How Histone Post-Translational Modifications Function When They Are Buried Under DNA

Michael Guy PoirierAssistant Professor

Department of PhysicsThe Ohio State University

DNA is Highly Wrapped and Compacted in Eukaryotes

Luger et al, Nature 1997

Alberts et al, 2002

Interphase

Mitosis

How can Wrapped DNA be Biologically Active

Bushnell, et al Science 2004 Richmond and Davey, 2002

Preinitiation Complex Nucleosome MSH2-MSH6

Warren et al, 2007

Nucleosomes must be altered for DNA Repair and RNA Transcription

1. Thermal fluctuations (i.e. site exposure)

2. Chromatin remodeling (i.e. SWI/SNF)

3. Histone Variants (i.e. CENP-A)

4. Chromatin associated proteins (i.e. HP1)

5. Post-translational modifications

Histone Post-Translational Modifications

Histone tails vs.

Histone fold domains

From Millipore’s website

Modifications in the Histone Fold Domains

Cosgrove et al, 2004

Key paper: Zhang, Eugeni, Parthun and Freitas, 2003

Histone H3 Modifications near the Nucleosome Dyad

Acetylation of H3-K115 and H3-K122.

NH3+

HN

O

O NH2

Mimicking Acetylation with K to Q mutation.

•Acetylation of K115 and K122 occur individually and together (Zhang, Eugeni, Parthun and Freitas, 2003).•These residues are important for both transcriptional regulation and DNA repair.

H3 GrowthrDNA

SilencingTelomeric Silencing

HU Resistance

Zeocin Resistance

PHO5 Induction

WT +++ +++ +++ +++ +++ ++++

K115A +++ ++ ++ ++ - ++

K115R +++ +++ +++ +++ nd nd

K115Q +++ + + ++ nd nd

K122A + + + +++ - ++

K122R +++ +++ + +++ nd nd

K122Q + + + +++ - ++++

Data is from: Hyland et al 2005 & English et al 2006

Biological Relevance of H3-K115Ac and H3-K122Ac

Hypothesis

Lysine acetylation in the nucleosome dyad disrupts DNA-histone interaction.

This facilitates nucleosome disassembly, repositioning and/or DNA unwrapping.

Test Hypothesis By:Preparing Nucleosomes with Acetylated H3-K115 and H3-K122

Using Biochemical and Biophysical tools to determine how nucleosomes are altered

InteinH3(1-109)

S

O

SO2-Na+H3(1-109)

HN

O

HN

O

SH

HN

O

HN

O

SH

Preparing Acetylated H3 by Expressed Protein Ligation

H3 - Intein

Intein

H3 thioesterH3

H3 peptide

Cleavage Ligation

WT

K115-

AcK11

5-Ac

K122-

AcK12

2-Ac

Dual M

odDua

l Mut

Purified Histone Octamer

60008000

10000

1200014000

160006000

800010000

1200014000

16000

m/Z m/Z

1394913948

13491

11237

15273

11239

13488

15362

Unmodified K115-Ac and K122-Ac

H3H2A/H2B

H4

H3H3 thioester

Purified H3

Regular Nucleosome Reconstitute with Acetylated H3-K115 and H3-K122

Fraction Number

Flu

ores

cenc

e In

tens

ityUnmod

ified

H3-K11

5Ac

H3-K12

2Ac

Dual M

od.

DNA

Nuc

H3-K11

5Q

H3-K12

2Q.

Dual M

ut.

DNA

UnmodifiedNucleosomesDual Mod.Nucleosomes

DNAPeak

NucPeak

Cy3

Nucleosome Positioning Sequence (147 bp)DNA arm (50bp) DNA arm (50bp)

Nucleosome positioning Sequence (147 bp)DNA arm(30bp)

DNA arm(10bp)

mp2-192

mp2-247 Cy5

Cy5Cy5

Nucleosome Competitive Reconstitutions

•Develop by Jonathan Widom and Coworkers.

•Two DNA sequences compete for a limited amount of histone octamer during graduate salt dialysis.

•At an intermediate salt concentration, an equilibrium is setup between octamer bound DNA and free DNA.

•This equilibrium is frozen in as the salt is fully dialyzed away.

•The ratio of the nucleosomes to free DNA is proportional to the equilibrium constant, Keq = [nucleosomes]/[DNA].

•This is compared to a control reconstitution with unmodified histone octamer.

•From this a relative free energy of binding is determined:

ΔΔG = -RT[ln(Keq modified) – ln(Keq unmodified)]

0.10.20.30.40.50.60.70.80.9

1

Kequ

DNA

Nuc

WT Dual Mod

Acetylation of K115 and/or K122 Reduces DNA-Histone Binding

K115Ac

Δ Δ G = 0.35 ± 0.23 kcal/mol

K122AcΔ Δ G =0.18 ± 0.26

kcal/mol

K115Ac & K122AcΔ Δ G = 0.45 ± 0.25

kcal/mol

ΔΔG Depends on DNA Sequence

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

5S WT 5S K115Ac &K122Ac

mp2 WT mp2 K115Ac& K122Ac

mp2 DNA

Δ Δ G = 0.45 ± 0.25 kcal/mol

5S DNA

Δ Δ G = 0.15 ± 0.05 kcal/mol

Mp2DNA

Nuc

WT Dual Mod

Kequ5S

DNA

Nuc

WT Dual Mod

Measuring Site Exposure with Restriction Enzymes

Hae

III

Hin

d III

Taq

α I

Hha

I

Pm

l I

mp2 DNA (192 bp)

Hind III

Hae III

Pml I

Taqα I

Hha I

nakedDNA

nakedDNAobs

nucleosome

nucleosomeobs

confequ

Ek

Ek

k

kK

0

0

21

12somemononucleo

equ

arraynucleosomeequrelative

equ K

KK

_

Time (min)

DN

A F

ract

ion

Unc

ut

Position from dyad (bp)

0 1 2 4 8 16 24 32

Ke

qu

K1

15

Ac

& K

12

2A

c

Ke

qu

un

mo

difi

ed

Hae

III

Hin

d III

Taq

α I

Hha

I

Pm

l I

Uncut

Cut

Uncut

Cut

mp2 DNA (147 bp)

0 1 2 4 8 16 24 32

K115Ac & K122AcUnmodified

DNA site exposure is not altered by the acetylation of K115 and K122

DNA digestion with Taqα I

Nucleosomes thermally reposition more rapidly with K115 and K122 acetylated.

DNA

0 0.5 1 2.5 5 10 15 20

K115Ac & K122AcUnmodified0 0.5 1 2.5 5 10 15 20

Time(min)

Time (min)

Ban

d F

ract

ion

DNA

Manipulating Nucleosomes with Magnetic Tweezers

CCD

Laser

Flow cell

Objective

Moveable permanent magnetApplies the force with field gradient

Dichroicmirrors

CCD or APDs

Dichroics and Band

Pass Filters

Lens

Lens

NS

Nucleosomes

Force

Biotin-Streptavidin

Dig.-Antidig.

MagneticBead

Glass Surface

Lamp

Magnetic Tweezers

L

L

2x

2x

2x

TLkF B

Measurements are done at fixed force.

Determine force from thermal fluctuations

Force vs. extension for a single DNA molecule

Extension (um)

For

ce (

pN)

Nucleosome arraysDNA template

BiotinStreptavidin

BamHIAvaI AvaI

17 nucleosome positioning sequences (3009 bp)

DNA arm (1500 bp) DNA arm (1500 bp)

0 .5 .6 .8 1 1.2 1.5 0 .5 .6 .8 1 1.2 1.5 0 .5 .6 .8 1 1.2 1.5[NPS][HO]

Native Composite GelNative Acrylimide Gel

Ava I digestionDNA Acrylimide Gel

BamHI digestion

Time (sec)

Be

ad H

eig

ht (

mic

rons

)Unwrapping Nucleosomes by Force

Extension Number

Fra

ctio

n of

Nuc

leos

omes

Rem

aini

ng

K115 and K122 Acetylation increase fraction of histone octamer force

induced disassociation

Apply 20 pN of force

Wait 8 minutes and count the number of

unfolding events

Relax to zero force

Wait 3 minutes

Rep

eat

•DNA-histone binding is reduced by up to ~0.5 kcal/mol.

•Reduction in binding affinity depends on DNA sequence.

•K to Q mimic does capture all of the effects of acetylation.

•Steric bulk is more important than change in charge.

•Do not alter site accessibility.

•Facilitate nucleosome repositioning.

•Facilitate nucleosome disassociation following DNA unwrapping.

What have we learned about K115Ac and K122Ac?

How does MSH2-MSH6 function around nucleosomes?

90º

?

Nuc + MM DNA

hMSH2-hMSH6 (nM)

Streptavidin

0 0 50 100 200

- + + + +

Nuc + MM DNA+ Strept

hMSH2-hMSH6+ Nuc + MM DNA

+ Streptavidin

MM DNA

1 2 1 2

Nucs

DNA

Nucleosomes and DNA Mismatch Construct

5S nucleosome positioning sequence

mismatchbiotin

167 bp 71 bp

Sucrose Gradient Purification

MM DNA

MM DNA + SA

MM Nuc DNA

MM Nuc DNA + SA+ hMSH2/6

MM DNA + SA+ hMSH2/6

MM Nuc DNA + SA

0 10 20 30 40 50 60

5S mismatch biotin

nucleosome

StreptavidinhMSH2/6M

M N

uc D

NA

MM

Nuc

DN

A +

SAM

M N

uc D

NA

+ hM

SH2/

6Time (min)

MSH2-MSH6 drives off nucleosomes

Acetylation of K115 and K122 Facilitates Nucleosome Removal by

MSH2-MSH6

WT MSH2/MSH6unmodified nucleosomes

WT MSH2/MSH6H3-K115Ac-K122Ac

MSH2-K675A/MSH6-K1140AH3-K115Ac-K122Ac

Summary of MSH2-MSH6 and Nucleosomes

•MSH2-MSH6 can drive off the histone octamer from a DNA positioning sequence.

•The nucleosome disassociation is ATP dependent

•Acetylation of K115 and K122 facilitates nucleosome removal

Acknowledgements

Funding

Ottesen Lab:

Jennifer Ottesen

Mridula Manohar

Annick Edon

Fishel Lab:

Richard Fishel

Sarah Javaid

Poirier Lab:

Alex Mooney

Justin North

Marek Simon

Robin Nakkula

Mark Parthun and Jonathan Widom