12-1 Action at a Distance Bacterial and eukaryotic enhancers stimulate transcription even though...

12-1

Action at a Distance

• Bacterial and eukaryotic enhancers stimulate transcription even though located some distance from their promoters

• Four hypotheses attempt to explain the ability of enhancers to act at a distance– Change in topology ie. supercoiling– Sliding – Looping – Facilitated tracking

12-2

Hypotheses of Enhancer Action

Change in topology

Sliding

Looping

Facilitated tracking

12-5

Complex Enhancers

• Many genes can have more than one activator-binding site permitting them to respond to multiple stimuli

• Each of the activators that bind at these sites must be able to interact with the preinitiation complex assembling at the promoter, likely by looping out any intervening DNA

12-6

Control Region of the Metallothionine Gene

• The metallothionine gene product helps eukaryotes cope with heavy metal poisoning

• Turned on by several different agents• Complex enhancers enable a gene to respond

differently to different combinations of activators• This gives cells exquisitely fine control over their

genes in different tissues, or at different times in a developing organism

After identifying individual regions in the 5’ UTR of the Endo16 gene that bind nuclear proteins, the isolated binding regions were fused to a reporter cassette and reintroduced into sea urchin.

Expression was monitored and it was determined that some regions act alone and others in combination with each other.

Very important for the fine control of gene expression required during development..

12-9

Architectural Transcription Factors

Architectural transcription factors are those transcription factors whose sole or main purpose seems to be to change the shape of a DNA control region so that other proteins can interact successfully to stimulate transcription.

Important when control regions are in very close proximity to one another.

• Short DNA acts as rigid rod

• Long DNA behaves more like a string and can be easily manipulate

12-11

Example of Architectural Transcription Factor: Control region of the human T-cell receptor

alpha chain (TCR gene

• Within 112 bp upstream of the start of transcription are 3 enhancer elements

• These elements bind to:– Ets-1, LEF-1, CREB– LEF-1 alone cannot activate gene. Role?

• bends DNA at the minor grove by 130deg

• Used synthetic DNA containing LEF-1 binding site in electrophoretic assay to show DNA bending

• When the site was positioned in the middle of the synthetic DNA, movement through a gel was greatly retarded

12-13

DNA Bending Aids Protein Binding

• The activator LEF-1 binds to the minor groove of its DNA target through its HMG domain and induces strong bending of DNA

• LEF-1 does not enhance transcription by itself

• Bending it helps other activators bind and interact with activators and general transcription factors

12-14

Enhanceosome

• An enhanceosome is a nucleoprotein complex containing a collection of activators bound to an enhancer in such a way that stimulates transcription

• Ex. IFN-beta contains 8 binding sites which must all be occupied by activators. The other end of the simple/complex enhancer spectrum

• only activated when a cell is under attack by a virus.

Dilema:Some enhancers act at great distances from their promoters. ie. Drosophila cut locus is 85kb from promoters.

Enhancer is likely to come in proximity to other genes, how does the cell prevent inappropriate activation?

12-17

Insulators

• Insulators can shield genes from activation by enhancers (enhancer blocking activity)

• Insulators can shield genes from repression by silencers (barrier activity). Prevent condensing

• Insulators define regions between DNA domains. ie. Between enhancers and promoters

12-18

Mechanism of Insulator Activity

• One mechanism which can be ruled out is that insulators induce the condensation of DNA upstream of their location.– If a gene were placed upstream of such an insulator, it would

always be silenced

– Experiments in Drosophila show that such genes can still be active and can be activated by their own enhancers.

12-19

Mechanism of Insulator Activity

• Sliding model– Activator bound to an

enhancer and stimulator slides along DNA from enhancer to promoter

• Looping model– Two insulators flank an

enhancer, when bound they interact with each other isolating enhancer

12-20

Mechanism of Insulator Activity

• Sliding model– Activator bound to an

enhancer and stimulator slides along DNA from enhancer to promoter

• Looping model– Two insulators flank an

enhancer, when bound they interact with each other isolating enhancer

• However…some insulators work as single copies.

• ie. The Drosophila hairy-wing insulator

- single insulator = some insulator activity

- two insulators = no insulator activity

- insulator-enhancer-insulator = increased insulator activity

12-23

Model of Multiple Insulator Action

Canceling of insulator activity

12-24

Summary

• Some insulators have both enhancer-blocking and

barrier activities, but some have only one or the

other

• Insulators may do their job by working in pairs that

bind proteins that can interact to form DNA loops

that would isolate enhancers and silencers so they

can no longer stimulate or repress promoters

• Insulators may establish boundaries between DNA

regions in a chromosome

12-25

12.6 Regulation of Transcription Factors

• Several activators do not active transcription by contacting the basal transcription apparatus directly. Rely on:

• Coactivators = no activator function on its own, but collaborates with one or more activators to stimulate the expression of a set of genes

Activator

Coactivator

12-27

12.6 Regulation of Transcription Factors

• Phosphorylation of activators can allow them to interact with coactivators that in turn stimulate transcription

12-28

Model for the Activation of a Nuclear Receptor-Activated Gene

12-29

12.6 Regulation of Transcription Factors

• Sometimes genes are inactivated by the destruction of their activators

• Ubiquitylation of transcription factors can mark them for – Destruction by proteolysis

– Stimulation of activity

• Sumoylation is the attachment of the polypeptide SUMO which can target for incorporation into compartments of the nucleus

• Methylation and acetylation can modulate activity

12-30

Ubiquitylation

• Normal function of ubiquitylation is to mark proteins for destruction by the proteasome. ie. Aprx 20% of all proteins are made incorrectly and need to be quickly disposed of

• A fine balance may exist between coactivators and corepressors which have ubiquitylating ability- ie. A corepressor may mark a coactivator for destruction, tipping the scale towards repression

12-32

Activator Sumoylation(SUMO or small ubiquitin like modifier)

• Sumoylation is the addition of one or more copies of the 101-amino acid polypeptide SUMO (Small Ubiquitin-Related Modifier) to lysine residues on a protein

• Process is similar to ubiquitylation

• Results quite different – sumoylated activators are targeted to a specific nuclear compartment that keeps them stable

12-33

Activator Acetylation

• Nonhistone activators and repressors can be acetylated by HATs

• HAT is the enzyme histone acetyltransferase which can act on nonhistone activators and repressors

• Such acetylation can have either positive or negative effects- ex. p53 acetylation by coactivator p300 results in increased DNA binding

12-34

Signal Transduction Pathways

• Signal transduction pathways begin with a signaling molecule interacting with a receptor on the cell surface

• This interaction sends the signal into the cell and frequently leads to altered gene expression

• Many signal transduction pathways rely on protein phosphorylation to pass the signal from one protein to another

• This leads to signal amplification at each step

12-35

Three pathways that use CBP/p300 to mediate transcription activation

12-36

Ras and Raf Signal Transduction

Lecture key words- Cell cycle- Transcription factors- Phosphorylation- Heterodimerization- Immunohistochemistry- Immunprecipitation- Growth factors- Apoptosis- Colony formation- Nuclear localization - Consensus sequences- Motifs

CDCA7 | a case study in cellular regulation

• Cell cycle control is the endgame of cellular regulation- critical balance between proliferation and

apoptosis CANCER

CDCA7 | a case study in cellular regulation

• Cell cycle control is the endgame of cellular regulation- critical balance between proliferation and

apoptosis CANCER

• Modes: -phosphorylation, -subcellular localization - heterodimerization

CDCA7 | What is known

• Novel member of cell division cycle-associated gene family

• Myc and E2F target gene with peak expression at G1-S

• Frequently overexpressed in human tumors

• JPO2 binds Myc and promotes Myc dependent transformation• JPO2 and CDCA7 share cysteine rich C-term which may bind DNA

• Not known if CDCA7 interacts with Myc

Myc | Just the facts• Discovered in Burkitt’s lymphoma

patients• Member of bHLH-LZ family of transcription factors

• Requires heterodimerization with Max to transactivate

• Regulates the expression of ~10-15% of genes

• Role in development, cell division, cell growth, metabolism, angiogenesis

• Driving force of cell cycle and malignant transformation

• Active in 70% of human cancers

• ~100,000 cancer deaths per year in the US due to changes in Myc

• Early response gene induced by growth factors, levels peak at G0-G1

14-3-3

14-3-3

P

P P

PPIP2

PIP3

rictor

TORTOR

PDK1

P

P

P

P

14-3-3

14-3-3

Transcription Pro-apoptotic Genes ?

P

P

14-3-3

14-3-3

14-3-3

14-3-3

14-3-3

14-3-3

CDCA7CDCA7

14-3-3

14-3-3

CDCA7CDCA7

CDCA7CDCA7

Growth Factors

ReceptorTyrosineKinase

P

P

Cytoplasm

Nucleus

CDCA7 | a case study in cellular regulation

• Cell cycle control is the endgame of cellular regulation- critical balance between proliferation and

apoptosis CANCER

• Modes: -phosphorylation-subcellular localization -heterodimerization

humCDCA7

1 371

361261zinc finger

AKT consensus site R X R X X T/S F/LCDCA7 T163 R P R R R T F

T163

AKT kinase 0.005%

24 49

>90% conservedhuman monkeydogmousechickenfrogzebrafish

69 78 112 261 363190

CDCA7 | Conservation

CDCA7 is phosphorylated at t163

WT

T163A

CD

CA

7

CD

CA

7+

CIP

T16

3A

T16

3A +

CIP

Vec

tor

-FLAG

-P-T163

• Custom made antibody against phospho-T163

• Many ways to prove phosphorylation

Radioactivity and mutational analysis

Phosphotase

-FLAG

-P-T163

Ve

cto

r0 5 15 45 120 360 PDGF (min)

Merge

Ratio P-T163/Total CDCA7

1.0 2.2 3.6 4.7 4.0 3.9

T= 0’ T= 20’ T= 30’

T= 40’ T= 50’ T= 60’

CDCA7 is phosphorylated at t163

Treatmentsw/ growth factors

Immunohistochemistry

Vec

tor CDCA7

Akt inh VIII

IP: -FLAGBlot: -P-T163

IP: -FLAGBlot: -FLAG

CDCA7 is phosphorylated at t163 by AKT

Inhibitors

CDCA7 | a case study in cellular regulation

• Cell cycle control is the endgame of cellular regulation- critical balance between proliferation and

apoptosis CANCER

• Modes: -phosphorylation-subcellular localization -heterodimerization

T163A CDCA7

CDCA7

-Flag DAPI

Where is cdca7 found?

humCDCA7

1 371

361261zinc finger

T163

24 49

>90% conservedhuman monkeydogmousechickenfrogzebrafish

69 78 112

NLS?

NLS?

261 363190

157-186 RRPRRRTFPGVASRRNPERRARPLTRSRSR

CDCA7 | Conservation

How do we test for a nuclear localization signal?Isolate region in question and test its ability to target an

innocuous protein to the nucleus

CDCA7 contains an NLS

157-167 CDCA7

167-188 CDCA7 157-188 (T163A)

157-188 CDCA7

SV40 SV40 KE 157-188 R171E

R176E

R184ER176/184E

R171/176E

157-RRPRRRTFPGVASRRNPERRARPLTRSRSRIL-188

• Passive diffusion into nucleus <45 KDa

Phosphorylation alters localization-CDCA7 Nuclei Merge

Unstimulated

PDGF

PDGF + LY

CDCA7 | a case study in cellular regulation

• Cell cycle control is the endgame of cellular regulation- critical balance between proliferation and

apoptosis CANCER

• Modes: -phosphorylation-subcellular localization -heterodimerization

humCDCA7

1 371

361261zinc finger

14-3-3 consensus binding site R-[S/F/Y]-X-pS/T -X -P

cdcA7 T163 R R R T F PMekk2 T283 G R K T F P

T163

24 49

>90% conservedhuman monkeydogmousechickenfrogzebrafish

69 78 112

NLS?

NLS?

261 363190

157-186 RRPRRRTFPGVASRRNPERRARPLTRSRSR

CDCA7 | Conservation

14-3-3 | Just the facts

• Large family of highly conserved, small, acidic polypeptides of 28-33 kDa

• Seven different isoforms in humans, 14-3-3σ directly implicated in cancer

• Binds to protein ligands at defined phospho-serine/threonine motif RSXpS/TXP

• 14-3-3 regulates process relevant to cancer biology: cell-cycle progression, apoptosis and mitogenic signaling

• Over 200 known ligands

14-3-3 | Modes of influence

• 14-3-3 exists as a dimer and offers two binding sites for phospho-S/T motifs• Can function as adaptor protein for:a) two proteins that would otherwise not associateb) one protein with two 14-3-3 motifs = high affinity

• Affects change by:• Alteration of enzymatic activity – maintains RAF1 in

inactive state• Alteration of DNA-binding activity – increases p53

DNA-binding after DNA damage• Sequestration - BAD, FKHRL1, HDAC5 and CDC25C

are localized to cytoplasm• Altering protein-protein interactions - reduced affinity

of CDC25A to CDC2• Adaptor protein functions – bridging of RAF1 to BCR

• Sequestration • Altering protein-protein interactions

Adapted from Hermeking, 2005

CDCA7 binds14-3-3 and is phospho dependent

-FLAG

-P-T163

-14-3-3

Vec

tor

P16

5A

F16

4AT

163A

R16

2A

R16

1A

R16

0A

P15

9A

R15

8A

Wild

type

Blot:

14-3-3 consensus site - - R X pT X PS/F

/Y

Western blots

14-3-3 alters CDCA7 localization

R161A CDCA7

T163A CDCA7

R161A/T163A CDCA7

CDCA7

-Flag DAPI

Is 14-3-3 masking the NLS within the T163 region?

CDCA7 | What is known

• Novel member of cell division cycle-associated gene family

• Myc and E2F target gene with peak expression at G1-S

• Frequently overexpressed in human tumors

• JPO2 binds Myc and promotes Myc dependent transformation• JPO2 and CDCA7 share cysteine rich C-term which may bind DNA

• Not known if CDCA7 interacts with Myc

WTT163A

(112-137)

(1-146)

(1-172)(1-202)

(1-234)

(260-370)

(230-370)

(170-370)

(153-370)

++++

+++-

---

CDCA7

His-Myc PulldownBlot: -FLAG

InputBlot: -FLAG

WT

CD

CA

7

T16

3A C

DC

A7

(11

2-13

7) C

DC

A7

(1-

146)

CD

CA

7(

1-17

2) C

DC

A7

(1-

202)

CD

CA

7(

1-23

4) C

DC

A7

(26

0-37

0) C

DC

A7

(23

0-37

0) C

DC

A7

(17

0-37

0) C

DC

A7

(15

3-37

0) C

DC

A7

CDCA7 binds the transcription factor Myc

Co-immunoprecipitation

So how does cdca7 affect phenotype?

Apoptosis

proliferation

14-3-3/CDCA7 binding influence Myc-induced transformation

Colony formation assay

Rat1

Myc-Rat1

Sh1-Myc-Rat1

14-3-3/CDCA7 binding influence Myc-induced apoptosis

Trypan blue exclusion

14-3-3

14-3-3

P

P P

PPIP2

PIP3

rictor

TORTOR

PDK1

P

P

P

P

14-3-3

14-3-3

Transcription Pro-apoptotic Genes ?

P

P

14-3-3

14-3-3

14-3-3

14-3-3

14-3-3

14-3-3

CDCA7CDCA7

14-3-3

14-3-3

CDCA7CDCA7

CDCA7CDCA7

Growth Factors

ReceptorTyrosineKinase

P

P

Cytoplasm

Nucleus

summary• CDCA7 is a novel target of AKT required for Myc-

dependent apoptosis• Phosphorylation of T163 inhibits CDCA7/Myc apoptosis by:• Promoting 14-3-3 binding• Disruption of Myc binding

• Shuttling to the cytoplasm

• Potential for medical intervention in Myc tumors where AKT is dysregulated