how histone post-translational modifications function when they are buried under dna michael guy...
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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