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
60
80 kb
DNase HSs

Domain opening?
G A 


b
LCR
Embryonic Fetal >
Adult
Embryonic
Locus region:
Control Regionis needed to:
Locus control
• openglobin
a chromatin
domain in erythroid
cells cells.
Activate linked
gene expression
in erythroid
• express of linked globin genes at a high level
Overcome• position
manyinintegration
sites
override effects
positionat
effects
transgenic mice
in transgenic mice.
Role in switching expression?
Yes
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
3
CH2
CH2
O
CH2 O
CH2
... NH CH C NH CH C ...
2
Gly
Lys
Histone acetyl
AcCoA
CH3
transferases
O C
NH
CH2
CH2
CoA
O
CH2 O
CH2
... NH CH C NH CH C ...
2
Ac
Positive charge on amino group
No charge on amide group
Histone deacetylases
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