HISTONE MODIFICATION

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HISTONE MODIFICATION REGULATION OF
TRANSCRIPTION BCH 6415 Spring 2006 (Part I)
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Schematic of ChIP
PCR of genomic fragments
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ADP-ribosylation Sumoylation Biotinylation Citrull
ine..
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Covalent Modifications of Histone Tails
Turner, BM Cell 2002
Lusser Curr Opin Plant Biol 54372002
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Fr. Peterson Laniel Curr Biol. 2005
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Histone Acetylation/Deacetylation
Kuo Allis Bioessays 1998
K lysine
Turner, BM 1999
Known acetylation sites in core histone N-term.
tails
Wade, PA Hum Mol Genet. 2001
Roth, et al. Ann Rev. Biochem. 7081 2001
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Two types of HATs Type A localized in
nucleus acetylates
nucleosomal histones Type B localized in
cytoplasm probably acetylates
newly synthesized histones prior to chromatin
assembly Five Familes of
HATs GNAT show seq similarity to Gcn5
HAT MYST p300/CBP GTF HATs (e.g.,
TAF250) Nuclear hormone-related (e.g., SCR1,
ACTR) Most HATs multisubunit complexes
HAT Families
Acetylation sites of various HATs
Mamorstein Roth Curr Op Genet. Devel.
2001 (see Yang, XJ NAR 2004 for updated list)
Some HATs can acetylate other proteins (e.g.,
TFIIE, TFIIF)
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Motifs in found in various HATs
GNAT
MYST
Fr. Marmorstein Roth Curr Opin 2001
Green bromodomains Orange chromodomains Red
GNAT HAT domain Pink MYST homology region
HAT Light Blue Green Zn fingers
Fr. Carrozza et al. Trends Genet 2003
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Distribution of histone acetylation across loci
and genes
Histone acetylation pattern across the mouse
ß-globin locus in DMSO-induced MEL cells
(ßmaj and minor genes expressed)
Forsberg et al. PNAS 9714494 2000
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Histone acetylation levels across the human p16
locus
Filled bars p16 expressing cells Open bars
non-expressing cells
Conclusion Highest H3k9/14Ac levels localized to
5 region of genes in higher eukaryotes.
Fr. Liang et al. PNAS 2004
In yeast, H3K9/14 acetylation extends across
coding region
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How does histone acetylation open
nucleosomes/chromatin?
Old Model Acetylation of core histones at
lysine sidechains neutralizes
positive charges and loosens
interaction between DNA and histone
octamer tails
BUT,
1) Tail-less reconstituted nucleosomes leads to
1.5- to 14-fold increase in accessibility
compared to normal nucleosomes Polach et al.
Effects of core histone tail domains on the
equilibrium constants for dynamic DNA site
accessibility in nucleosomes. J. Molec. Biol.
298211 2000 2) Nucleosomes reconstituted w/
hyperacetylated core histones leads to only 1.1-
to 1.8- fold increase in accessibility (ave.
1.4-fold) to restriction enzyme cleavage
Anderson et al. Effects of histone acetylation
on the equilibrium accessibility of nucleosomal
DNA targets. J. Molec. BIol. 307977 2001
Is this fairly small effect of histone
acetylation on increased accessibility to
nucleosomal DNA sufficient to
facilitate binding of regulatory proteins and
activate transcription?? ALSO acetylated
nucleosomes have hydrodynamic properties similar
to unmodified nucleosomes, and hyperacetylation
of histones does not appear to release histone
tails from nucleosomal DNA Therefore, the role
histone acetylation in modulating nucleosome
structure is not clear
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Other Possible Roles for Histone Acetylation in
Gene Activation 1) Decondensation and
opening of higher order chromatin structure to
facilitate access of trans-acting
transcriptional activators. 2) Regulating the
binding of regulatory proteins to chromatin
modulation of protein-protein interactions
between N-term. tails of core histones in
nucleosomes and regulatory proteins (e.g., via
bromodomains)
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HISTONE DEACETYLASES (HDACs) Common components
of co-repressor (e.g., N-CoR) and chromatin
remodeling (e.g., NURD) complexes Generally
associated with transcriptionally silent chromatin
Related to yeast RPD3 transcriptional
regulator homology in catalytic site nuclear
localization
Different HDACs have different specificities,
tissue distributions, developmental
stage specificities
Similarity to yeast HDA1 HDAC homology in
C-term catalytic site and N-term. regulatory
region nuclear cytoplasmic localization
Sir2 family of NAD- dependent HDACs histones
may not be primary substrate
Class I and II HDACs inhibited by trichostatin A
(TSA) (Class III not inhibited by TSA)
Fr. Marks et a. Curr Opin Pharma 2003
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Histone acetyltransferase complexes
stabilize SWI/SNF binding to promoter
nucleosomes AH Hassan, KE Neely, JL Workman Cell
104817-27 2001
1) Biotinylated DNA from linearized plasmid
contained 5 Gal4 binding sites upstream of
adenovirus2 E4 minimal promoter. 2) Nucleosomal
arrays reconstituted w/ purified non-acetylated
(or hyperacet. by butyrate treating cells) HeLa
histones. 3) Added Gal4-VP16, then added
purified SWI/SNF complex (also cold random
nucleosomal arrays as competitor) 4) In vitro
ChIP assay Cross-linked nucleosomal arrays
w/ formaldehyde Nucleosomal arrays digested
w/ micrococcal nuclease to generate mostly mono-
and di-nucleosomes Immunoprecipitated w/ Ab
against HA-tagged Swi/Snf2 subunit of SWI/SNF
Reversed cross-links, purified DNA, and slot
blotted Hybridized blots w/ various
32P-labelled probes (A, B, C, D, -B, -A)
autoradiography
Not IPd
Unacetylated histones
IPd
Conclusions 1) In the absence of Gal4-VP16,
SWI/SNF bound non-specifically along the length
of the array 2) In the presence of competitor
chromatin, GAl4-VP16 recruited SWI/SNF to array
(away from cold competitor chromatin) and
localized SWI/SNF to region of the Gal4 binding
sites (irrespective of promoter position
in template). 3) Also, used restriction enzyme
accessibility assay to show localized nucleo.
disruption by SWI/SNF (data not shown here).

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S supernatent B bound to beads
(Ab against SWI/SNF subunit)
(Ab against Gal4 binding domain)
SWI/SNF gone after removing Gal4
1) Used nucleosomal template (incl. 5 gal4
binding sites and nucleosome positioning seq)
w/ one end biotinylated and attached for
immobilization on streptavidin magnetic beads 2)
Added Gal4-VP16, then SWI/SNF (and competitor
chromatin) 3) Then competed off Gal4-VP16 by
adding oligo w/ Gal4 binding site 4)
Assayed by Western blot to determine if SWI/SNF
was retained on array in the absence of
bound Gal4-VP16. Conclusion In the
presence of competitor chromatin, stable binding
of SWI/SNF to unacetylated nucleosomal array
requires continued interaction and binding of
the activator that recruited it (Gal4-VP16).
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• 1) Assembled nucleosomal array with
  hyperacetylated histones from
• butyrate (inhibits histone deacetylase)
  -treated HeLa cells.
• 2) Added Gal4-VP16, then added SWI/SNF and
  unacetylated
• competitor chromatin
• 3) Added competitor oligo containing Gal4 binding
  site to remove
• bound Gal4-VP16
• 4) Assayed by Western blot to determine if
  SWI/SNF was
• retained on array in the absence of bound
  Gal4-VP16 (see lane 6).
• Conclusion After Gal4-VP16 was removed, SWI/SNF
  was
• retained more efficiently on hyperacetylated
  nucleosomal
• templates compared to control unacetylated
  templates.
• Demonstrates potential functional link between
  histone
• acetylation and SWI/SNF function.
• Acetylating nucleosomal templates in vitro with
  SAGA or
• NuA4 histone acetylase complexes also enhances
  stability

SWI/SNF still bound to Ac nucleosomes after Gal4
removed
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Conclusion Acetylated histones enhance SWI/SNF
retention on promoter-proximal (i.e., near
Gal4-VP16 binding sites) nucleosomes (i.e.,
SWI/SNF doesnt move/slide along array after
is activator removed).
Used hyperacetylated histones purified from
butyrate-treated HeLa cells
Used reconstituted nucleosomes acetylated in
vitro by the SAGA complex
1) Biotinylated DNA from linearized plasmid
contained 5 Gal4 binding sites upstream of
adenovirus2 E4 minimal promoter. 2) Nucleosomal
arrays reconstituted w/ purified hyperacetylated
HeLa histones, or SAGA-acetylated nucleosomes 3)
Added Gal4-VP16, then added purified SWI/SNF
complex (also cold random nucleosomal arrays as
competitor) 4) In vitro ChIP assay
Cross-linked nucleosomal arrays w/ formaldehyde
Nucleosomal arrays digested w/ micrococcal
nuclease to mostly mono- and di-nucleosomes
Immunoprecipitated w/ Ab against HA-tagged
Swi/Snf2 subunit of SWI/SNF Reversed
cross-links, purified DNA, and slot blotted
Hybridized blots w/ various 32P-labelled
probes (A, B, C, D) autoradiography