Chromatin and Transcription

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Chromatin and Transcription Topic 4
Why dont transcription factors bind to all their
sites? How do transcription activation domains
work? What is the influence of chromatin on
transcription?
Nucleosomes and chromatin structure Transcription
initiation Transcription elongation
Histone modifications Histone remodeling
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How to achieve a high packing ratio
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From Luger et al. 1997 Nature 389251
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Chromatin Regulatory Complexes
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Taken from Fry and Peterson. 2001. Curr. Biol.
11R185-R188
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Remodeling Complexes
1980s Ira Herskowitz mating type
switching and HO expression in S.
cerevisiae Mutations that lower expression and
induction of HO Switch Genes SWI1, SWI2,
SWI3 Marian Carlson Sucrose Non-Fermentation
pathway Mutations that lower expression and
induction of Suc2 invertase gene Sucrose
Non-Fermenting SNF2, SNF5, SNF6 Mutations of
these genes reduced expression and induction of
the target genes and SWI2 SNF2 SWI/SNF
genes were found to be part of a multi-subunit
complex now referred to as a SWI/SNF co-activator
complex
Some activation domains, such as VP16, bind to
SWI/SNF complexes
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Screen for suppressors of SWI1 deletion
Get SIN mutations, SIN1 and SIN2 What are
these two proteins? SIN1 is a HMG1/2-like
protein in yeast thought to interact with the H1
linker histone SIN2 is Histone H3 (later other
SIN mutations were found in H4)
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Suppressor mutations destabilize DNAnucleosome
interactions
Alan Wolffe. Curr Biol. 1994 Jun 14(6)525-8.
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• What do the ATPase-remodeling complexes do?
• SWI/SNF complexes use ATP to drive changes in
  helical twist of DNA - thought to be the
  biomechanical force that enables transcription
  factor accessibility. They
• Alter the rotational path of DNA on the surface
  of the nucleosome
• Lose 40bp of associated DNA from the nucleosome
• Lose supercoils
• Displace histones from DNA
• Displace nucleosomes?

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Chromatin Remodeling Complexes
Non-covalent
nucleosome positioning nucleosome
conformation nucleosome composition (variant
histones)
SWI/SNF complex -Brg subunit (ATPase) -hBrm
subunit (ATPase)
Brg and Brm are bromodomain proteins
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SWR1
SWR1 a SWI/SNF-related complex
From Kingston and Narlikar. 1999. Genes
Dev.132339-2352
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From Kingston and Narlikar. 1999. Genes
Dev.132339-2352
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Fig. 4. Long Nucleosome Free Regions are common
in promoters
G.-C. Yuan et al., Science 309, 626 -630
(2005)
Published by AAAS
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Fig. 5. Nucleosome Free Regions are pervasive in
promoters
G.-C. Yuan et al., Science 309, 626 -630
(2005)
Published by AAAS
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Chromatin Modification Complexes
variant histones H2A.X H2A.Z H3.1 H3.3
SWI/SNF (SWR1)
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Chromatin Modification Complexes
SWI/SNF
Acetylation Phosphorylation Methylation Ubiquitina
tion
ACF chaperones ATP variant histones
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Two theories for the functional results
of Chromatin Modification
Histone Code David Allis vs. Structural Changes
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Bromodomains - binds acetylated
lysines Chromodomains - binds methylated lysines
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Li, Carey, Workman. 2007. Cell 128707-719
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BM Turner. 2007. Nat. Cell Biol. 92-6.
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Bivalent domains in stem cells developmental
transcription factors that carry a repressive
(PcG) mark and an activating mark
Known H3K27me3 (PRC2), then H2A
monoUbiquitination (PRC1). H3K4me3
(trxG) Bivalent genes are expressed/silent Bivalen
cy predisposes to activation OR inactivation
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Bivalent Marks in ES cells identify Developmental
Regulators
Pietersen and van Lohuizen. 2007.
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Chromatin Modification
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GCN5 found to work with other proteins in a
complex Genetic screens Biochemical
isolation of GCN5 complex led to Spt Ada
GCN5 Acetyl transferase SAGA Other components
are the TAFs!
Known transcription activation domains such as
VP16 bind to components of SAGA.
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Histone H4-K16Ac is incorporated into nucleosomal
arrays
M. Shogren-Knaak et al., Science 311, 844
-847 (2006)
Published by AAAS
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Dense, compacted nucleosomes rely on Histone H4
tail interactions with neighboring nucleosomes
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Loose open chromatin is enabled by H4K16
acetylation
H4K16 acetylation inhibits formation of 30nm
chromatin fibers H4K16 acetylation prevents
SWI/SNF-mediated nucleosome sliding
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Chromatin Modification Complexes
Covalent Modifications
Acetylation Methylation
Phosphorylation Ubiquitination
HDAC Complexes
NuRD - HDACs 1 2 - Mi-2 ATPase
Sin3 - HDAC1 RPD3
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Chromatin Modification Complexes
Covalent Modifications
Acetylation Methylation
Phosphorylation Ubiquitination
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Phospho-histone H3 (serine10) is a marker of
mitotic cells
Crosio et al. 2002. Mol. Cell. Biol.22874-885
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Chromatin Modification Complexes
Covalent Modifications
Acetylation Methylation
Phosphorylation Ubiquitination
HMTs Methylating Complexes
SET domain proteins (Su(var)3-9, Enhancer of
zeste and Trithorax, Set2) functions in
activation and repression for repression
tightly linked to DNA CpG methylation and HP1
(heterochromatin protein 1) binding
heterochromatin has methylation on H4K9, H4K27
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Wysocka et al. 2004. Cell 122654-658
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Histone Methylation and Gene Repression
methylated K9, K20, K27, K36 are recognized by
repressor complexes repressor complexes
recruit HDACs, chromatin remodeling machines
DNA methyltransferases can also be recruited
HP1
Eaf3 contains a chromodomain which
recognizes methylated lysines RPD3 is a
histone deacetylase DNMT is a DNA methyl
transferase for CpG dinucleotides
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Heterochromatin
Constitutive always condensed
Facultative regulated
Composition DNA methylation Histone
methylation Repressor Complexes siRNA
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Chromatin Modification Complexes
ACF chaperones ATP variant
histones H2A.Z
TAFII250 GCN5 RPD3 CBP/p300 NURD
SET domain proteins
SWI/SNF
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Gene in Heterochromatin
Gene in euchromatin
DNAse I sensitivity Assay
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DNAase1 hypersensitivity assay
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Micrococcal Nuclease Digestion
Regular Nucleosomal Array
Irregular Nucleosomal Array
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From Kadonaga.1998. Cell 92307-313
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Primary Reading Feldman et al.
Chromatin Modification Complexes
Methylation
G9a
RPD3 (HDAC) complex
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The Epigenetics Behind The Shut-Down of Oct4
Expression
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NY Times, Nov. 2007
Oct4 Sox2 KLf4 C-MYC (Nanog)
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Oct4, Sox2, Klf4, C-MYC, (Nanog)
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Activation marks
Repression marks
HhaI (Hh) GCGC HpaI (Hp) CCGG
Figure 1
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Kidney
Embryo
Rex-1 in P19 cells
Typical non-expressed genes in ES cells
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DNMT-/- No DNA methylation
Oct4 still repressed (data stated in text)
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G9a-/- No K9 methylation
G9a starts off the whole cascade
G9a is required for DNA methylation and for
repression of Oct4
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ES cells differentiated for 2wks (RA treatment),
then cultured in LIF
G9a-
DNMT-
Wt ES cells
G9a transgene
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