Regulation by changes in histones, nucleosomes and chromatin

1
Regulation by changes in histones, nucleosomes
and chromatin

• Opening and activation
• Movement from heterochromatin to euchromatin
• Nucleosomes and transcription factors
• Chromatin remodeling activities
• Histone acetyl transferases and deacetylases
• Thanks Dr. Jerry Workman

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Human b-globin gene cluster
Domain opening?
Locus control region Activate linked globin gene
expression in erythroid cells. Overcome position
effects at many integration sites in transgenic
mice. Role in switching expression?
3
Domain opening and gene activation are separable
events
Loca- tion, hetero- chrom- atin
General histone hyper- Acn
Human HBB complex
wildtype N-MEL
DNase sensi- tive
H3 hyper Acn
LCR HSs
Txn
d
g
b
e
g
ORGs




away
Del. HS2-HS5
-
-


away
T-MEL, Hisp. del.
-
-
-
-
close
Reik et al. (1988) Mol. Cell. Biol.
185992-6000. Schübeler et al. (2000) Genes
Devel. 14940- 950
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Chromosome localization in interphase
In interphase, chromosomes appear to be localized
to a sub-region of the nucleus.
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Gene activation and location in the nucleus

• Condensed chromatin tends to localize close to
  the centromeres
• Pericentromeric heterochromatin
• Movement of genes during activation and silencing
• High resolution in situ hybridization
• Active genes found away from pericentromeric
  heterochromatin
• Silenced genes found associated with
  pericentromeric heterochromatin

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Domainopening is associated with movement to
non-hetero-chromatic regions
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Proposed sequence for activation

• 1. Open a chromatin domain
• Relocate away from pericentromeric
  heterochromatin
• Establish a locus-wide open chromatin
  configuration
• General histone hyperacetylation
• DNase I sensitivity
• 2. Activate transcription
• Local hyperacetylation of histone H3
• Promoter activation to initiate and elongate
  transcription

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A scenario for transitions from silenced to open
to actively transcribed chromatin
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From silenced to open chromatin
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Movement from hetero- to euchromatin
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Nucleosome remodelers and HATs further open
chromatin
12
Assembly of preinitiation complex on open
chromatin
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Transcription factor binding to DNA is inhibited
within nucleosomes

• Affinity of transcription factor for its binding
  site on DNA is decreased when the DNA is
  reconstituted into nucleosomes
• Extent of inhibition is dependent on
• Location of the binding site within the
  nucleosome.
• binding sites at the edge are more accessible
  than the center
• The type of DNA binding domain.
• Zn fingers bind more easily than bHLH domains.

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Stimulate binding of transcription factors to
nucleosomes

• Cooperative binding of multiple factors.
• The presence of histone chaperone proteins which
  can compete H2A/H2B dimers from the octamer.
• Acetylation of the N-terminal tails of the core
  histones
• Nucleosome disruption by ATP-dependent remodeling
  complexes.

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Binding of transcription factors can destabilize
nucleosomes

• Destabilize histone/DNA interactions.
• Bound transcription factors can thus participate
  in nucleosome displacement and/or rearrangement.
• Provides sequence specificity to the formation of
  DNAse hypersensitive sites.
• DNAse hypersensitive sites may be
• nucleosome free regions or
• factor bound, remodeled nucleosomes which have an
  increased accessibility to nucleases.

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Nucleosome remodeling
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Chromatin remodeling ATPases are large complexes
of multiple proteins

• Yeast SWI/SNF
• 10 proteins
• Needed for expression of genes involved in
  mating-type switching and sucrose metabolism
  (sucrose non-fermenting).
• Some suppressors of swi or snf mutants are
  mutations in genes encoding histones.
• SWI/SNF complex interacts with chromatin to
  activate a subset of yeast genes.
• Is an ATPase
• Mammalian homologs hSWI/SNF
• ATPase is BRG1, related to Drosophila Brahma
• Other remodeling ATPase have been discovered.

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Chromatin remodeling ATPases catalyze stable
alteration of the nucleosome
II form a stably remodeled dimer, altered DNAse
digestion pattern III transfer a histone octamer
to a different DNA fragment
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Covalent modification of histones in chromatin
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Histones are acetylated and deacetylated
Histone acetyl transferases
Histone deacetylases
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Covalent modification of histone tails
N-ARTKQTARKSTGGKAPRKQLATKAARKSAP...- H3
4
9 10
14
23
27 28
18
N-SGRGKGGKGLGKGGAKRHRKVLRDNIQGIT...- H4
5
8
12
16
20
1
acetylation
phosphorylation
methylation
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Two types of Histone Acetyltransferases (HATs).

• Type A nuclear HATs acetylate histones in
  chromatin.
• Type B cytoplasmic HATs acetylate free histones
  prior to their assembly into chromatin.
• Acetylate K5 and K12 in histone H4

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Acetylation by nuclear HATs is associated with
transcriptional activation

• Highly acetylated histones are associated with
  actively transcribed chromatin
• Increasing histone acetylation can turn on some
  genes.
• Immunoprecipitation of DNA cross-linked to
  chromatin with antibodies against Ac-histones
  enriches for actively transcribed genes.
• Acetylation of histone N-terminal tails affects
  the ability of nucleosomes to associate in
  higher-order structures
• The acetylated chromatin is more open
• DNase sensitive
• accessible to transcription factors and
  polymerases
• HATs are implicated as co-activators of genes in
  chromatin, and HDACs (histone deacetylases) are
  implicated as co-repressors

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Nuclear HAT As are coactivators

• Gcn5p is a transcriptional activator of many
  genes in yeast. It is also a HAT.
• PCAF (P300/CBP associated factor) is a HAT and is
  homologous to yeast Gcn5p.
• P300 and CBP are similar proteins that interact
  with many transcription factors (e.g. CREB, AP1
  and MyoD).
• P300/CBP are needed for activation by these
  factors, and thus are considered coactivators.
• P300/CBP has intrinsic HAT activity as well as
  binding to the HAT PCAF.

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HAT complexes often contain several trancription
regulatory proteins.

• Example of the SAGA complex components
• Gcn5 catalytic subunit, histone acetyl
  transferase
• Ada proteins
• transcription adaptor proteins required for
  function of some activators in yeast.
• Spt proteins (TBP-group)
• regulate function of the TATA-binding protein.
• TAF proteins
• associate with TBP and also regulate its
  function.
• Tra1
• homologue of a human protein involved in cellular
  transformation.
• May be direct target of activator proteins.

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Yeast SAGA interacting with chromatin
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Roles of histone acetylation

• Increase access of transcription factors to DNA
  in nucleosomes.
• Decondense 30nm chromatin fibers
• Serve as markers for binding of non-histone
  proteins (e.g. bromodomain proteins).

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Histone deacetylases are associated with
transcriptional repression
A mammalian histone deacetylase
Histone deacetylases Are recruited by
inhibitors of transcription. Are inhibited by
trichostatin and butyrate.
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Repression by deacetylation of histones
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Methylated DNA can recruit HDACs
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Connections in eukaryotic transcriptional
activation

• Transcriptional activators
• Coactivators
• Nucleosome remodeling
• Histone modification
• Interphase nuclear localization

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The functions of SWI/SNF and the SAGA complex are
genetically linked.

• Some genes require both complexes for activation.
• Other genes require one or the other complex.
• Many genes require neither - presumably utilize
  different ATP-dependent complexes and/or HATs

33
The yeast HO endonuclease gene requires both
SWI/SNF and SAGA

• The order of recruitment at the promoter
• 1. SWI5 activator sequence recognition
• 2. SWI/SNF complex remodel nucleosomes
• 3. SAGA acetylate histones
• 4. SBF activator (still at specific sequences)
• 5. general transcription factors
• Cosma, Tanaka and Nasmyth (1999) Cell 97299-311.
• The order is likely to differ at different genes