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

Histone acetyl transferases

Histone deacetylases

Covalent modification of histone tails

N-ARTKQTARKSTGGKAPRKQLATKAARKSAP...- H34 9 10 14 18 23 27 28

N-SGRGKGGKGLGKGGAKRHRKVLRDNIQGIT...- H45 8 12 16 201

acetylationphosphorylation 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.

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