Transcript No Slide Title

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 b-globin gene cluster
0
20
40

G A 
60
80 k b
DNase HSs
Domain opening?


b
Yes
LCR
Embryonic Fetal >
Embryonic
Adult
Locus Control
Region is needed to:
Locus control
region:
• openglobin
a chromatin
in erythroid
cells
Activate linked
genedomain
expression
in erythroid
cells.
• express of linked globin genes at a high level
Overcome• position
effectseffects
at many
integration
override position
in transgenic
micesites
in transgenic mice.
Role in switching expression?
Domain opening and gene activation are
separable events
wildtype
N-MEL
ORGs
Location,
DNase heterosensi- chromtive
atin
Human
HBB
complex
LCR
HSs     b
Del. HS2-HS5
General
histone H3
hyper- hyper
Ac’n
Ac’n Txn
+
away
+
+
+
+
away
+
-
-
-
close
-
-
-
T-MEL, Hisp. del.
x
x
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 appear
to be localized to a sub-region of the
nucleus.
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
Domain
opening is
associated
with
movement
to nonheterochromatic
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 nonfermenting).
– 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 pattern
III: transfer a histone octamer to a different DNA fragment
Covalent modification of histones in
chromatin
Histones are acetylated and deacetylated
+NH
Histone acetyl
3
CH2
CH2
AcCoA
O
CH2 O
CH2
... NH CH C NH CH C ...
2
Gly
Lys
Ac
Pos itive char ge on amino gr oup
CH3
transferases
O C
NH
CH2
CH2
CoA
O
CH2 O
CH2
... NH CH C NH CH C ...
2
Histone deacetylases
No char ge on amide gr oup
Covalent modification of histone tails
N-ARTKQTARKSTGGKAPRKQLATKAARKSAP...- H3
4
9 10
14
23
18
27 28
N-SGRGKGGKGLGKGGAKRHRKVLRDNIQGIT...- H4
1
5
8
phosphorylation
12
16
20
acetylation
methylation
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
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.
Yeast SAGA interacting with chromatin
SAGA Complex
TAF90p
Tra1p
TAF25/23p
Ada3p
Spt7p
Ada1p
TAF68/61p
TAF60p
Ada2p
Act.
Spt20/
Ada5
p
Gcn5p
HAT
TAF20/17p
Spt8p
Spt3p
TBP
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
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
A mammalian histone deacetylase:
HD1
RbAp48
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 97:299311.
• The order is likely to differ at different genes