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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Regulation by changes in histones, nucleosomes and chromatin Opening and activation Movement from heterochromatin to euchromatin Nucleosomes and transcription.
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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
Human -globin gene cluster
ε γ γ ψη δ G A
0 20 40 60 80 kb
LCR
DNase HSs
Yes
Embryonic Fetal >Embryonic
Adult
Locus Control Region is needed to: • open a chromatin domain in erythroid cells • express of linked globin genes at a high level • override position effects in transgenic mice
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?
Domain opening and gene activation are separable events
LCR HSs δγ ε
Human HBB complex
ORGs γ
wildtypeN-MEL
DNasesensi-tive
Generalhistonehyper-Ac’n
H3 hyperAc’n
Loca-tion,hetero-chrom-atin Txn
+
-
Del. HS2-HS5
T-MEL, Hisp. del.
x x - -close -
away
away
--+ +
+ + +
Reik et al. (1988) Mol. Cell. Biol. 18:5992-6000.Schübeler et al. (2000) Genes & Devel. 14:940- 950
Chromosome localization in interphase
In interphase, chromosomes appearto be localized to a sub-region of thenucleus.
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
Domainopening is associated
with movement
to non-hetero-
chromatic regions
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
A scenario for transitions from silenced to open to actively
transcribed chromatin
From silenced to
open chromatin
Movement from hetero- to euchromatin
Nucleosome remodelers and HATs
further open chromatin
Assembly of preinitiation complex on
open chromatin
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.
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.
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.
Nucleosome remodeling
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.
Chromatin remodeling ATPases catalyze stable alteration of the nucleosome
II: form a stably remodeled dimer, altered DNAse digestion patternIII: transfer a histone octamer to a different DNA fragment
Covalent modification of histones in chromatin
Histones are acetylated and deacetylated
AcCoA
C
O
CHNHCH2
C
O
NH... ...CH2
CH2
CH2
CH2
NH 3+
C
O
CHNHCH2
C
O
NH... ...CH2
CH2
CH2
CH2
NH
Gly Lys
CCH3
O
CoA
AcPositive charge on amino group No charge on amide group
• 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
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
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.
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.
Ada2p
Ada3p
Spt8p
Spt20/Ada5p Gcn5p
HAT
Ac
Ac
Ac
Ac
Ac
Ac
Ac
AcTBP
Act.
SAGA Complex
Spt7p
Tra1p
Spt3p
TAF68/61p
TAF60p
TAF20/17p
Ada1p
TAF90p
TAF25/23p
Yeast SAGA interacting with chromatin
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).
Histone deacetylases are associated with transcriptional repression
HD1
RbAp48
A mammalian histone deacetylase:
Histone deacetylases:Are recruited by inhibitors of transcription.Are inhibited by trichostatin and butyrate.
Repression by deacetylation of histones
Methylated DNA can recruit HDACs
Connections in eukaryotic transcriptional activation
• Transcriptional activators
• Coactivators
• Nucleosome remodeling
• Histone modification
• Interphase nuclear localization
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
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 97:299-311.
• The order is likely to differ at different genes