Chromatin Structure and Function

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Chromatin Structure and Function
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The formation of chromatin is the first step in
a series of folding events that reduce the length
of DNA 10,000 fold from its extended form into a
compact mitotic chromosome. An average human
mitotic chromosome 5 microns in length contains 5
cm of DNA.
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The Organization of Chromatin
Hansen, J.C., Annu Rev Biophys Biomol Struct. 2002
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Core Histones
There are four other histones called H2A, H2B,
H3, and H4. These histones are among the most
highly conserved protein sequences known. The H4
sequence form cows and pea plants differs by
only 3 out of 104 residues (conservative
substitutions). This speaks to the important
nature of these proteins.
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Histone-histone associations
The four core histones tend to associate with
each other in very specific patterns of
interactions. Histones H3 and H4 interact very
strongly with each other and form a specific
complex, a tetramer. Histones H2A and H2B
also interact with each other and can form
primarily dimers and some higher oligomers. The
pattern of association is
H3
H4
H2A
H2B
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The crystal structure of the nucleosome core
particle consisting of histone proteins H2A , H2B
, H3 and H4. (Luger Richmond)
Nature. 1997 Sep 18 389 (6648) 251-60.
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Nucleosome Crystal Structure
Blue H3, Green H4, Orange H2A, Red H2B
Luger, K. et. al., Nature 1997.
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The nucleosome core particle shown in the figure
consists of about 146 bp of dsDNA wrapped in 1.65
left-handed superhelical turns around four
identical pairs of proteins individually known as
histones and collectively known as the histone
octamer. The remaining 50 bp of the repeating
unit consists of "linker DNA", dsDNA which
separates the core particles.
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X-ray Structure of the core particle H3/H4 and
H2A/H2B fold
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Epigenetic Control of Cell Function
Histone Tails are chemically modified by many
types of enzymes
DNA methylation
Histone Proteins (core) H2A,H2B, H3 H4
Nucleosome
DNA
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One genome but many cell types
epigenetics
Neuronal cell
genetics/epigenetics
epigenetics
Breast cancer cell
Chromosome
gt20 Disrupted DNA-methylation and
histone-modification genes in cancer Nature
Reviews Genetics 8, 286-298 (2007)
genetic information
Liver cell
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Histone Modifications Alter Chromatin Structure
and Gene Activation
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Prevalent Epigenetic PTMs of Histones
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Histone Code Hypothesis

• As proposed by Allis and Strahl that multiple
  histone modifications, acting in a combinatorial
  or sequential fashion on one or multiple histone
  tails, specify unique downstream functions

Strahl, B.D. and Allis, C.D., Nature. 2000
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Acetylation of Lysine 16 of histone H4 completely
abolishes the ability of the tail domains to
mediate nucleosome-nucleosome interactions, which
are required for chromatin condensation. Science
311, 844-847 (2006)
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Correlations
Phosphorylation of serine residues 10 and 28 on
histone H3 is a marker for chromosomal
condensation. The combination of phosphorylation
of serine residue 10 and acetylation of a lysine
residue 14 on histone H3 correlate with active
transcription.
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Methylated lysine 9 and lysine 27 on histone H3
are associated with repressed genes. Methylated
lysine 4, 36 and 79 of H3 are associated with
active chromatin
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Combinatorial histone modifications
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Code Readers
Protein code readers bind to specific sets of
modifications leading to downstream effects such
as gene transcription, DNA synthesis and repair.
Cellular Decisions
Modified Histones
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Modular domains found in various histone
acetyltransferases
GCN5
Green bromodomains Orange chromodomains Red
GNAT HAT domain Pink MYST homology region
HAT Light Blue Green Zn fingers
MYST
Fr. Carrozza et al. Trends Genet 2003
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GCN5 superfamily of HATs
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Trievel et al. 1999
Dutnall et al., 1998
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Ligand Bound form of a GCN5-type HAT
Rojas et al., 1999
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Yeast Esa1, a MYST family HAT

• Essential SAS2-related acetyltransferase 1
• Linked to rDNA transcription, DNA damage repair,
  cell cycle control
• Reported to be an acetyltransferase of histones
  H4, H2A, Htz1

Bird, et al. 2002 Clarke, et al. 1999 Smith, et
al., 1998 Boudreault, et al., 2003
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Esa1 is part of a large protein complex called
NuA4
(Nucleosomal Acetyltransferase of H4)
Doyon, et al., 2004
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Structure of Esa1 with acetyl-cysteine Catalytic
Intermediate?
CoA
Cys 304
Glu 338
Yan, et al., 2002
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GCN5 Family
Ternary complex (sequential) mechanism
Ac-Histone
Histone
CoA
AcCoA
E
EAcCoA
EAcCoAHistone
ECoA
E
ECoAAc-Histone
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Is Cys-304 involved in catalysis with the highly
active trimeric enzyme (piccolo NuA4)?
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Cys-304 does not mediate acetyl-transfer
Berndsen, et al., Biochemistry 2007
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Proposed mechanism of protein acetylation by Esa1
Berndsen, Albaugh et al., Biochemistry 2007
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HDAC (Histone Deacetylase) Families

• Class I and II
• Zinc-hydrolase
• 11 human homologs
• Inhibited by hydroxamic acid containing
  compounds (TSA, SAHA)
• Class III (Sir2 or sirtuins)
• No structural/mechanistic similarity to Class I
  and II
• NAD requiring
• 7 human homologs
• Inhibited by nicotinamide

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HDACs and cancer HDAC inhibitors show potent
anti-tumor activity. Inhibition of HDAC activity
induces growth arrest, differentiation, and
apoptosis of transformed or several cancer
cells. Thought to de-repress expression of tumor
suppressor genes via direct increases in histone
acetylation, but not really clear if this is
direct histone acetylation or non-histone
acetylation.
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Sir2 proteins are found in all forms of life and
are implicated in many biological processes
Gene silencing lifespan extension insulin
secretion protection from cell stress glucose
homeostasis fatty acid metabolism
7 human homologs sirtuins

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Other reported activity Sir2 catalyzed
mono-ADP-ribosylation
Do Sir2 enzymes catalyze protein
mono-ADPribosylation?
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J.A. Garcia-Salcedo et al. EMBO. Vol 22 (21).
5851-5862, 2003.
ADP-ribosyltransferase activity of TbSIR2
Incorporation of label 32P-NAD over time
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Conclusion TbSir2 is a proper protein deacetylase
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Can we detect ADP-ribosylation?
Reaction Conditions3 mM TbSIR225 mM 32P-NAD
(1 mCi)20 mg Histones10 mM DTT150 mM NaCl50
mM Tris-HCl (pH 8.0) t 4hr, t 37C
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• Conclusions
• Incubation of 32P-NAD with TbSir2 or histones,
  separately, results in 32P-incorporation.
• Incubation with 32P-NAD and calf thym histones
  in the presence of TbSir2 yields markedly more
  32P-incorporation, whereas recombinant histones
  show no additional 32P with TbSir2.
• Whats different?

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Is it acetylation?
Reaction Conditions3 mM TbSIR225 mM 32P-NAD
(1 mCi) 20 mg Histones10 mM DTT150 mM NaCl50
mM Tris-HCl (pH 8.0) t 4hr, t 37C
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Does human SIRT1 display ADPribosylation
activity?
Reaction Conditions3 mM SIRT125 mM 32P-NAD
(1 mCi) 20 mg Histones10 mM DTT150 mM NaCl50
mM Tris-HCl (pH 8.0) t 4hr, t 37C
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• Conclusions
• The OAADPr-dependent labeling of histones does
  not require enzyme.
• OAADPr non-enzymatically reacts more readily than
  does NAD
• The above observation may account for some of the
  reported sirtuin-dependent ADPribosylation
  activity.
• In addition to the non-enzymatic 32P
  incorporation from OAADPr, there is also
  enzyme-dependent activity that requires NAD and
  acetylated histones.

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Reaction mechanism Direct evidencefor the
proposed intermediate
Jackson and Denu, J. Biol. Chem. 2002 Sauve et
al., Biochemistry 2002. Tanner et al. PNAS 2000
Tanny and Moazed, PNAS 2001 Smith and Denu
Biochemistry 2006
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Summary of 18O-labeling experiments
Brian Smith
Smith Denu, Biochemistry (2006) 45(1)272-82.
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Analogs as Inhibitors of Sirtuins
Hst2
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Steady-state kinetic inhibition analysis
reveals potency of thioacetyl peptide
ThioAcetyl Ki 17 nM Kd 4.7 µM
Trifluoroacetyl Ki 4.8 µM Kd 3.3 µM
- Ki for thioacetyl-lysine peptide is 300 and
1200-fold lower than the Km and the Kd
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Acetyl 6.7 s-1 Thioacetyl 4.5 s-1
Stalled intermediate
Acetyl kcat 0.2 s-1 Thioacetyl kcat 0.0024 s-1
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Class Selective Substrates
6-orders of magnitude difference
Analogs vary in the electron-withdrawing
potential of X
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Mechanistic Differences among Deacetylase Classes
Class III e.g. Hst2
Class I/II e.g. HDAC8
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General chemical mechanism of AdoMet-dependent
histone lysine methyltransferases. Both SET
domain and Dot1 methyltransferases use a similar
mechanism of methyl transfer.
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Proposed chemical mechanism of LSD1/KDM1, lysine
demethylases
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Lysine demethylase Reaction catalyzed by JHDM
enzymes, 2-oxoglutarate-dependent Fe(II)
dioxygenases
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Proposed mechanism for JHDM enzymes
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Proposed chemical mechanism of PAD enzymes,
protein arginine deiminases