Histone Modifications

Outline
1.

Overview of histone modifications:

a. Types of modifications and modifiers
b. General roles of modifications
c. Techniques used to study histone modifications

2.

Specific modifications (acetylation, methylation, etc):

a. Residues/positions that are frequently modified
b. Enzymes that add/remove the modification
c. Biological roles

3.

Cross-talk between histone modifications

4.

Summary

5.

A histone code?

Quick Overview
Modifications and
modifiers
General roles
Techniques

Types of Histone Modifications

Bhaumik, Smith, and Shilatif

Features of Histone
Modifications

Covalently attached groups (usually to histone tails)

Methyl

Acetyl

Phospho

Ubiquitin

Reversible and Dynamic

Enzymes that add/remove modification
Signals

Have diverse biological functions

SUMO

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

Features of Histone
Modifications

Small vs. Large groups

One or up to three groups per residue

Ub = ~8.5 kDa
H4 = 14 kDa

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

Histone Modifications and

Modifers

Writers: enzymes that add a mark

Readers: proteins that bind to and interpret the mark

Erasers: enzymes that remove a mark

Tarakhovsky, A., Nature Immunol

Histone Modifications and

Modifers
Residue

Modification

Modiying Enzyme

Lysine

Acetylation
Deacetylation

HAT
HDAC

Lysine

Methylation
Demethylation

HMT
HDM

Lysine

Ubiquitylation
Deubiquitylation

Ub ligase
Ub protease

Phosphorylation
Serine/Threonine Dephosphorylatio
n

Kinase
Phosphatase

PRMT
Methylation
Arginine
Deiminase/Demeth
Demethylation
ylase
Others: Sumoylation (Lysine), ADP Ribosylation
(Glutamate)

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 coregulators

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
- Prevent
binding:
H3S10P prevents Heterochromatin
14-3-3
bind phosphorylation
Proteinproteins
1 binding

Kouzarides, Cell 2007

General Roles of Histone

Modifications
Gene Regulation

Wade P A Hum. Mol.

DNA Damage

Moggs and Orphanides, Toxicological Science

General Roles of Histone

Modifications
Chromatin Condensation

Kruhlak M J et al. J. Biol.

Spermatogenesis

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

ChIP-seq

Local amplification

Massively parallel sequencing

ChIP-seq

Depth: the
number of mapped
sequence tags

Genome

Coverage: how much of the genome the reads can be

mapped to

ChIP-seq vs. ChIP-chip

Mikkelsen et al.,

Outline
1.

Overview of histone modifications:

a. Types of modifications and modifiers
b. General roles of modifications
c. Techniques used to study histone modifications

2.

Specific modifications (acetylation, methylation, etc):

a. Residues/positions that are frequently modified
b. Enzymes that add/remove the modification
c. 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 Shilatif

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.)

Histone Acetyl Transferases

(HATs/KATs)
Two general types:
Type B: cytoplasmic (newly synthesized
histones)
HAT1

Type B

Kouzarides, Cell,

HAT SuperfamiliesType

A: nuclear

1. GNAT

2. MYST

3. P300/CBP
(metazoan)
4. Rtt109

Other HATs exist as

well:
e.g. associated with
nuclear receptors or
Pol III

HAT Superfamilies

Similarities
Structurally conserved central core
Can acetylate non-histone proteins

Differences
Sequence divergence
Catalytic mechanisms
Interacting proteins (regulate specificity)

GNAT

MYST

P300/CBP

Rtt109

Marmorstein and Trievel

Histone Deacetylases (HDACs)

Multi-enzyme complexes

Targeted by transcriptional repressors

Deactylate histone tails

HDAC Superfamilies
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

HDACs are in Complexes

Butler and Bates, Nature Reviews

Roles of Acetylation
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)

Robinson et al., J. Mol.

PCAF

3. May occur at same residues H3K27ac

as
methylation with repressive effect
(competitive antagonism)
H3K27me3
Yang and Chen, Cell Research, 2011.

Mujtaba et al., Oncogene,

Roles
of
Acetylation
-Continued
Roles of Acetylation
4. Highly correlated with active transcription
i.e. enriched at TSS of actively transcribed genes
H4Ac

H3Ac

H3

RNAPII

Expression:
P1<P2<P3<P4
208 TSS investigated

Heintzman N et al., Nature Gene

Roles of Acetylation
5. Correlated with binding of activating transcription
factors
i.e. enriched at promoters and enhancers
H4Ac
H3Ac
p300

Expression:
E1<E2<E3
74 enhancers
(distal p300 binding sites

Heintzman N et al., Nature Gene

Lysine Methylation

Bhaumik, Smith, and Shilatif

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. Plan

Lysine Methyltransferases: KMTs

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

Only room
for one
methyl
group

Set7/9

Xiao et al., Nature

KMTs

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

***Most contain SET Domains

Roles
of
Lysine
methylation
Roles of Lysine Methylation
1. Recruitment of other chromatin proteins through
specific domains:
Chromodomain (CHD ATPases, HP1, PC)
Chromo-like
Tudor (some histone demethylases)
(Royal)
PhD (many chromatin regulators BPTF,
ING2)
MBT (in some polycomb proteins)
WD-40 (WDR5)

Roles of Lysine Methylation

Bannister and Kouzarides Cell Research

2011

Roles of Lysine Methylation

2. Different readout depending on level of methylation

Liu et. al, Annu. Rev. Plan

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
Promoters
Enhancers
H3K4me3: PhD ING2 recognizes during DNA damage and
shuts down active transcription through mSin3aHDAC1

Heintzman N et al., Nature

Roles of Lysine Methylation

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

Li et al.,

Roles of Lysine Methylation

3. Transcriptional activation
H3K36me3 marks actively transcribed genes

PolII

H3K36me3

Guenther et al.,

Roles of Lysine Methylation

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.,

Co-occurrence of activating and

repressive
lysine methylation
Bivalency marks poised genes

Mikkelsen et al.,

Position of histone
modifications

H3K9me3 and H4K20me3 can occur in actively transcribed g

Thought to prevent access to cryptic transcription

initiation
Mikkelsen
et al.,

Lysine Demethylation
LSD1: H3K4

Jumonji family: H3K9

(JHDM2A:me1 and me2)

JHDM1A demethylase: H3K36 me1 and me2

Wysocka et al., Cel

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., NS

Lysine Demethylation

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

Wolf et al., EMBO Report

Lysine Methylation/Demethylation
Individual lysines can be targeted by different
methyltransferases/demethylases

Shi, Nature Reviews,

Other Histone Modifications

Kouzarides, Cell,

Arginine Methylation

Bhaumik et al., NSMB,

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

Arginine Demethylation

Involves a different mechanism than

Lysine demethylation
Lysine demethylation:
removal of methyl groups

Arginine demethylation:
1. different amino acid

2. Also possible simple de-me

JMJD6, H3R2 and H4R3

Bannister and Kouzarides, Nat

Serine/Threonine Phosphorylation

Bhaumik et al., NSMB,

Serine/Threonine Phosphorylation

Kinases phosphorylate
Phosphatases remove
example H3S10P during mitosis
Kinases: Aurora B
Phosphatase: PP1

Roles of Ser/Thr Phosphorylation

Cheung et al., Cel

Roles of
Ser/Thr
Phosphorylatio
n

H2AX-p
DAPI

UV
Laser

Fernandez-Capetilly et al.,

Hirota et al., Nature, 2005.

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 modifiers
b. General roles of modifications
c. Techniques used to study histone modifications

2.

Specific modifications (acetylation, methylation, etc):

a. Residues/positions that are frequently modified
b. Enzymes that add/remove the modification
c. Biological roles

3.

Cross-talk between histone modifications

4.

Summary

5.

A histone code?

Crosstalk among Modifications

Bannister and Kouzarides, Cell Re

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
In
In
H3S10P GCN5 H3K14ac
cis
trans

Lee et. al, Cell

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. C

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 recruitment
PHF8 binds H3K4me3 most strongly if H3K9ac and H3K14ac

Crosstalk among Modifications

One complex may contain both demethylase and methyltransferase

targeting different residues with opposing functions

Crosstalk among Modifications

One modification affects the generation of another

H3K9me3

Pericentric heterochr

H4K20me
Schotta et al., Genes and

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)

Sois there a histone code?

Euchromatin

Heterochromatin

Kharchenko et al., Nature, 2011.

Sois there a histone code?

Iwase et al.,

Sois there a histone code?

Discuss.

Summary: Histone PTMs and

readout
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