG value was 40.2±5.4, which is in

contrast to ONPG value 95.7±3.1 when GAL4AD-AhRJunD and GAL4AD-ArntFos were

coexpressed in the same reporter strain. This indicates that attachment of an AD to both

monomers increases the transcription potency of the reporter gene through synergistic activation,

thereby potentially increasing the sensitivity of the system.

Figure C.1 The HIS3 reporter assay for detection of the AhRJunD/ArntFos:XRE interaction by use of protein expression vector pCETT in strain YM4271[pHISi-1/XRE-6]. The transformants that coexpress both GAL4AD-AhRJunD and ArntFos proteins, as well as their corresponding controls, were plated on both SD/-H/-L control plates (left) and SD/-H/-L/30 mM 3-AT test plates (right) and incubated at 30 °C, 5 days. The transformants on the same plate are labeled according to the following order: upper left: pCETT/AhRJunD/ArntFos; upper right: pCETT/AhRJunD; lower left: pCETT//ArntFos; lower right: pCETT.

Figure C.2 The X-gal colony-lift filter assay. YM4271[pLacZi/XRE-6] cells were transformed with A) pCETT/AhRJunD/ArntFos; B) pCETT/AhRJunD; C) pCETT//ArntFos; D) pCETT and plated on SD/-L/-U plates. The colony-lift assay was performed after 4 days growth, 30 °C. Photos were taken after 60 min incubation at 30 °C.

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Figure C.3 ONPG measurements for detection of AhRJunD/ArntFos:XRE interactions by use of protein expression vector pCETT in strain YM4271[pHISi-1/XRE-6]. Vertical axis indicates the mean values in β-galactosidase units. Error bars represent standard error measurement from at least three independent trials conducted in triplicate.

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Appendix D Table of Oligonucleotides

Table D.1 Oligonucleotides used in this thesis* No. SEQUENCE APPLICATION

2-1 ACTATCTATTCGATGATGAAGATACCCCACCAAACCCAAA

2-2 GGCGCTCGCCCTATAGTGAGTCGTATTAAAGATCTCTTTTTTTGGGTTTGGTGGGGTATC

2-3 CTCACTATAGGGCGAGCGCCGCCATCATGGAGGAGCAGAAGCTGATCTCAGAGGAGGACC

2-4 GCGCGCACCTTGTCGACCGCGGCCTCCATGGCCATATGCAGGTCCTCCTCTGAGATCAGC

2-5 GCGGTCGACAAGGTGCGCGCTCTAGATGATCATGAATCGTAGATACTGAAAAACCCCGCA

2-6 ATGCACAGTTGAAGTGAACTTGCGGGGTTTTTCAGTATCT

Assembly of CE4MCS fragment to construct pGAD424-MCS II by homologous recombination.

2-7 AGAAAGGTCGAATTGGGTACCGCCGCCAATAAAGAGATCTTTAAT

2-8 CTCGCCCTATAGTGAGTCGTATTAAAGATCTCTTTATTGGCGGCG

Assembly of FINALREC fragment to construct pGAD424-MCS IIΔAD and pGADT7ΔAD by homologous recombination.

2-9 AAAGACGTCGCATGCAACTTCTTTTCTTT Forward primer for amplification of T2 fragment.

2-10 ATTGACGTCAAGCTTGCATGCCGGTAGAGGT Reverse primer for amplification of T2 and F2 fragments.

2-11 AAAGACGTCCCTGCAGGTCGAGATCCGGGA Forward primer for amplification of F2 fragment.

2-12 AAAAGTCGACCCTGTCACCGAGACCCCTGG

2-13 ACGCTCTAGATCAGTCTGAGTCAGGCCCCA

Forward and reverse primers for amplification of p53 fragment.

2-14 AAAGAATTCGGAACTGATGAATGGGAGCAG

2-15 AAAGGATCCTTATGTTTCAGGTTCAGGGGGAG

Forward and reverse primers for amplification of LTAg fragment.

2-16 AAAGAATTCCCGCCTGAAATCACCGGGCAG

2-17 AAAGGATCCTCAGGCCAAGCTCCTTTGTCTT

Forward and reverse primers for amplification of 53BP2 fragment.

3-1 AAAGAATTCTTGCGTGTTGCGTGTTGCGTG Forward primer for three-copy XRE target sequence assembly.

3-2 AAATCTAGACACGCAACACGCAACACGCAA Reverse primer for three-copy XRE target sequence assembly (Xba I).

3-3 AAAGTCGACCACGCAACACGCAACACGCAA Reverse primer for three-copy XRE target sequence assembly (Sal I).

3-4 AAAGAATTCTTGCGTGTTGCGTGTTGCGTGTTGCGTGTTGCGTGTTGCGTG Forward primer for six-copy XRE target sequence assembly.

3-5 AAATCTAGACACGCAACACGCAACACGCAACACGCAACACGCAACACGCAA Reverse primer for six-copy XRE target sequence assembly (Xba I).

3-6 AAAGTCGACCACGCAACACGCAACACGCAACACGCAACACGCAACACGCAA Reverse primer for six-copy XRE target sequence assembly (Sal I).

3-7 ATGCGTCGACGCCAACATCACCTACGCCAG

3-8 ATGCTCTAGATCAACTAGTGCCATTTTTAGTC

Forward and reverse primers for amplification of NAhR fragment (Sal I and Xba I).

3-9 ATGCGGATCCATAGCTCTGCGGATAAAGAGAG

3-10 ATTTTCCCTGGCAAGTCTCTCTTTATCCGCAGAG

3-11 AGAGAGACTTGCCAGGGAAAATCACAGTGA

3-12 ATGCCTCGAGTCACACATTGGTGTTGGTACAGA

26 and 29: forward and reverse primers for amplification of NArnt fragment. 27and 28: intermediate primers for the construction of NArnt fragment.

4-1 TGCGTCGACCAGAAAACAGTAAAGCCAAT Forward primer for amplification of AhRbHLH, AhRJunD and AhR(ΔL)JunD fragments (Sal I).

4-2 TATTCTAGAGGAGGATTTTAATGCAACA Reverse primer for amplification of AhRbHLH fragment (Xba I).

4-3 ACTGAATTCAGCTCTGCGGATAAAGAGAG Forward primer for amplification of ArntbHLH and ArntFos fragments.

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No. SEQUENCE APPLICATION

4-4 ATACTCGAGGGATGTGTTGCCAGTTCCCC Reverse primer for amplification of ArntbHLH fragment (Xho I).

4-5 AGCTTCTTTGATGTTGCATTAGAAGAAAAGGTTAAGACC Intermediate primer for the construction of AhRJunD fragment.

4-6 CAAAGAAGCGGTAGAAGCCAATTCGGTGTTTTGAGACTTCAAGGTCTTAACCTTTTCTTC

4-7 GCTTCTACCGCTTCTTTGTTGAGAGAACAAGTTGCTCAATTGAAGCAAAAGGTTTTGTCT

Intermediate primers for the construction of AhRJunD and AhR(ΔL)JunD fragments.

4-8 GTCCTCTAGAAACGTGAGACAAAACCTTTTGCTT Reverse primer for amplification of AhRJunD and AhR(ΔL)JunD fragments (Xba I).

4-9 TGCGGGGAACTGGCAACACACAAGCTGAAACTGACCAA

4-10 CAACAAGTTAGCAATTTCGGTTTGCAAAGCAGACTTTTCGTCTTCCAATTGGTCAGTTTC

4-11 GAAATTGCTAACTTGTTGAAGGAAAAGGAAAAGTTGGAGTTTATCTTGGCTGCTCACAGA

Intermediate primers for the construction of ArntFos fragments.

4-12 AAAGGATCCTGGTCTGTGAGCAGCCAAGAT Reverse primer for amplification of ArntFos fragment.

4-13 AGCTTCTTTGATGTTGCAGAAGAAAAGGTTAAGACC Intermediate primer for the construction of AhR(ΔL)JunD fragment.

4-14 TGCGAATTCCAGAAAACAGTAAAGCCAAT Forward primer for amplification of AhRbHLH, AhRJunD and AhR(ΔL)JunD fragments (EcoR I).

4-15 TATGGATCCGGAGGATTTTAATGCAACA Reverse primers for amplification of AhRbHLH fragment (BamH I).

4-16 ATACTGCAGGGATGTGTTGCCAGTTCCCC Reverse primer for amplification of ArntbHLH fragment (Pst I).

4-17 GTCCGGATCCAACGTGAGACAAAACCTTTTGCTT Reverse primers for amplification of AhRJunD and AhR(ΔL)JunD fragments (BamH I).

*Oligonucleotide sequences are shown in 5′ to 3′ direction. Restriction sites used for cloning are in bold.

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