histone modifications

Histone Modifications

Outline

1. Overview of histone modifications:a. Types of modifications and modifiersb. General roles of modificationsc. Techniques used to study histone modifications

2. Specific modifications (acetylation, methylation, etc):a. Residues/positions that are frequently modifiedb. Enzymes that add/remove the modificationc. Biological roles

3. Cross-talk between histone modifications

4. Summary

5. A histone code?

Quick Overview

• Modifications and modifiers• General roles• Techniques

Bhaumik, Smith, and Shilatifard, 2007.

Types of Histone Modifications

• Covalently attached groups (usually to histone tails)

• Reversible and Dynamic– Enzymes that add/remove modification– Signals

• Have diverse biological functions

Cell, 111:285-91, Nov. 1, 2002

Methyl Acetyl Phospho Ubiquitin SUMO

Features of Histone Modifications

• Small vs. Large groups

• One or up to three groups per residue

Features of Histone Modifications

Jason L J M et al. J. Biol. Chem. 2005

Ub = ~8.5 kDaH4 = 14 kDa

• Writers: enzymes that add a mark

• Readers: proteins that bind to and “interpret” the mark

• Erasers: enzymes that remove a mark

Tarakhovsky, A., Nature Immunology, 2010.

Histone Modifications and Modifers

• Others: Sumoylation (Lysine), ADP Ribosylation (Glutamate)

Histone Modifications and Modifers

Residue Modification Modiying Enzyme

Lysine AcetylationDeacetylation

HAT HDAC

Lysine MethylationDemethylation

HMT HDM

Lysine UbiquitylationDeubiquitylation

Ub ligaseUb protease

Serine/Threonine PhosphorylationDephosphorylation

KinasePhosphatase

Arginine MethylationDemethylation

PRMTDeiminase/

Demethylase

Histone Modifiers

• Do not bind to DNA themselves– Can be recruited by:

• Histone modifications (through chromodomains, bromodomains, etc.)

• Transcription factors• RNA (fission yeast, mammals, plants)• DNA damage

• Act as transcriptional co-regulators

• Enhance activities of transcriptional repressors or activators– Co-repressor: ex. HDACs– Co-activator: ex. HATs

General Roles of Histone Modifications• Intrinsic

– Single nucleosome changes– Example: histone variant specific modifications (H2AX)

• Extrinsic – Chromatin organization: nucleosome/nucleosome interactions– Alter chromatin packaging, electrostatic charge– Example: H4 acetylation

• Effector-mediated – Recruitment of other proteins to the chromatin via

• Bromodomains bind acetylation• Chromo-like royal domains (chromo, tudor, MBT) and PHD bind methylation• 14-3-3 proteins bind phosphorylation

Kouzarides, Cell 2007

- Prevent binding: H3S10P prevents Heterochromatin Protein 1 binding

General Roles of Histone Modifications

Wade P A Hum. Mol. Genet. 2001.

Gene Regulation

Moggs and Orphanides, Toxicological Sciences, 2004.

DNA Damage

General Roles of Histone Modifications

Chromatin Condensation Spermatogenesis

Kruhlak M J et al. J. Biol. Chem. 2001.

Techniques to Study Histone Modifications

• Chromatin immunoprecipitation (ChIP): Strategy for localizing histone marks

• ChIP-qPCR: if you know the target gene

• ChIP-chip, ChIP-seq, or other genome-wide techniques: unbiased

• Limitations: » Antibody specificity» Inherent biases of localization methods

• Mass Spectrometry:• Requires digestion of histones

ChIP-chip

Massively parallel sequencing

Local amplification

ChIP-seq

ChIP-seq

Genome

“Depth”: the number of mapped sequence tags

“Coverage”: how much of the genome the reads can be mapped to

ChIP-seq vs. ChIP-chip

Mikkelsen et al., 2007

Outline

1. Overview of histone modifications:a. Types of modifications and modifiersb. General roles of modificationsc. Techniques used to study histone modifications

2. Specific modifications (acetylation, methylation, etc):a. Residues/positions that are frequently modifiedb. Enzymes that add/remove the modificationc. Biological roles

3. Cross-talk between histone modifications

4. Summary

5. A histone code?

Specific Histone Modifications

• Positions that are modified• Modifying enzymes• Functions of the modification

Lysine Acetylation

Bhaumik, Smith, and Shilatifard, 2007.

Acetylation

• Many lysine residues can be acetylated• mainly on histone tails (sometimes in core)

• Can be part of large acetylation domains

• Modifying enzymes: • often multi-enzyme complexes • can modify multiple residues

• Well correlated with transcriptional activation

• Other roles (chromatin assembly, DNA repair, etc.)

• Two general types:• Type B: cytoplasmic (newly synthesized histones)

• HAT1

Kouzarides, Cell, 2007.

Histone Acetyl Transferases (HATs/KATs)

Type B

1. GNAT

2. MYST

3. P300/CBP (metazoan)

Other HATs exist as well:e.g. associated with nuclear receptors or Pol III

4. Rtt109 (fungal)

HAT SuperfamiliesType A: nuclear

Marmorstein and Trievel, 2009

GNAT MYST P300/CBP Rtt109

HAT Superfamilies

• Similarities• Structurally conserved central core• Can acetylate non-histone proteins

• Differences• Sequence divergence• Catalytic mechanisms• Interacting proteins (regulate specificity)

• Multi-enzyme complexes

• Targeted by transcriptional repressors

• Deactylate histone tails

Histone Deacetylases (HDACs)

• Class I HDACs• RPD3-like (HDAC 1, 2, 3)• most cell types• in nucleus

• Class II HDACs• HDA1- like (HDAC 4, 5, 6, 7, 9, 10)• HDAC and N-term repressor motif• restricted expression • shuttle in/out nucleus

• Class III HDACs• Sir-2 (NAD-dependent)• sirtuins

HDAC Superfamilies

HDACs are in Complexes

Butler and Bates, Nature Reviews Neuroscience, 2006.

1. Opens up chromatin: – Reduces charge interactions of histones with DNA

(K has a positive charge)– Prevents chromatin compaction (H4K16ac

prevents 30nm fiber formation)

2. Recruits chromatin proteins with bromodomains (SWI/SNF, HATs: GCN5, p300)

3. May occur at same residues as methylation with repressive effect (competitive antagonism)

Roles of Acetylation

Robinson et al., J. Mol. Biol., 2008.

Mujtaba et al., Oncogene, 2007.

PCAF

Yang and Chen, Cell Research, 2011.

H3K27ac

H3K27me3

4. Highly correlated with active transcription i.e. enriched at TSS of actively transcribed genes

Roles of Acetylation -- Continued

Expression: P1<P2<P3<P4

Heintzman N et al., Nature Genetics, 2007.

Roles of Acetylation

H4Ac H3Ac H3 RNAPII

208 TSS investigated

5. Correlated with binding of activating transcription factors i.e. enriched at promoters and enhancers

Roles of Acetylation

Heintzman N et al., Nature Genetics, 2007.

H4Ac H3Ac p300

Expression: E1<E2<E3 74 enhancers

(distal p300 binding sites)

Lysine Methylation

Bhaumik, Smith, and Shilatifard, 2007.

Lysine Methylation

• Many lysine residues can be methylated• Mainly on histone tails (sometimes in core)• Can be mono-, di-, or tri-methylated

• Depending on residue and number of methyl groups, can be associated with active or repressive transcription

• Other roles• Transcriptional elongation• Pericentromeric heterochromatin• X chromosome inactivation

Liu et. al, Annu. Rev. Plant Biol., 2010.

• Enzymes very specific• Target a certain lysine on a certain histone

• Put on mono, di, and/or tri methyl (me, me2, me3)• Many contain SET domains (me-transferase)• ‘Readout’ is very specific

• Ex. H3K4me1 vs. H3K4me3

Lysine Methyltransferases: KMTs

Xiao et al., Nature, 2003.

Only room for one methyl

group

Set7/9

H3K4me: trithorax/Set1, KMT2

H3K9me: Suv3-9, KMT1

H3K27me: polycomb Group (E(z)), KMT6

H3K36me: Set2, KMT3

H3K79me: Dot1 (nonSET) KMT4

H4K20me: Suv4-20, KMT5

KMTs

***Most contain SET Domains

1. Recruitment of other chromatin proteins through specific domains:

Chromodomain (CHD ATPases, HP1, PC)Tudor (some histone demethylases)PhD (many chromatin regulators BPTF, ING2)MBT (in some polycomb proteins)

WD-40 (WDR5)

Roles of Lysine methylation

Chromo-like(Royal)

Roles of Lysine Methylation

Bannister and Kouzarides Cell Research 2011

Roles of Lysine Methylation

Roles of Lysine Methylation

Liu et. al, Annu. Rev. Plant Biol., 2010.

2. Different readout depending on level of methylation

Roles of Lysine Methylation

2. Different readout depending on level of methylation

– Methylation status of H3K4 is recognized by different domains• H3K4me1: chromodomain in CHD1 (ATPase) = transcription activation• H3K4me2: WD-40 domain in WDR5 in MLL (Trx) = transcription activation• H3K4me3: PhD domain of BPTF in NURF (ISWI) = transcription activation• H3K4me3: PhD ING2 recognizes during DNA damage and shuts down

active transcription through mSin3a-HDAC1

Promoters Enhancers

Heintzman N et al., Nature Genetics, 2007.

Li et al., Cell, 2007.

Roles of Lysine Methylation

3. Transcriptional activation– H3K4me3: euchromatin promoter, 5’ end activates transcription (Set1)– H3K36me3: in gene (ORF), transcriptional elongation (Set2)

3. Transcriptional activation– H3K36me3 marks actively transcribed genes

Guenther et al., Cell 2007

H3K36me3

Roles of Lysine Methylation

PolII

4. Transcriptional repression• H3K9me (Suv39H1)• In promoter, represses transcription• In larger domains, heterochromatin formation• H3K27me (EZH2)• Repression of transcription • Polycomb group silencing• H4K20me (Suv420H1)• Some forms of silencing and repression of gene expression• In repetitive elements, similar localization as H3K9me3 in ES cells

Li et al., Cell, 2007.

Roles of Lysine Methylation

Co-occurrence of activating and repressivelysine methylation

Bivalency marks “poised” genes

Mikkelsen et al., Nature 2007

Position of histone modifications

Mikkelsen et al., Nature, 2007.

H3K9me3 and H4K20me3 can occur in actively transcribed genes

Thought to prevent access to cryptic transcription initiation

Wysocka et al., Cell, 2005.

Jumonji family: H3K9(JHDM2A:me1 and me2)

LSD1: H3K4

JHDM1A demethylase: H3K36 me1 and me2

Lysine Demethylation

Lysine Demethylation

• LSD1 – first histone demethylase– amine oxidase– only me1 and me2 can serve as substrates

• Different domain structure from other demethylase• Complex determines specificity (H3K4me vs. H3K9me)

Stavropoulos et al., NSMB, 2006.

• JmjC– JmjC-domain containing oxygenases– 27 family members– Catalytic JmjC domain can accommodate me3 as substrate

Lysine Demethylation

Wolf et al., EMBO Reports, 2007.

Lysine Methylation/Demethylation

Shi, Nature Reviews, 2007.

Individual lysines can be targeted by different methyltransferases/demethylases

Kouzarides, Cell, 2007.

Other Histone Modifications

Arginine Methylation

Bhaumik et al., NSMB, 2007.

Arginine Methylation

Can be symmetric or asymmetric

Zhang et al., EMBO Journal, 2000.

Protein Arginine Methyltransferases (PRMTs)

Arginine methylation can be activating or repressive

Kouzarides, Curr. Opin. G&D, 2002.

Arginine demethylation:1. different amino acid

Involves a different mechanism than Lysine demethylation

Lysine demethylation:removal of methyl groups

Arginine Demethylation

Bannister and Kouzarides, Nature, 2005.

2. Also possible simple de-methylationJMJD6, H3R2 and H4R3

Serine/Threonine Phosphorylation

Bhaumik et al., NSMB, 2007.

Serine/Threonine Phosphorylation

• Kinases phosphorylate

• Phosphatases remove

example H3S10P during mitosisKinases: Aurora BPhosphatase: PP1

Roles of Ser/Thr Phosphorylation

Cheung et al., Cell, 2000.

Hirota et al., Nature, 2005.

Fernandez-Capetilly et al., JEM, 2004.

H2AX-pDAPI

Laser

UV

Roles of Ser/Thr Phosphorylation

Histone Ubiquitination

• Ubiquitination– H2A K119:

• Function: Polycomb repression (Ring1a, PCG)– H2B K123: activation (Rad6+Bre yeast; RNF20/RNF40 and UbcH6 in mam)

• Function: FACT recruitment, transcriptional elongation– H3 and H4: DNA repair (CUL4)

• De-ubiquitination– H2A: Dub (PCAF)

• Function: counteract Polycomb– H2B: Ubp8 (SAGA)

• Function: transcription elongation

Outline

1. Overview of histone modifications:a. Types of modifications and modifiersb. General roles of modificationsc. Techniques used to study histone modifications

2. Specific modifications (acetylation, methylation, etc):a. Residues/positions that are frequently modifiedb. Enzymes that add/remove the modificationc. Biological roles

3. Cross-talk between histone modifications

4. Summary

5. A histone code?

Crosstalk among Modifications

Bannister and Kouzarides, Cell Research, 2011.

Crosstalk among Modifications

• Mutually exclusive: a position can be modified either with an activating or repressive mark (competitive antagonism)– H3K9ac vs. H3K9me

• One modification recruits a modifying enzyme that places/removes another modification on the same/different histone tail– H3S10P GCN5 H3K14ac

In cis In trans

Lee et. al, Cell, 2010.

Crosstalk among Modifications

• Mutually exclusive: a position can be modified either with an activating or repressive mark (competitive antagonism)– H3K9ac vs. H3K9me

• One modification recruits a modifying enzyme that places/removes another modification on the same/different histone tail– H3S10P GCN5 H3K14ac

• The binding of a protein may be disrupted by modification of an adjacent position – HP1 (binds to H3K9me) by H3S10P : “phospho switch”

Badia et al., Curr. Med. Chem., 2007.

Crosstalk among Modifications

• Mutually exclusive: a position can be modified either with an activating or repressive mark – H3K9ac vs. H3K9me

• One modification recruits a modifying enzyme that places/removes another modification on the same/different histone tail– H3S10P GCN5 H3K14ac

• The binding of a protein may be disrupted by modification of an adjacent position – HP1 (binds to H3K9me) by H3S10P

• Cooperative recruitmentPHF8 binds H3K4me3 most strongly if H3K9ac and H3K14ac

One complex may contain both demethylase and methyltransferasetargeting different residues with opposing functions

Crosstalk among Modifications

Crosstalk among Modifications

One modification affects the generation of another

Schotta et al., Genes and Dev., 2004.

Pericentric heterochromatin

H3K9me3

H4K20me

Summary: Histone PTMs

• Covalent and reversible • Usually occur on histone tails• Modifying enzymes:

• Redundancy: A single position can be modified by multiple different enzymes

• Specificity: Some enzymes (like HMTs) can target only one residue and some (like HATs) can target many

• Histone PTMs recruit other proteins to DNA via specific domains• Bromo (Ac)• Chromo/PHD (Me)• 14-3-3 (Ph)

• Participate in regulation of many processes– transcription– DNA repair– chromatin assembly – long-range packaging (heterochromatin formation, silencing)

• Readout frequently depends on the context

So…is there a histone code?

Kharchenko et al., Nature, 2011.

Euchromatin

Heterochromatin

So…is there a histone code?

Iwase et al., NSMB, 2011.

• Discuss.

So…is there a histone code?

1. Type of modification– Which amino-acid– Number of modifications (me)

2. Position in genome– Promoter: H3K36me, H3K9me are repressive– Coding region: H3K36me, H3K9me are activating and prevent cryptic initiation of transcription in ORF

3. Other histone modifications– combinatorial (occur together)– H3K4me + H3K9me: transcriptional activation– H4K20me + H3K9me: heterochromatin formation– H3K27me + H3K4me: “bivalent” mark in stem cells

4. Size of histone modification domain– large: heritable (can be copied more easily)

• H3K27me can recruit PRC2 has H3K27me3 activity• H3K4me recruits WDR5 (MLL thrithorax): H3K4me

5. Cycles of modifications– H2Bub H2B required for transcriptional elongation

Summary: Histone PTMs and readout