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The interplay of histone modifications - Writers that read

October 2015
EMBO Reports 16(11)

October 2015
16(11)

DOI:10.15252/embr.201540945

License
CC BY 4.0

Authors:

Tianyi Zhang

London Institute of Medical Sciences

Sarah Cooper

University of Greenwich

Histones are subject to a vast array of posttranslational modifications including acetylation, methylation, phosphorylation, and ubiquitylation. The writers of these modifications play important roles in normal development and their mutation or misregulation is linked with both genetic disorders and various cancers. Readers of these marks contain protein domains that allow their recruitment to chromatin. Interestingly, writers often contain domains which can read chromatin marks, allowing the reinforcement of modifications through a positive feedback loop or inhibition of their activity by other modifications. We discuss how such positive reinforcement can result in chromatin states that are robust and can be epigenetically maintained through cell division. We describe the implications of these regulatory systems in relation to modifications including H3K4me3, H3K79me3, and H3K36me3 that are associated with active genes and H3K27me3 and H3K9me3 that have been linked to transcriptional repression. We also review the crosstalk between active and repressive modifications, illustrated by the interplay between the Polycomb and Trithorax histone-modifying proteins, and discuss how this may be important in defining gene expression states during development.

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Review

“Histones and Chromatin” Review series

The interplay of histone modifications – writers

that read

Tianyi Zhang

†

, Sarah Cooper

*,†

& Neil Brockdorff

Abstract

Histones are subject to a vast array of posttranslational modifi-

cations including acetylation, methylation, phosphorylation, and

ubiquitylation. The writers of these modifications play important

roles in normal development and their mutation or misregulation

is linked with both genetic disorders and various cancers. Readers

of these marks contain protein domains that allow their recruit-

ment to chromatin. Interestingly, writers often contain domains

which can read chromatin marks, allowing the reinforcement of

modifications through a positive feedback loop or inhibition of

their activity by other modifications. We discuss how such positive

reinforcement can result in chromatin states that are robust and

can be epigenetically maintained through cell division. We

describe the implications of these regulatory systems in relation to

modifications including H3K4me3,H3K79me3, and H3K36me3 that

are associated with active genes and H3K27me3 and H3K9me3 that

have been linked to transcriptional repression. We also review the

crosstalk between active and repressive modifications, illustrated

by the interplay between the Polycomb and Trithorax histone-

modifying proteins, and discuss how this may be important in

defining gene expression states during development.

Keywords chromatin; histone modifications; Polycomb; Trithorax

DOI 10.15252/embr.201540945 | Received 29 June 2015 | Revised 4 September

2015 | Accepted 16 September 2015 | Published onl ine 16 October 2015

EMBO Reports (2015) 16: 1467–1481

See the Glossary for abbreviations used in this article.

Introduction

In eukaryotes, DNA is packaged in the form of chromatin. The basic

unit of chromatin, the nucleosome, is comprised of 147 bp of DNA

wrapped around a histone octamer made of two dimers of H2A and

H2B and a tetramer of H3 and H4 proteins. The N- and C-terminal

histone tails protrude from the nucleosome core and have the poten-

tial to interact with adjacent nucleosomes and the linker DNA. All

histones can be posttranslationally modified, and the sites of modifi-

cation are often on the histone tails. These modifications can

regulate chromatin structure directly and frequently act as binding

sites for the recruitment of other non-histone proteins to chromatin.

The most abundant histone modifications are acetylation, phospho-

rylation, methylation, and ubiquitylation, although many other

modifications have been reported (reviewed recently in [1]).

Transcriptionally active and silent chromatin is characterized by

distinct posttranslational modifications on the histones or combina-

tions thereof. Active genes typically carry high levels of lysine acety-

lation on the H3 and H4 tails, trimethylation of H3 lysine 4,

trimethylation of H3 lysine 79, ubiquitylation of H2B, and trimethy-

lation of H3 lysine 36 (Fig 1). Marks associated with repressed genes

include trimethylation of lysine 27, ubiquitylation of H2A on lysine

119, and trimethylation of H3 lysine 9 (Fig 1). The chromatin-modi-

fying enzymes that catalyze these marks can be recruited to target

sites by sequence-specific DNA-binding transcription factors that

regulate transcriptional states of particular genes. However, other

more general features of the DNA such as its global CG content and

DNA methylation status can be read by the DNA-binding Zn-finger

CxxC domain present in many chromatin-modifying enzymes [2].

Equally, the act of transcription can direct the recruitment of writers

that associate with the transcriptional machinery, leading to the

accumulation of specific marks such as H3K4me3 and H3K36me3.

Given the large number of different histone modifications, the

potential combinatorial complexity is vast. Advances in technology

over the past decade such as ChIP-sequencing have allowed us to

map the distribution and co-localization of histone marks at high-

resolution genome wide, while mass spectrometry, often in combi-

nation with stable isotope labeling, enables the analysis of histone

marks and dynamics at the level of a single histone tail. Interest-

ingly, mass spectrometric data suggest that there are many combina-

tions of modifications that are either more likely to occur together,

or are mutually exclusive, suggesting crosstalk between these

marks. Such crosstalk can occur in cis between distinct modifi-

cations on the same histone tail, or in trans either on neighboring

histones within the same nucleosome or on neighboring nucleo-

somes in a chromatin domain.

The patterns of histone marks associated with distinct transcrip-

tional states are established through a dynamic interplay between

histone readers, writers, and erasers. Importantly, the writers that

place these marks contain chromatin-reading domains that can bind

preexisting histone marks. Studies have shown that such crosstalk

between histone marks can both positively and negatively regulate

binding and catalytic activity of writers, resulting in positive and

Developmental Epigenetics, Department of Biochemistry, University of Oxford, Oxford, UK

*Corresponding author. Tel: +44 1865 613230; E-mail: sarah.cooper@bioch.ox.ac.uk

†

These authors contributed equally to this work

ª 2015 The Authors. Published under the terms of the CC BY 4.0 license EMBO reports Vol 16 |No 11 | 2015

1467

negative feedback loops. Therefore, writers that can also read the

histone modifications are required for the establishment and mainte-

nance of chromatin states at active and repressed genes and may

play important roles in the memory and switching of gene

expression states.

In this review, we will focus on several examples of the positive

and negative feedback mechanisms that regulate the formation,

reinforcement, and maintenance of the distinct patterns of histone

marks associated with active and repressed transcriptional states

(Fig 2). However, such features are likely to be more general

features of chromatin states, and the principles seen in these exam-

ples are likely to be applicable to the plethora of other chromatin

modifications whose function is still unclear.

Active histone modifications

In eukaryotic organisms, gene expression is regulated through the

synergistic actions of multiple factors, including but not limited to,

transcription factors, the transcriptional machinery, chromatin

remodelers, and the presence of specific histone variants and

histone modifications. Active chromatin domains are characterized

by a distinct array of histone marks. H3K27ac and H3K4me1 are

associated with active enhancers [3], and high levels of H3K4me3

and H3 and H4 acetylation are found at the promoters of active

genes [4–6]. The bodies of active genes are enriched in H3 and H4

acetylation [7], H3K79me3 [8], and H2BK120u1 [9,10], and increas-

ing H3K36me3 toward the 3

end [11]. These histone marks may

regulate transcription by creating an open chromatin structure and

recruit effectors that mediate a transcriptionally competent state.

While the function of many active histone modifications is not fully

understood, there is abundant evidence that their deposition is

required for the proper regulation of gene expression. Positive

crosstalk mechanisms between different histone modifications play

an important role in the recruitment and maintenance of active

histone modifications at active genes.

Establishment and maintenance of H3K4me3

H3K4me3 is a highly conserved histone modification and its associa-

tion with transcription is evolutionarily conserved in eukaryotes.

Glossary

AEPB2 AE-binding protein 2

ASH2L absent, small, or homeotic-like

ATRX5/6 Arabidopsis Trithorax-related protein 5 /6

BEND3 Ben domain containing 3

BLOCS broad local enrichments

BRE1 Brefeldin-A sensitivity protein 1

CBX chromobox

CDYL chromodomain protein, Y-like

CFP1 CXXC finger protein 1

ChIP-sequencing chromatin immunoprecipitation followed by DNA

sequencing

CpG cytosine-phosphate-guanine

CTCF CCCTC-binding factor

CTBP2 C-terminal-binding protein 2

CTD C-terminal domain

DNMT3A/B DNA methyltransferase 3A/B

DOT1 disruptor of telomeric silencing 1

DOT1L DOT1-like

DPY30 dumpy-30 protein homolog

EAF3 Esa1p-associated factor 3

EED embryonic ectoderm development

ESC embryonic stem cell

EZH2/1 enhancer of zeste homolog 2/1

FRAP fluorescence recovery after photobleaching

G9a/GLP G9a and G9

a-like protein

H2AK119u1 histone H2A lysine 119 monoubiqutination

H2BK120u1 histone H2B lysine 120 monoubiqutination

H2BK34u1 histone H2B lysine 34 monoubiqutination

H3K27me1/2/3 histone H3 lysine 27 mono/di/trimethylation

H3K36me1/2/3 histone H3 lysine 36 mono/di/trimethylation

H3K4me1/2/3 histone H3 lysine 4 mono/di/trimethylation

H3K9me1/2/3 histone H3 lysine 9 mono/di/trimethylation

HAT histone acetyltransferase

HBO1 histone acetyltransferase bound to ORC 1

HDAC

histone deactylase complex

HMT histone methyltransferase

Hox gene homeobox-containing gene

HP1 heterochromatin protein 1

JARID2 jumonji, AT-rich interactive domain 2

KDM2A/B lysine demethylase protein 2A/B

MBD methyl binding domain

MES-4 mesoderm expressed 4

MLL1/2/3/4 mixed-lineage leukemia 1/2/3/4 complex

MSL1/2 male-specific lethal 1/2

NO66 nucleolar protein 66

NSD1/2/3 nuclear receptor-binding SET domain

protein 1/2/3

NuA3/4 nucleosomal acetyltransferase of histone H3/H4

NURD nucleosome remodeling and deacetylase

P300/CBP P300- and CREB-binding protein

PAF polymerase-associated factor

PCGF1/2/3/4/5/6 Polycomb group ring finger 1/2

/3/4/5/6

PCL1/2/3 Polycomb-like 1/2/3

PH polyhomeotic

PHD finger plant homeodomain finger

Pol II RNA polymerase II

PRC1 Polycomb repressive complex 1

PRC2 Polycomb repressive complex 2

RAD6 ras-related associated with diabetes protein 6

RBBP5 retinoblastoma-binding protein 5

RING1A/B really interesting new gene 1A/B

RNF20/40 ring finger protein 20/40

RpAb46/48 Rb-associated protein 46/48

RPD3S reduced potassium dependency 3S complex

RYBP RING1- and YY1-binding protein

SAGA Spt-Ada-Gcn5 acetyltransferase

SET domain

Su(var)3-9, enhancer-of-zeste and Trithorax

domain

SETD1A/B SET domain containing 1A/B

SETD2 SET domain containing 2

SETMAR SET domain and mariner transposase fusion

containing protein 2

SMYD2 SET and MYND domain-containing protein 2

SUV3-9 H1/H2 suppressor of variegation 3-9 homolog 1/2

SUZ12 Suppressor of zeste 12 homolog

TrxG Trithorax group

TSS transcription start site

WDR5 WD repeat-containing protein 5

YAF2 YY1-associated factor 2

ZMYND11 zinc finger, MYND-type containing 11

EMBO reports Vol 16 |No11 | 2015 ª 2015 The Authors

EMBO reports The interplay of histone modifications Tianyi Zhang et al

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In mammals, H3K4 methylation is catalyzed by six related homologs

of the yeast SET1—SETD1A, SETD1B, MLL1, MLL2, MLL3, and

MLL4 [12]. These complexes are comprised of the catalytic SETD1/

MLL subunits and four core subunits WDR5, RBBP5, ASH2L, and

DPY30, and as well many other complex-specific subunits [13–15].

H3K4me3 is a hallmark of active genes and is distributed along the

promoter and TSS regions [6,16,17]. Work in yeast shows that SET1

associates with the PAF complex and the Ser5-phosphorylated initi-

ating form of Pol II and is co-transcriptionally deposited [18] (Fig 3).

Additionally, the recruitment of SETD1 and MLL to specific target

genes is mediated by many cell type-specific transcription factors or

transcriptional coactivators [19–23]. However in higher organisms

especially, more general mechanisms of H3K4me3 recruitment and

establishment are also at play.

Notably, the distribution of H3K4me3 is highly coupled to the

presence of CpG islands, regions of CpG- and GC-dense DNA that

are predominately unmethylated and found at 50–70% of vertebrate

promoters [24]. A biochemical link between CpGI promoters and

H3K4me3 was eluciated with the discovery of the Zn-finger CxxC

domain which specifically binds nonmethylated CpGs and is present

in MLL1/2 and the CFP1 subunit of SETD1A/B [2] (Fig 3). All CpGI

promoters are marked with H3K4me3, and the level of H3K4me3 is

correlated to gene activity [25,26]. Emerging evidence suggests that

in vivo, MLL2 is responsible for maintaining H3K4me3 at CpGI

promoters with low expression [27,28], while the SETD1-specific

subunit CFP1 is preferentially enriched at active gene promoters

with higher levels of H3K4me3 [29]. In ESCs, CpGI promoters linked

to developmentally regulated genes are bivalent and harbor the

repressive H3K27me3 mark as well as H3K4me3 [30]. Importantly,

it has been suggested that the ability of H3K4 writers to sample

CpGIs genome wide and the presence of H3K4me3 at CpGI promo-

ters may poise silent genes for rapid activation upon differentiation.

SETD1/MLL complexes may reinforce their binding through

recognition of their own mark, H3K4me3. The PHD finger domain

of CFP1 is known to read H3K4me3 and mediates SETD1 interaction

with H3K4me3 [31–33]. The third PHD domain in MLL1 is impor-

tant for H3K4me3 binding and MLL1 recruitment to target sites in

the Hox locus [34]. Other PHD domains within SETD1/MLL may

also interact with H3K4me3 but remain to be further characterized

[35]. The ability of SETD1/MLL to sample promoters and bind

H3K4me3 may be involved in the maintenance of this mark at active

genes. These mechanisms of H3K4me3 binding by H3K4 writers

suggest that once established, this mark may positively reinforce its

own deposition.

Crosstalk between H2BK120u1,H3K4me3, and H3K79me3

One of the best-studied pathways of positive histone crosstalk is the

stimulation of H3K4me3 and H3K79me3 by H2BK120u1 (or

H2BK123u1 in yeast). In yeast, H2BK123u1 is established by the

ubiquitin ligase RAD6/BRE1 during transcriptional initiation and

localizes to the TSS and along the bodies of active genes [36]. Deple-

tion of RAD6/BRE1 or mutation of H2BK123 causes severe loss of

H3K4me3 and H3K79me3 [37,38]. This positive crosstalk between

H2BK123u1 and H3K4me3 and H3K79me3 is specific and does not

extend to the regulation of H3K36me3, another mark associated

with transcription [37,38].

H2BK123 lies in close proximity to H3K79 on the exposed

nucleosome surface, and the H3K79 methyltransferase DOT1 in

yeast has been shown to be influenced by deletion and mutation of

residues on the H2B tail [39]. In humans, the situation is more

complex, as H3K79me3 and DOT1L distribution is not solely depen-

dent on H2BK120u1. Human DOT1L localizes at active genes and

peaks around the TSS and moreover has been shown to bind both

Active genes

Repressed genes

Positive

feedback

Negative

feedback

Writers

Active

marks

Positive

feedback

Writers

Repressive

marks

Figure 2. Crosstalk between chromatin writers and histone marks at

active and repressed genes.

Chromatin writers and chromatin marks associated with active genes positively

reinforce each other through various positive feedback mechanisms. The same

holds true for writers and marks associated with repressed genes. Additionally,

negative feedback mechanisms and mutual inhibition between writers and

marks associated with the opposite gene expression state also reinforce distinct

transcriptional states.

Repressed genes

H3K9me3

H2AK119ub1

H3K27me3

Promoter

Active genes

H3/H4 acetylation

H2BK120ub1

H3K79me3

H3K36me3H3K4me3

Promoter

TSS

Figure 1. The distribution of histone modifications over active and

repressed genes.

ª 2015 The Authors EMBO reports Vol 16 |No11 | 2015

Tianyi Zhang et al The interplay of histone modifications EMBO reports

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Ser5- and Ser2-phosphorylated forms of the Pol II CTD [40]. As

such, H3K79me3 is a marker of active genes, yet its exact role in

transcriptional regulation remains to be discovered.

The crosstalk between H2BK120u1 and H3K4me3 is conserved in

mammals, as knockdown of the BRE1 homologs RNF20/40 leads to

global reduction in H3K4me3 [41] (Fig 3). More recently, studies on

the MSL1/MSL2 E3 ligase that catalyzes H2BK34u1 have also

revealed a crosstalk between H2BK34u1 and H3K4me3 [42]. Both

H2BK120u1 and H2BK34u1 are now known to allosterically stimu-

late the activity of the MLL complex through binding to the ASH2L

subunit [43]. Sites of ubiquitylation at H2BK120 and H2BK34 reside

on the nucleosome surface and may provide a more favorable

substrate for SET1 or MLL complex binding and activity [43]. As

ASH2L is a core subunit of all writers of H3K4 methylation, H2B

ubiquitylation may be one mechanism of H3K4me3 maintenance at

active promoters through a positive feedback loop whereby tran-

scription results in deposition of H2Bub, which subsequently

activates the H3K4 methyltransferases.

H3K4me3 and histone acetylation

Histone lysine acetylation is a highly abundant mark and is known

to regulate many cellular processes including transcription. Acetyla-

tion of histones H3 and H4 is highly correlated with gene

expression. A unique structural motif, the bromodomain, specifi-

cally recognizes acetylated lysines and is present in many proteins

involved in transcriptional regulation [44]. Besides the direct

recruitment of effectors, histone acetylation has also been proposed

to physically alter chromatin structure by neutralizing the positive

charge of lysines and disrupting intra- and internucleosomal interac-

tions, which lead to an open chromatin environment permissible to

transcription. Lysine acetylation of three residues on the H3 globular

domain H3K56, H3K64, and H3K122, all of which lie at the H3–DNA

interface, may disrupt electrostatic interactions within the nucleo-

some and have been linked to gene activation [45–47]. H3K122ac

has been shown to directly promote in vitro transcription through

stimulating histone eviction [47]. H3 and H4 histone tail acetyla-

tions enhance DNA unwrapping, while H3 acetylation sensitizes

nucleosomes to salt-induced dissociation [48].

H3K4me3 and H3/H4 acetylation coexist at the promoter and

TSS of active genes, and there are many studies that suggest

H3K4me3 promotes downstream H3/H4 acetylation through recruit-

ment of HATs (Fig 4). H3K4me3 readers have been identified in

many HAT complexes. SGF29, a component of the SAGA HAT

complex, contains a tandem tudor domain that binds H3K4me3 and

overlaps with H3K4me3 at gene promoters. SGF29 deletion causes

loss of H3K9ac and loss of SAGA complex at target sites [49]. Simi-

larly, yeast NuA3 [50] and NuA4 [51], and mammalian HBO1 [52]

provide other examples of HAT complexes that contain PHD fingers

that preferentially bind H3K4me3. Dynamic turnover of H3 lysine

acetylation through the combinatorial action of the HAT p300/CBP

and HDAC has been shown to occur on histone H3 tails with pre-

existing H3K4me3, but not other modifications associated with

active gene expression such as H3K79me3 or H3K36me3 [53]. This

H3K4me3-linked acetylation is conserved in higher eukaryotes

including fly, mouse, and human. Loss of H3K4me3 upon CFP1

deletion leads to loss of CpGI-associated H3K9ac in ESCs [29].

Further work using the Dictyostelium discoideum model shows that

upon knockout of SET1 and loss of H3K4me3, dynamic H3 acetyla-

tion was lost [54]. The dynamic turnover of acetylation rather than

the modification itself may be key in transcriptional activation

(reviewed in [55]). In support of this, many members of the

H3K4me3-binding PHD fingers are associated with HDACs as well

as HATs [56]. As H3K4me3 has been found to be promoter-

associated before transcription initiation, H3K4me3-dependent

co-targeting of both HATS and HDACs may facilitate the dynamic

turnover of histone acetylation. The above examples illustrate that

positive crosstalk between H3K4me3 and histone acetylation is an

evolutionarily conserved pathway and that the cooperativity

Pol II

DPY30

ASH2L

SETD1/

MLL

WDR5

RbBP5

SETD1

CFP1

WDR5

RbBP5

DPY30

ASH2L

MLL1/2

WDR5

RbBP5

DPY30

SETD1

CFP1

WDR5

RbBP5

DPY30

ASH2L

CxxC

Methylated

CpG site

Unmethylated

CpG site

H3K4me3

H2BK120u1

TSS

Figure 3. Establishment of H3K4me3 and interplay with H2BK120u1.

The SETD1 complex associates with Pol II, and H3K4me3 is deposited co-transcriptionally. CFP1 (associated with SETD1) and MLL1/2 can be recruited to promoters de novo via

CxxC domain binding to CpG islands. H2BK120u1 can recruit H3K4 writers, possibly through recognition of H2BK120u1 by the ASH2L subunit.

EMBO reports Vol 16 |No11 | 2015 ª 2015 The Authors

EMBO reports The interplay of histone modifications Tianyi Zhang et al

1470

between H3K4me3 and hyperacetylation as well as the dynamic

turnover of acetylation is important in ensuring proper transcrip-

tional regulation.

H3K36me3 and histone deacetylation

Methylation at histone H3K36 is an abundant histone mark highly

conserved in eukaryotes. H3K36 mono-, di-, and trimethylation exist

in yeast and all of these states are catalyzed by SET2. Mammals on

the other hand have multiple writers of H3K36 methylation, includ-

ing the NSD1/2/3 family, ASH1L, SMYD2, SETMAR, and SETD2,

but SETD2 is the sole enzyme responsible for H3K36 trimethylation

in vivo (reviewed in [57]) (Fig 4). Interestingly, the uncoupling of

H3K36me3 activity from H3K36me1/2 over evolution alludes to

specific biologically distinct roles of each methylation state.

H3K36me3 is highly correlated with the transcribed regions of

active genes and levels of H3K36me3 increase toward the 3

end of

genes [11]. This particular distribution results from the association

of Set2 with the elongating Ser2-phosphorylated CTD of Pol II,

which is predominant over the bodies and 3

ends of active genes

[58–60]. Like H3K4me3, H3K36me3 has also been linked to regula-

tion of histone acetylation. H3K36me3 recruits HDACs to sites of

active transcription (Fig 4). In yeast, recognition of H3K36me2/3 by

the bromodomain-containing EAF3 complex recruits the HDAC

RPD3S complex, which deacetylates histones and prevents spurious

transcription initiation from within gene bodies [61–63]. H3K4me3

and histone hyperacetylation at gene promoters may regulate tran-

scriptional initiation from the TSS, while H3K36me2/3-mediated

deacetylation is required in the wake of the transcriptional

machinery to prevent initiation from aberrant sites within the gene

body. This mutual exclusivity of H3K4me3 and H3K36me3 may be

important for maintaining transcriptional integrity. This idea is

supported by work showing that promoters lack the H3K36me2/3

mark, and the H3K36me2 demethylases KDM2A/B co-localize with

H3K4me3 at CpGI promoters, ensuring active removal of H3K36me2

from transcriptional start sites [64,65].

H3K36me2/3 is recognized by a protein motif, the PWWP

domain, found in many nuclear chromatin-binding proteins [66–

69]. Notably, all three members of the NSD family of H3K36 methyl-

transferases that catalyze H3K36me1/2 each contain two PWWP

domains [70] and have been shown to preferentially bind H3

peptides containing H3K36me3 [69]. This implies that H3K36me2/3

recognition by its writers may be important for the propagation of

H3K36me1 and H3K36me2 at certain sites. Mono-/dimethylation of

H3K36 is more pervasive than H3K36me3 and not restricted to sites

of active transcription or euchromatin domains [71,72]. The biologi-

cal function of mono-/dimethylation is unknown, though an

increase in H3K36me2 levels as a result of mutations in NSD2 has

been linked to upregulation of gene expression profiles in cancers

[73–75]. H3K36me2 may have an important biological function in

its own right or may be required to serve as a substrate for subse-

quent SETD2-mediated H3K36 trimethylation. The broad distribu-

tion of H3K36me2 and H3K36me3 over active chromatin may also

prevent the spreading and accumulation of silencing marks such as

H3K27me3 through direct inhibition of the Polycomb complex PRC2

[76,77], which will be discussed below.

Repressive histone modifications

The methylation of residues lysine 27 and lysine 9 of H3 and the

ubiquitinylation of H2A on lysine 119 are hallmarks of repressive

chromatin and are often found at silent gene loci. H3K27me3 and

H2AK119u1 are associated with the formation of facultative hetero-

chromatin, whereas H3K9me2/3, as well as having important roles

in the formation of constitutive heterochromatin, also plays a part

in regulating gene expression during development.

H3K27me3 and H2AK119u1 crosstalk

The Polycomb Repressive Complex 2 (PRC2) is responsible for the

methylation of lysine 27 and contains four core subunits, EZH2/1,

SUZ12, EED, and RBAP46/8 [78]. The catalytic subunit is the SET

domain-containing protein EZH2 (or the related EZH1), although

these enzymes are only functional in the context of the full core

complex [79–81]. There are also accessory proteins that can asso-

ciate with the core PRC2 complex to form two types of PRC2:

PRC2.1 which includes a Polycomb-like subunit (PCL1/2/3) and

PRC2.2 which includes the JARID2 and AEBP2 subunits [82]. The

function of these accessory proteins remains unclear, although

HDAC

Pol II

HATS

SETD2

ASH1L

SMYD2

NSD1/2/3

SETMAR

Methylated

CpG site

Unmethylated

CpG site

TSS

H3K4me3

H3/H4ac

H3K36me2

H3K36me3

Figure 4. Interplay between H3K4me3,H3K36me3, and H3/H4 acetylation.

H3K4me3 reinforces H3 and H4 acetylation at the promoters of active genes. Various H3K36 writers catalyze H3K36me1/2 and SETD2 associates with elongating Pol II and

catalyzes H3K36me3 co-transcriptionally. H3K36me2/3 recruits HDACs that deacetylate histones over gene bodies.

ª 2015 The Authors EMBO reports Vol 16 |No11 | 2015

Tianyi Zhang et al The interplay of histone modifications EMBO reports

1471

they have been shown to modulate activity of PRC2 and may also

play a role in targeting PRC2 to chromatin. PRC2 is able to mono-,

di-, and trimethylate H3K27, although there is some dispute if

PRC2 is the only H3K27me1 methyltransferase. These different

methylation states have very different roles, and although

H3K27me3 is linked to gene repression, recent studies have

suggested that H3K27me1 may be important for gene activation

and is enriched over the bodies of genes [83]. The H3K27me2

modification is very prevalent in the genome, with MS/MS analy-

sis demonstrating that it accounts for 60–80% of all nucleosomes

in mESCs [84], although little is known about its function or bind-

ing proteins. H3K27me3 is the most well-characterized mark in

terms of facultative heterochromatin formation and is critical for

the repression of key transcriptional regulators during develop-

ment. Therefore, in terms of gene silencing, we will focus on the

trimethylation state of H3K27.

In ES cells, H3K27me3 is present at the promoters of several

thousand genes, including the Hox gene clusters, where it is associ-

ated with heritable gene silencing [85]. H3K27me3 modification is

also highly enriched on the inactive X chromosome suggesting a role

in facultative heterochromatin formation [86]. In more differentiated

cell types, larger domains of H3K27me3, termed BLOCS, are often

visualized over silent loci in the genome [87]. As described above,

for many of the enzymes associated with active gene expression,

there are also positive feedback loops important for the establish-

ment and spreading of repressive domains. The PRC2 component

EED contains an aromatic cage that is able to specifically bind to

H3K27me3 [88]. It has been shown that the binding of PRC2 to the

modification it deposits is required for the full establishment of

H3K27me3 domains, and such a positive feedback mechanism could

also be important for the inheritance of the H3K27me3 mark

through cell division [89]. PRC2 has also been shown to be stimu-

lated by dense chromatin via an interaction of the SUZ12 subunit

with the H3 tail (A31-R42) [90]. In this way, positive feedback from

the local chromatin structure will also allow robust domains of

H3K27me3 to be maintained over repressed genes.

The Polycomb repressive complex 1 (PRC1) is an E3 ubiquitin

ligase complex that can modify chromatin by monoubiquitylation of

H2A on lysine 119. All PRC1 complexes contain the catalytic

RING1A/B subunit, and one of six PCGF proteins [91]. The presence

of different PCGF subunits is thought to define the class of PRC1

complex, for example, PCGF2 (MEL18) and PCGF4 (BMI) make up

the canonical PRC1 complexes which also contain CBX (2,4,6,7,8)

and polyhomeotic subunits [92]. Variant complexes include either

the RYBP or YAF2 protein, the presence of which is mutually exclu-

sive with the CBX component [91,93]. These variant complexes,

such as the complex containing RING1B/PCGF1/RYBP/BCOR/

KDM2B, have been implicated in recruitment of PRC1 and have

been shown to have higher H2AK119u1 activity compared with

canonical PRC1 complexes [91,94]. Interestingly, RYBP also

contains a ubiquitin-binding domain and has been shown to bind

H2AK119u1 [95]. This suggests that a positive reinforcement mech-

anism could be important to establish or maintain high levels of

H2AK119u1 at PRC1 target domains, in a similar manner to PRC2

where EED binds to H3K27me3.

Both PRC1 and PRC2, along with their associated chromatin

modifications, H2AK119u1 and H3K27me3, have been shown to co-

localize at many regions of the genome, such as the promoters of

developmentally regulated genes and the inactive X chromosome

[96–99]. A hierarchical recruitment model, whereby the H3K27me3

modification placed by PRC2 is read by PRC1, has been proposed to

explain this co-recruitment of both PRC1 and PRC2 to chromatin

[100]. This occurs by a specific interaction of the H3K27me3 modifi-

cation with the chromodomain of the CBX protein found in canoni-

cal PRC1 complexes [101]. Hence, all PRC2 targets would also

become PRC1 targets and a repressive domain would be estab-

lished. However, this hierarchical model is not able to account for

all PRC1 recruitment to chromatin since even in the absence of

PRC2, the variant RYBP-containing complexes still localize to the

correct regions of the genome [93,102]. More recently, data from

three laboratories have shown that the reverse mechanism is also

possible, whereby PRC1 is recruited first, followed by PRC2. In this

model, the H2AK119u1 placed by a variant PRC1 complex is recog-

nized by PRC2 [103–105]. At present, we do not know how

H2AK119u1 is recognized by PRC2, but it has been shown that the

PRC2.2 complex (containing the accessory factors AEBP2 and

JARID2) is enriched in chromatin containing the H2AK119u1 modi-

fication [105]. Additionally, in vitro, this PRC2.2 complex is more

active on an H2AK119u1 nucleosome substrate compared with

unmodified nucleosomes [105]. A remaining question is whether

this PRC2 recruitment to H2AK119u1 is via a direct recruitment

mechanism, similar to CBX binding to H3K27me3, or by a change in

chromatin state or structure associated with the large H2AK119u1

modification.

In summary, the establishment of Polycomb repressive domains

may require these enzymes to read not only their own mark, for

example, EED-binding H3K27me3 or RYBP-binding H2AK119u1, but

also the marks placed by their partner complex. In this way,

H3K27me3 can establish or reinforce H2AK119u1 modifications,

and H2AK119u1 can establish or reinforce H3K27me3 deposition

(Fig 5). Which modification or Polycomb complex is initiating this

recruitment is still a matter of debate although recent work has

implicated the variant PRC1-KDM2B-containing complex in initia-

tion [106–108]. Polycomb target sites overlap with regions of dense

unmethylated DNA, CpG islands, and the CxxC domain of KDM2B

is able to recognize unmethylated CpGs, providing a plausible

mechanism for PRC1 recruitment. Further work is needed to fully

understand how both PRC1 and PRC2 complexes are initially

recruited to CpG islands. However, once this is established, the posi-

tive feedback mechanisms described above involving the histone

modifications that these enzymes place will be important to

maintain and reinforce their activity at these target sites.

H3K27me3 and H3K9me2/3 crosstalk

Generally, methylation of H3K9 is associated with constitutive

heterochromatin formation and transcriptional silencing. Recently,

there has been some evidence that H3K9 methylation can crosstalk

with the Polycomb H3K27me3 modification to cooperate in gene

repression or as mutually exclusive pathways present at constitutive

heterochromatin.

The heterodimeric complex of G9a and GLP catalyzes H3K9me1

and H3K9me2 modifications [109], which are mainly associated with

transcriptional silencing but also occur in euchromatic regions [110].

Both proteins contain ankyrin repeat domains that can bind to

H3K9me1/2 modifications, allowing the enzymes to read their own

marks and therefore allow spreading of the H3K9me2 modification

EMBO reports Vol 16 |No11 | 2015 ª 2015 The Authors

EMBO reports The interplay of histone modifications Tianyi Zhang et al

1472

[111]. The enzyme SETDB1 can place both H3K9me2/me3 and has

roles in repression of transposons, in gene silencing and at pericen-

tric heterochromain [112–115]. The SUV3-9H1/H2 enzymes deposit

H3K9me2 and H3K9me3 modifications and contain a chromo-

domain which can recognize these marks [116]. A major site of the

H3K9me3 modification is at pericentric heterochromatin, where there

are self-reinforcing feedback loops involving the chromodomain-

containing protein HP1 which can bind to H3K9me3, and recruit de

novo DNA methyltransferases (DNMT3A/B) [117,118]. The resulting

DNA methylation can be recognized by MECP2, a protein containing

a MBD (methyl binding domain) which can also bind to and recruit

SUV3-9 enzymes to pericentric heterochromain [119] (Fig 6A). Inter-

estingly at mitosis, H3 is phosophorylated by the kinase Aurora B at

H3S10, and this modification next to the H3K9me3 mark causes HP1

to be displaced from the pericentric heterochromatin during this

phase of the cell cycle [120].

Several reports have demonstrated that H3K9me3 and H3K27me3

modifications are mutually exclusive [87,103,121,122]. In differenti-

ated cells, H3K27me3 BLOCS, which form over silent gene loci, are

mutually exclusive with H3K9me3 domains over features such as

transposons [87]. In SUV3-9H1/H2 knockout cells, there is a loss of

H3K9me3 at the pericentric heterochromatin, and a subsequent gain

of H3K27me3 [103,121], suggesting that not only can these two

marks compensate for each other, but that normally H3K9me3

prevents H3K27me3 establishment. A recent study isolating proteins

associated with pericentric heterochromatin has shown that a

chromatin-associated protein, BEND3, is recruited to pericentric

chromatin in the absence of H3K9me3 (or DNA methylation) and is

important for recruiting H3K27me3 [122]. Lack of DNA methylation

can also cause H3K27me3 to be recruited to pericentric hetero-

chromatin, but in this case H3K9me3 is still present and forms mutu-

ally exclusive domains with H3K27me3, despite both modifications

now being enriched at DAPI-dense pericentric regions [103]. Recruit-

ment of H3K27me3 to pericentric heterochromatin has also been

shown to occur during early mouse development. In the one-cell

stage embryo, H3K27me3 can be visualized specifically on the male

pericentric heterochromatin, but not the female heterochromatin,

which contains H3K9me3 [123]. In this system, it has recently

been shown that it is not the presence of H3K9me3 on the mater-

nal pericentric heterochromatin itself that prevents H3K27me3

recruitment, but rather the presence of HP1b which binds to

H3K9me3 [124].

Despite reports that H3K27me3 and H3K9me3 are mutually

exclusive, a number of ChIP-sequencing studies in ES cells [115],

extra-embryonic lineages [125], and differentiated cells [126] have

shown that both H3K9me2 and H3K9me3 modifications can coexist

with the PRC2 modification H3K27me3 at developmentally

repressed genes. Given that both marks are associated with gene

repression, it has been suggested that they may cooperate with each

other. A mass spectrometry study, in which H3K27me3-containing

nucleosomes were purified from HeLa cells, showed that H3K9me2

modifications, and to a lesser extent H3K9me3, were also present

with H3K27me3 [127]. Large-scale proteomic screens have identi-

fied several Polycomb proteins as readers of H3K9me3 modifications

[31,69]. However, the authors suggest that this may be due to the

affinity of CBX proteins to H3K27me3 and the high degree of

sequence identity surrounding H3K9 and H3K27 (T

ARKST and

ARKSA, respectively). In vitro there is no difference in PRC2 activ-

ity on nucleosomes that contain H3K9 methylation compared to WT

nucleosomes [76]. However, a recent paper has found a direct inter-

action of PRC2 with the G9a/GLP complex and that G9a enzymatic

activity (H3K9me2) modulates PRC2 genomic recruitment [128]. In

addition, studies have reported that PRC2 is necessary for the bind-

ing of HP1 to chromatin [129–131]. Both H3K27me3 and H3K9me2

modifications have been shown to be present on the inactive X

chromosome where the two modifications play complementary

roles [132]. Here, the molecular mechanisms of crosstalk have been

elucidated by the discovery of CDYL, a protein that can bind both

H3K9me2 and H3K27me3. CDYL can interact with G9a to propagate

the H3K9me2 modification [133]. Interestingly, the loss of PRC2,

and subsequent loss of H3K27me3, reduces the amount of H3K9me2

present on the Xi, suggesting that CDYL is a link between these two

enzymatic activities allowing the combinatorial reading and writing

of both modifications (Fig 6B).

RbAP48

RYBP

RING1B

PCGF

PRC1

JARID2

SUZ12

EZH2

EED

AEBP2

PRC2.2

PCGF2/4

RING1B

CBX

RbAP48

SUZ12

EZH2

EED

PRC2

PRC1

Unmethylated

CpG site

H2AK119u1 H3K27me3

TSS

Figure 5. Crosstalk between the Polycomb complexes PRC1 and PRC2.

PRC2 reinforces its own mark through binding of EED to H3K27me3. PRC1 may also reinforce its own mark through binding of RYBP to H2AK119u1. Establishment of PRC1 can

be reinforced by the presence of PRC2, through recognition of H3K27me3 by the CBX subunit of PRC1. PRC2 establishment can also be reinforced by PRC1 through the

recognition of H2AK119u1 by the JARID2/AEBP2 PRC2.2 complex.

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Tianyi Zhang et al The interplay of histone modifications EMBO reports

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The mutually exclusive distribution of H3K27me3 and H3K9me3

described above has predominantly been shown in studies of consti-

tutive heterochromatin loci, and in all cases, this clear separation of

domains has been seen for trimethylation marks. There are also

many examples in which the H3K9me2 modification, and in some

cases H3K9me3, can act in concert with H3K27me3, suggesting posi-

tive crosstalk between these two mechanisms of heterochromatin

formation. In the best-documented examples, this positive crosstalk

appears to be between H3K27me3 and H3K9me2. Crosstalk may

also vary in different cell types or different differentiation states, in

which chromatin structures or the balance of different enzymes may

be altered. What is clear is that at least in some circumstances

through, for example, CDYL, HP1, or DNA methylation, both the

Polycomb system and the H3K9 methylation systems are able to

read the chromatin state placed by each other and to write their

own modifications accordingly.

As discussed above, both H3K27me3 and H3K9me3 repressive

marks can exist as extensive domains within cells. These domains

can spread through positive feedback of the writers and their ability

to recognize and propagate their marks. Given this, the establish-

ment of boundaries is critical to isolate heterochromatin from

euchromatin domains. These boundary elements have been found

in multiple eukaryotic organisms, ranging from yeast to human

(recently reviewed in [134]). Boundaries can be formed by equilib-

rium between heterochromatin-promoting factors (remodelers, Poly-

comb proteins, H3K9me3 machinery) and euchromatin-promoting

factors (remodelers, transcriptional machinery, Trithorax proteins).

Such boundaries could vary in position and this concept forms the

basis of position effect variegation (PEV) in which the spread of

heterochromatin, for example, domains of H3K9me3, results in the

stochastic silencing of a neighboring gene (reviewed in [135]). Addi-

tionally, cis-regulatory elements and the binding of insulator

proteins such as CTCF can also determine boundaries. For instance,

H3K27me3 domain boundaries within the Hox gene cluster are

bound by CTCF; deletion of these CTCF-binding sites results in the

infringement of Pol II and H3K4me3 into adjacent heterochromatin

territories and disruption of Hox gene silencing [136].

Interplay between repressive and active

chromatin modifications

The main theme that emerges from the data we have described is

that chromatin writers, associated with either active or repressed

states, are positively regulated by their own marks or marks associ-

ated with the same transcriptional state. However, there are clear

data that show these chromatin writers can also be negatively influ-

enced by marks associated with the opposite transcriptional state.

These negative feedback loops reinforce the maintenance of distinct

chromatin states, and may play an important role for switching of

gene expression during differentiation and development by creating

and reinforcing a bistable state.

Historically, the best-characterized example of an interplay

between chromatin complexes in regulating gene expression is the

antagonism between Polycomb and Trithorax complexes. Drosophila

genetics first established Polycomb and Trithorax proteins as two

groups having opposing function on Hox gene expression, and sub-

sequently on the regulation of many important developmental

genes. Histone crosstalk is important in this interplay, and some of

the molecular mechanisms that govern this mutual antagonism

between Polycomb and Trithorax proteins and marks have been

elucidated.

Schmitges et al [76] were the first to show a mechanism of direct

inhibition of PRC2 by the TrxG modifications H3K4me2/3 and

H3K36me2/3. The catalytic activity of the PRC2 core complex was

greatly reduced on recombinant nucleosomes carrying trimethyl-

lysine analogs at H3K4 and H3K36 on the H3 tail. This study and

further work demonstrated inhibition of PRC2 activity when the

marks H3K4me2/3 and H3K36me2/3 exist in cis (on the same

histone tail) as the target H3K27. It has been suggested that the

allosteric inhibition of the catalytic subunit EZH2 occurs through

the recognition of the H3K4me2/3 and H3K36me2/3 marks by the

SUZ12 VEFS domain [76]. Inhibition of PRC2 by H3K36me2/3 is

consistent with mass spectrometry data of the histone H3 peptide

fragment K27-R40 isolated from total chromatin from mESCs and

transformed cell lines [73,77,137]. This shows that trimethylation

at H3K27 and H3K36 do not coexist on the same H3 tail or are

DNMT3A/B

HP1

A Positive feedback at repressive pericentric heterochromatin

CDYL

G9A

GLP

G9A

GLP

CDYL

PRC2

B Co-occupancy of H3K9me2 and H3K27me3 on

the inactive X chromosome

SUV3–9

MECP2

Methylated

CpG site

H3K9me2

H3K9me3

H3K27me3

Figure 6. Interplay between H3K9me3, DNA methylation, and

H3K27me3.

(A) At the pericentric heterochromatin, DNA methylation and H3K9me3

positively reinforce each other. HP1 binds H3K9me3 and recruits the de novo

DNA methyltransferases DNMT3A/B. MECP2 can bind methylated DNA and

recruit the H3K9me3 methyltransferase SUV3-9. (B) H3K27me3 and H3K9me2

coexist on the inactive X chromosome. CDYL may recruit G9a to the inactive

X chromosome through its ability to recognize H3K9me2 and H3K27me3.

CDYL may reinforce the prop agation of H3K9me2 at the Xi.

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EMBO reports The interplay of histone modifications Tianyi Zhang et al

1474

present at very low levels. Furthermore, removal of SETD2, the

only HMT capable of placing H3K36me3, leads to an increase

of H3K27me2 over bodies of active genes and reduces levels of

expression [83].

It is important to note that although PRC2 is inhibited in cis by

H3K4me2/3 and H3K36me3, PRC2 is active on nucleosomes harbor-

ing these modifications on only one of the two H3 tails, thereby

allowing the formation of asymmetrically modified nucleosomes

[127]. Such nucleosomes have been identified in vivo and may

represent the nucleosomes present at bivalent promoters in mESCs

(promoters that harbor both active and repressive marks, see later).

This negative feedback mechanism has also been shown to oper-

ate at a chromosomal level in C. elegans. Normally H3K36me3 and

H3K27me3 occupy mutually exclusive domains on the autosomes

[138]. However, removal of the H3K36me1/2 writer MES-4 in the

germ line results in a global loss of H3K36me3 leading to the redis-

tribution of H3K27me3 to exogenous sites at germ line-expressed

genes, which are normally modified by H3K36me3. This redistribu-

tion causes a titration of the H3K27me3 mark from its endogenous

sites, including the X chromosomes, and an inability to maintain

normal gene expression states [138–140].

As well as the inhibition of Polycomb activity by Trithorax

marks, Polycomb marks have also been shown to inhibit the activity

of some Trithorax proteins. The best-studied examples are the inhi-

bition of H3K36 methyltransferases by the PRC1 modification

H2AK119u1. Yuan et al [141] show that the catalytic domain of

H3K36 methyltransferases is inhibited by recombinant nucleosomes

containing H2AK119u1. Additionally, there is evidence that the PcG

PRC1 mark H2AK119u1 inhibits H3K4 methyltransferases MLL1 and

possibly MLL3. A study by Endoh et al shows that upon RING1A/B

knockout and subsequent depletion of H2AK119u1, H3K4me3 levels

at several PcG target genes increase [142]. Although not extensively

investigated, it is possible that H3K27me3 may also inhibit the depo-

sition of H3K4 methylation by the SETD1 and MLL3/4 complexes

[143].

These studies provide good evidence that PcG and TrxG marks

mutually inhibit the writers associated with the opposing group. It

was therefore unexpected when several groups showed that the

tudor domains of the PRC2-associated PCL1/2/3 proteins can

specifically bind H3K36me3 [144–147]. Structural and biochemical

analyses show that the tudor domain of PCL recognizes

H3K36me2/3 with high specificity; however, PCL co-localizes only

moderately with H3K36me3 in vivo by ChIP-sequencing [144,147].

One interpretation of this observation is that the role of this inter-

action may be important in the spreading of PRC2 and H3K27me3

to bodies of active genes, and perhaps during the switching of tran-

scriptional state by allowing the initial recruitment of PRC2 to

active genes. Consistent with the latter observation, it has been

shown that the H3K36 demethylase NO66 can be recruited by

PCL3, which would allow for the removal of H3K36me3 before the

subsequent acquisition of H3K27me3 [146].

H3K27me3 is associated with gene repression, while H3K27ac is

associated with gene activation and active enhancers. Since they act

on the same lysine residue, these marks are mutually exclusive, and

the switch between methylation and acetylation has been well

established. The removal of H3K27ac by the NURD complex facili-

tates the recruitment of PRC2 and accumulation of H3K27me3 at

promoters leading to gene repression [148]. This process occurs

during differentiation of ESCs, when CTBP2 in combination with NURD

initiates the silencing of genes that were originally active, through

H3K27 deacetylation, allowing deposition of H3K27me3 by PRC2[149].

Upon loss of H3K9 or DNA methylation, PRC2 accumulates at

the pericentric heterochromatin as discussed earlier. In this condi-

tion, BEND3 recruits NURD to the pericentric heterochromatin

[122], and thus, a similar mechanism of deacetylation of H3K27ac

could explain the subsequent PRC2 recruitment and accumulation

of H3K27me3 at these sites.

Activation of Polycomb-repressed genes requires a methylation

to acetylation switch at H3K27. The phosphorylation of H3S28 on

the residue neighboring H3K27 has been shown to mediate this

switch. H3S28p inhibits H3K27me3, allowing an accumulation of

H3K27 acetylation [150,151]. Similarly, the loss of PRC2 subunit

SUZ12 and H3K27me3 leads to the accumulation of H3K27ac at PcG

target genes [84]. It has been suggested that one role of H3K27me3

is to exclude the HATs p300 and CBP, preventing accumulation of

H3K27ac at enhancers that is important for gene activation [152].

H3K27me2 has been suggested to play a similar role to prevent the

firing of non-cell-type-specific enhancers. This idea is supported by

the increase of H3K27ac at these enhancers when H3K27

methylation is lost [83].

Discussion

It is clear that specific histone modifications are associated with the

transcriptional state. For many modifications, it is not well

established whether they directly influence transcription or their

placement is simply a consequence of the transcriptional state

present at a particular gene. There are reports that acetylation can

directly alter chromatin structure to a more accessible state allowing

the recruitment of transcription factors and the transcription

machinery. In addition, ubiquitylation of H2A has been shown to

inhibit the elongating form of Pol II, suggesting direct effects on the

transcriptional state. Conversely, there is substantial evidence to

support the idea that transcription factors determine and initiate

gene expression, and writers recognize this state and aid in the

maintenance of this state through multiple feedback mechanisms.

One such mechanism is the recruitment of the H3K4 HMT SETD1

and the H3K36 HMT SETD2 by Pol II itself to deposit histone modifi-

cations across the promoter and gene body. The emerging idea that

PcG and TrxG can sample CpG islands genome wide and establish

domains of repression or activation depending on the transcriptional

state at the target promoter also supports the idea that the transcrip-

tional state defines the chromatin modification landscape. This

mechanism requires extensive positive and negative crosstalk

between these modifications that we have discussed.

Many writers of chromatin modifications are positively regulated

by the marks that they place, as well as other marks associated with

the same transcriptional state, contributing to the reinforcement of

gene expression or silencing. This mechanism could also account

for the spreading of marks such as the repressive modifications

H3K9me3 and H3K27me3 over large domains in differentiated cells.

These reinforcing mechanisms may also play a role in cellular

memory by faithful propagation of the histone modifications that

allow gene expression profiles to be maintained epigenetically

through cell division. There is evidence to suggest that some writers

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Tianyi Zhang et al The interplay of histone modifications EMBO reports

1475

remain associated with chromatin during DNA replication, but the

exact molecular mechanisms of this epigenetic memory have yet to

be fully elucidated.

Negative histone crosstalk plays an equally important role in

dictating distinct chromatin environments. Writers, especially in the

case of PcG and TrxG proteins, are often negatively regulated by

marks associated with the opposing transcriptional state. As we have

seen in the case of H3K27 methylation and acetylation, negative

crosstalk is also involved in the switching of gene expression states.

It is known that in certain circumstances, marks associated with

positive and negative transcription can coexist. In ESCs, bivalent

promoters contain both H3K27me3 and H3K4me3, albeit on different

histone tails of the same nucleosome, and may represent a chro-

matin profile amenable to switching between transcriptional states.

Genes coding for master transcription factors often have bivalent

promoters in ESCs, and their expression is dynamically regulated

through development.

The ability of writers to read and place histone modifications is

important for the maintenance and regulation of specific transcrip-

tional states throughout development. This is evident by the fact

that mice deficient in chromatin-modifying enzymes display severe

developmental defects. Several of these enzymes have been known

to be involved in various genetic disorders such as Sotos syndrome

and Wolf–Hirschhorn syndrome related to translocation of several

members of the NSD family of H3K36 methyltransferases [153–155],

and cancers related to translocation or mutation of MLL, NSD2,

PRC2, SETD2, and countless others [156–158]. Most recently, an

H3K27M mutation in H3.3 found in pediatric glioma cancer has

been shown to deplete levels of H3K27me3 globally potentially

through a dominant negative mechanism [159].

In conclusion, the ability of chromatin writers to read preexisting

histone modifications contributes in two major ways. First, it allows

the maintenance of distinct and robust transcriptional states, which

could potentially be propagated through cell division, and therefore

act as epigenetically inherited features. Second, crosstalk between

modifications and enzymes of opposing transcriptional states can

allow the establishment of bistable switches that allow the dynamic

regulation of gene expression states. In the future, we hope to

understand the extent to which histone crosstalk plays a role in

defining the epigenetic landscape of a cell (Sidebar A), and address

the role of histone modifications and the crosstalk between them

during the processes of development and disease.

Acknowledgements

We thank David Brown, Vincenzo Di Cerbo, Benoit Moindrot, and Andrew

Bassett for critical reading of the manuscript. Work in the Brockdorff labora-

tory is funded by grants from the European Research Council (340081) and

Wellcome Trust (103768).

Conflict of interest

The authors declare that they have no conflict of interest.

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Sidebar A: In need of answers

i) How do chromatin modifications regulate gene transcription?

Although there is a clear correlation between chromatin modifi-

cations and gene expression states, it will be important to establish

the role that modifications play in regulating transcription and

indeed whether they are a cause or an effect of transcription.

ii) What is the role of highly prevalent chromatin modifications such

as H3K36me2 or H3K27me2 that mark up to 50%ofH3 in mESCs

and differentiated tissues [ 84,121 ,160]? Is it possible that these

marks are necessary to reduce noise, for example, by blocking inap-

propriate histone modifications?

iii) What is the role of histone variants? H3.3 and H2A.Z are enriched

over active genes and may have more specialized regulatory roles

compared to their more abundant canonical counterparts [161,162].

Histone readers and writers may be sensitive to the histone variant

status. For instance, the putative tumor suppressor ZMYND11 is an

H3.3 variant-specific reader of H3K36me3 [163], while the H3K27

methyltransferase in plants, ATRX5/6, is active on the canonical H3.1

but inhibited by H3.3 [164].

iv) How are epigenetic profiles established within a cell? What are the

relative contributions of direct targeting of histone-modifying activ-

ities and crosstalk between histone modifications or transcriptional

state? For example, are chromatin writers able to sample and read

the preexisting chromatin state to determine their activity or bind-

ing profiles, or are they directly recruited to their sites of action by

sequence-specific DNA-binding factors?

v) To what extent are epigenetic modifications maintained through

cell division, and do self-reinforcing feedback loops provide a model

for the mechanism of such inheritance? Unlike writers and readers,

the genomic location of histone modifications can be easily trans-

mitted through both mitosis and meiosis because they are an inte-

gral part of the packaging of DNA. However, self-reinforcing loops

might become essential after replication to overcome the dilution

of old, modified nucleosomes with new nucleosomes and maintain

an epigenetic code.

vi) Do the reciprocal feedback loops between positively and negatively

acting histone marks provide the basis for a bistable switch, in

which each state is positively reinforced and stable once the initial

decision has been made? In order to properly generate and validate

such mathematical models, it will be critical to obtain quantitative

data on the kinetics and dynamics of the catalytic and binding

processes that are involved. This will involve experiments such as

FRAP and in vitro binding and enzymatic assays, and importantly

determining the changes that occur when the system has been

perturbed.

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properly cited.

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Chromatin profiling and state predictions reveal insights into epigenetic regulation during early porcine development

Article

Full-text available

May 2024

Background Given their physiological similarities to humans, pigs are increasingly used as model organisms in human-oriented biomedical studies. Additionally, their value to animal agriculture across the globe has led to the development of numerous studies to investigate how to improve livestock welfare and production efficiency. As such, pigs are uniquely poised as compelling models that can yield findings with potential implications in both human and animal contexts. Despite this, many gaps remain in our knowledge about the foundational mechanisms that govern gene expression in swine across different developmental stages, particularly in early development. To address some of these gaps, we profiled the histone marks H3K4me3, H3K27ac, and H3K27me3 and the SWI/SNF central ATPase BRG1 in two porcine cell lines representing discrete early developmental time points and used the resulting information to construct predicted chromatin state maps for these cells. We combined this approach with analysis of publicly available RNA-seq data to examine the relationship between epigenetic status and gene expression in these cell types. Results In porcine fetal fibroblast (PFF) and trophectoderm cells (PTr2), we saw expected patterns of enrichment for each of the profiled epigenetic features relative to specific genomic regions. H3K4me3 was primarily enriched at and around global gene promoters, H3K27ac was enriched in promoter and intergenic regions, H3K27me3 had broad stretches of enrichment across the genome and narrower enrichment patterns in and around the promoter regions of some genes, and BRG1 primarily had detectable enrichment at and around promoter regions and in intergenic stretches, with many instances of H3K27ac co-enrichment. We used this information to perform genome-wide chromatin state predictions for 10 different states using ChromHMM. Using the predicted chromatin state maps, we identified a subset of genomic regions marked by broad H3K4me3 enrichment, and annotation of these regions revealed that they were highly associated with essential developmental processes and consisted largely of expressed genes. We then compared the identities of the genes marked by these regions to genes identified as cell-type-specific using transcriptome data and saw that a subset of broad H3K4me3-marked genes was also specifically expressed in either PFF or PTr2 cells. Conclusions These findings enhance our understanding of the epigenetic landscape present in early swine development and provide insight into how variabilities in chromatin state are linked to cell identity. Furthermore, this data captures foundational epigenetic details in two valuable porcine cell lines and contributes to the growing body of knowledge surrounding the epigenetic landscape in this species.

Analysis of singularly perturbed stochastic chemical reaction networks motivated by applications to epigenetic cell memory

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May 2024

Epigenetic cell memory, the inheritance of gene expression patterns across subsequent cell divisions, is a critical property of multi-cellular organisms. In recent work [10], a subset of the authors observed in a simulation study how the stochastic dynamics and time-scale differences between establishment and erasure processes in chromatin modifications (such as histone modifications and DNA methylation) can have a critical effect on epigenetic cell memory. In this paper, we provide a mathematical framework to rigorously validate and extend beyond these computational findings. Viewing our stochastic model of a chromatin modification circuit as a singularly perturbed, finite state, continuous time Markov chain, we extend beyond existing theory in order to characterize the leading coefficients in the series expansions of stationary distributions and mean first passage times. In particular, we characterize the limiting stationary distribution in terms of a reduced Markov chain, provide an algorithm to determine the orders of the poles of mean first passage times, and determine how changing erasure rates affects system behavior. The theoretical tools developed in this paper not only allow us to set a rigorous mathematical basis for the computational findings of our prior work, highlighting the effect of chromatin modification dynamics on epigenetic cell memory, but they can also be applied to other singularly perturbed Markov chains beyond the applications in this paper, especially those associated with chemical reaction networks.

SIRT3: A Potential Target of Different Types of Osteoporosis

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Mar 2024
CELL BIOCHEM BIOPHYS

Osteoporosis (OP) is a common age-related disease. OP is mainly a decrease in bone density and mass caused by the destruction of bone microstructure, which leads to an increase in bone fragility. SIRT3 is a mitochondrial deacetylase that plays critical roles in mitochondrial homeostasis, metabolic regulation, gene transcription, stress response, and gene stability. Studies have shown that the higher expression levels of SIRT3 are associated with decreased levels of oxidative stress in the body and may play important roles in the prevention of age-related diseases. SIRTs can enhance the osteogenic potential and osteoblastic activity of bone marrow mesenchymal stromal cells not only by enhancing PGC-1α, FOXO3, SOD2, and oxidative phosphorylation, but also by anti-aging and reducing mitochondrial autophagy. SIRT3 is able to upregulate antioxidant enzymes to exert an inhibitory effect on osteoclasts, however, it has been shown that the inflammatory cascade response can in turn increase SIRT3 and inhibit osteoclast differentiation through the AMPK-PGC-1β pathway. SIRT3 plays an important role in different types of osteoporosis by affecting osteoblasts, osteoclasts, and bone marrow mesenchymal cells. In this review, we discuss the classification and physiological functions of SIRTs, the effects of SIRT3 on OCs osteoblasts, and BMSCs, and the roles and mechanisms of SIRT3 in different types of OP, such as diabetic OP, glucocorticoid-induced OP, postmenopausal OP, and senile OP.

Epigenetic Switches in Retinal Homeostasis and Target for Drug Development

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INT J MOL SCI

Retinal homeostasis, a tightly regulated process maintaining the functional integrity of the retina, is vital for visual function. Emerging research has unveiled the critical role of epigenetic regulation in controlling gene expression patterns during retinal development, maintenance, and response to mutational loads and injuries. Epigenetic switches, including DNA methylation, histone modifications, and non-coding RNAs, play pivotal roles in orchestrating retinal gene expression and cellular responses through various intracellular, extracellular, and environmental modulators. This review compiles the current knowledge on epigenetic switches in retinal homeostasis, providing a deeper understanding of their impact on retinal structural integrity and function and using them as potential targets for therapeutic interventions.

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POULTRY SCI

Emerging Roles of Vitamin B12 in Aging and Inflammation

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INT J MOL SCI

Vitamin B12 (cobalamin) is an essential nutrient for humans and animals. Metabolically active forms of B12-methylcobalamin and 5-deoxyadenosylcobalamin are cofactors for the enzymes methionine synthase and mitochondrial methylmalonyl-CoA mutase. Malfunction of these enzymes due to a scarcity of vitamin B12 leads to disturbance of one-carbon metabolism and impaired mitochondrial function. A significant fraction of the population (up to 20%) is deficient in vitamin B12, with a higher rate of deficiency among elderly people. B12 deficiency is associated with numerous hallmarks of aging at the cellular and organismal levels. Cellular senescence is characterized by high levels of DNA damage by metabolic abnormalities, increased mitochondrial dysfunction, and disturbance of epigenetic regulation. B12 deficiency could be responsible for or play a crucial part in these disorders. In this review, we focus on a comprehensive analysis of molecular mechanisms through which vitamin B12 influences aging. We review new data about how deficiency in vitamin B12 may accelerate cellular aging. Despite indications that vitamin B12 has an important role in health and healthy aging, knowledge of the influence of vitamin B12 on aging is still limited and requires further research.

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Mar 2024

Histone H2B monoubiquitination (at Lys120 in humans) regulates transcription elongation and DNA repair. In humans, H2B monoubiquitination is catalyzed by the heterodimeric Bre1 complex composed of Bre1A/RNF20 and Bre1B/RNF40. The Bre1 proteins generally function as tumor suppressors, while in certain cancers, they facilitate cancer cell proliferation. To obtain structural insights of H2BK120 ubiquitination and its regulation, we report the cryo-electron microscopy structure of the human Bre1 complex bound to the nucleosome. The two RING domains of Bre1A and Bre1B recognize the acidic patch and the nucleosomal DNA phosphates around SHL 6.0–6.5, which are ideally located to recruit the E2 enzyme and ubiquitin for H2BK120-specific ubiquitination. Mutational experiments suggest that the two RING domains bind in two orientations and that ubiquitination occurs when Bre1A binds to the acidic patch. Our results provide insights into the H2BK120-specific ubiquitination by the Bre1 proteins and suggest that H2B monoubiquitination can be regulated by nuclesomal DNA flexibility.

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ATX1 and HUB1/2 promote recruitment of the transcription elongation factor VIP2 to modulate the floral transition in Arabidopsis

Article

Mar 2024
PLANT J

Histone 2B ubiquitination (H2Bub) and trimethylation of H3 at lysine 4 (H3K4me3) are associated with transcription activation. However, the function of these modifications in transcription in plants remains largely unknown. Here, we report that coordination of H2Bub and H3K4me3 deposition with the binding of the RNA polymerase‐associated factor VERNALIZATION INDEPENDENCE2 (VIP2) to FLOWERING LOCUS C ( FLC ) modulates flowering time in Arabidopsis . We found that RING domain protein HISTONE MONOUBIQUITINATION1 (HUB1) and HUB2 (we refer as HUB1/2), which are responsible for H2Bub, interact with ARABIDOPSIS TRITHORAX1 (ATX1), which is required for H3K4me3 deposition, to promote the transcription of FLC and repress the flowering time. The atx1‐2 hub1‐10 hub2‐2 triple mutant in FRIGIDIA ( FRI ) background displayed early flowering like FRI hub1‐10 hub2‐2 and overexpression of ATX1 failed to rescue the early flowering phenotype of hub1‐10 hub2‐2 . Mutations in HUB1 and HUB2 reduced the ATX1 enrichment at FLC , indicating that HUB1 and HUB2 are required for ATX1 recruitment and H3K4me3 deposition at FLC . We also found that the VIP2 directly binds to HUB1, HUB2, and ATX1 and that loss of VIP2 in FRI hub1‐10 hub2‐2 and FRI atx1‐2 plants resulted in early flowering like that observed in FRI vip2‐10 . Loss of function of HUB2 and ATX1 impaired VIP2 enrichment at FLC , and reduced the transcription initiation and elongation of FLC . In addition, mutations in VIP2 reduced HUB1 and ATX1 enrichment and H2Bub and H3K4me3 levels at FLC . Together, our findings revealed that HUB1/2, ATX1, and VIP2 coordinately modulate H2Bub and H3K4me3 deposition, FLC transcription, and flowering time.

Genome-wide methylation profiling of Peripheral T-cell lymphomas identifies TRIP13 as a critical driver of tumor proliferation and survival

Preprint

Full-text available

Feb 2024

Cytosine methylation of genomic DNA contributes to the regulation of gene expression and is involved in normal development including hematopoiesis in mammals. It is catalyzed by the family of DNA methyltransferases (DNMTs) that include DNMT1, DNMT3A, and DNMT3B. Peripheral T-cell lymphomas (PTCLs) represent a diverse group of aggressive mature T-cell malignancies accounting for approximately 10–15% of non-Hodgkin lymphoma cases in the US. PTCLs exhibit a broad spectrum of clinical, histological, and immunophenotypic features with poor prognosis and inadequately understood molecular pathobiology. To better understand the molecular landscape and identify candidate genes involved in disease maintenance, we used high-resolution Whole Genome Bisulfite Sequencing (WGBS) and RNA-seq to profile DNA methylation and gene expression of PTCLs and normal T-cells. We found that the methylation patterns in PTCLs are deregulated and heterogeneous but share 767 hypo- and 567 hypermethylated differentially methylated regions (DMRs) along with 231 genes up- and 91 genes downregulated in all samples suggesting a potential association with tumor development. We further identified 39 hypomethylated promoters associated with increased gene expression in the majority of PTCLs. This putative oncogenic signature included the TRIP13 (thyroid hormone receptor interactor 13) gene whose both genetic and pharmacologic inactivation, inhibited cellular growth of PTCL cell lines by inducing G2-M arrest accompanied by apoptosis suggesting that such an approach might be beneficial in human lymphoma treatment. Altogether we show that human PTCLs are characterized by a large number of recurrent methylation alterations, and demonstrated that TRIP13 is critical for PTCL maintenance in vitro.

KDM2B links the Polycomb Repressive Complex 1 (PRC1) to recognition of CpG islands

Article

Full-text available

Dec 2012
eLife

CpG islands (CGIs) are associated with most mammalian gene promoters. A subset of CGIs act as polycomb response elements (PREs) and are recognized by the polycomb silencing systems to regulate expression of genes involved in early development. How CGIs function mechanistically as nucleation sites for polycomb repressive complexes remains unknown. Here we discover that KDM2B (FBXL10) specifically recognizes non-methylated DNA in CGIs and recruits the polycomb repressive complex 1 (PRC1). This contributes to histone H2A lysine 119 ubiquitylation (H2AK119ub1) and gene repression. Unexpectedly, we also find that CGIs are occupied by low levels of PRC1 throughout the genome, suggesting that the KDM2B-PRC1 complex may sample CGI-associated genes for susceptibility to polycomb-mediated silencing. These observations demonstrate an unexpected and direct link between recognition of CGIs by KDM2B and targeting of the polycomb repressive system. This provides the basis for a new model describing the functionality of CGIs as mammalian PREs.DOI:http://dx.doi.org/10.7554/eLife.00205.001.

Transcription factor interaction with COMPASS-like complex regulates histone H3K4 trimethylation for specific gene expression in plants

Article

Full-text available

Feb 2015

Significance In metazoans, pausing of Pol II during early elongation is a widespread regulatory mechanism for transcription regulation. However, preinitiation complex (PIC) assembly is more important for transcription in plants. Chromatin remodeling and histone modifications are considered important for access of protein factors to the underlying DNA sequences. However, how histone modifications are specifically and timely generated at active promoters is less understood. COMPASS-like complex plays a critical role in PIC assembly and histone H3K4 trimethylation. We found that Arabidopsis transcription factors bZIP28/bZIP60 interact with COMPASS-like components both in vitro and in vivo. We present a general model on how histone H3K4 trimethylation is specifically formed during inducible gene expression by using the endoplasmic reticulum (ER) stress response system in Arabidopsis plants.

Acetylation of histone H3 at lysine 64 regulates nucleosome dynamics and facilitates transcription

Article

Mar 2014
eLife

Post-translational modifications of proteins have emerged as a major mechanism for regulating gene expression. However, our understanding of how histone modifications directly affect chromatin function remains limited. In this study, we investigate acetylation of histone H3 at lysine 64 (H3K64ac), a previously uncharacterized acetylation on the lateral surface of the histone octamer. We show that H3K64ac regulates nucleosome stability and facilitates nucleosome eviction and hence gene expression in vivo. In line with this, we demonstrate that H3K64ac is enriched in vivo at the transcriptional start sites of active genes and it defines transcriptionally active chromatin. Moreover, we find that the p300 co-activator acetylates H3K64, and consistent with a transcriptional activation function, H3K64ac opposes its repressive counterpart H3K64me3. Our findings reveal an important role for a histone modification within the nucleosome core as a regulator of chromatin function and they demonstrate that lateral surface modifications can define functionally opposing chromatin states. DOI: http://dx.doi.org/10.7554/eLife.01632.001.

CpG islands and the regulation of transcription

Article

Jan 2011

Deaton AM, Bird A

10.1101/gad.2037511

H3K27ME3 FORMS BLOCS OVER SILENT GENES AND INTERGENIC REGIONS AND SPECIFIES A HISTONE BANDING PATTERN ON A MOUSE AUTOSOMAL CHROMOSOME

Conference Paper

Jan 2009

Histone Variant H2A.Z Marks the 5′ Ends of Both Active and Inactive Genes in Euchromatin (DOI:10.1016/j.cell.2005.10.002)

Article

Jul 2008

Spectrum of NSD1 mutations in Sotos and Weaver syndromes

Article

Jun 2003
J MED GENET

M Rio

Sotos syndrome is an overgrowth syndrome characterised by pre- and postnatal overgrowth, macrocephaly, advanced bone age, and typical facial features. Weaver syndrome is a closely related condition characterised by a distinctive craniofacial appearance, advanced carpal maturation, widened distal long bones, and camptodactyly. Haploinsufficiency of the NSD1 gene has recently been reported as the major cause of Sotos syndrome while point mutations accounted for a minority of cases. We looked for NSD1 deletions or mutations in 39 patients with childhood overgrowth. The series included typical Sotos patients (23/39), Sotos-like patients (lacking one major criteria, 10/39), and Weaver patients (6/39). We identified NSD1 deletions (6/33) and intragenic mutations (16/33) in Sotos syndrome patients. We also identified NSD1 intragenic mutations in 3/6 Weaver patients. We conclude therefore that NSD1 mutations account for most cases of Sotos syndrome and a significant number of Weaver syndrome cases in our series. Interestingly, mental retardation was consistently more severe in patients with NSD1 deletions. Macrocephaly and facial gestalt but not overgrowth and advanced bone age were consistently observed in Sotos syndrome patients. We suggest therefore considering macrocephaly and facial gestalt as mandatory criteria for the diagnosis of Sotos syndrome and overgrowth and advanced bone age as minor criteria.

Ctbp2 Modulates NuRD-Mediated Deacetylation of H3K27 and Facilitates PRC2-Mediated H3K27me3 in Active Embryonic Stem Cell Genes during Exit from Pluripotency

Article

Apr 2015
STEM CELLS

For cells to exit from pluripotency and commit to a lineage, the circuitry of a core transcription factor (CTF) network must be extinguished in an orderly manner through epigenetic modifications. Yet, how this choreographed epigenetic remodeling at active ESC genes occurs during differentiation is poorly understood. In this study, we demonstrate that C-terminal binding protein 2 (Ctbp2) regulates nucleosome remodeling and deacetylation (NuRD)-mediated deacetylation of H3K27 and facilitates recruitment of polycomb repressive complex 2 (PRC2)-mediated H3K27me3 in active ESC genes for exit from pluripotency during differentiation. By genomewide analysis, we found that Ctbp2 resides in active ESC genes and co-occupies regions with ESC CTFs in undifferentiated ESCs. Further, ablation of Ctbp2 effects inappropriate gene silencing in ESCs by sustaining high levels of H3K27ac and impeding H3K27me3 in active ESC genes, thereby sustaining ESC maintenance during differentiation. Thus, Ctbp2 preoccupies regions in active genes with the NuRD complex in undifferentiated ESCs that are directed toward H3K27me3 by PRC2 to induce stable silencing, which is pivotal for natural lineage commitment. This article is protected by copyright. All rights reserved. © 2015 AlphaMed Press.

Cbx2 Targets PRC1 to Constitutive Heterochromatin in Mouse Zygotes in a Parent-of-Origin-Dependent Manner

Article

Mar 2015

Polycomb repressive complexes PRC1 and PRC2 regulate expression of genes involved in proliferation and development. In mouse early embryos, however, canonical PRC1 localizes to paternal pericentric heterochromatin (pat-PCH), where it represses transcription of major satellite repeats. In contrast, maternal PCH (mat-PCH) is enriched for H3 lysine 9 tri-methylation (H3K9me3) and Hp1β. How PRC1 is targeted to pat-PCH, yet excluded from mat-PCH, has remained elusive. Here, we identify a PRC1 targeting mechanism that relies on Cbx2 and Hp1β. Cbx2 directs catalytically active PRC1 to PCH via its chromodomain (CD(Cbx2)) and neighboring AT-hook (AT(Cbx2)) binding to H3K27me3 and AT-rich major satellites, respectively. CD(Cbx2) prevents AT(Cbx2) from interacting with DNA at PCH marked by H3K9me3 and Hp1β. Loss-of-function studies show that Hp1β and not H3K9me3 prevents PRC1 targeting to mat-PCH. Our findings indicate that CD(Cbx2) and AT(Cbx2) separated by a short linker function together to integrate H3K9me3/HP1 and H3K27me3 states. Copyright © 2015 Elsevier Inc. All rights reserved.

Transcription. CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation

Article

Feb 2015

Polycomb and Trithorax group proteins encode the epigenetic memory of cellular positional identity by establishing inheritable domains of repressive and active chromatin within the Hox clusters. Here we demonstrate that the CCCTC-binding factor (CTCF) functions to insulate these adjacent yet antagonistic chromatin domains during embryonic stem cell differentiation into cervical motor neurons. Deletion of CTCF binding sites within the Hox clusters results in the expansion of active chromatin into the repressive domain. CTCF functions as an insulator by organizing Hox clusters into spatially disjoint domains. Ablation of CTCF binding disrupts topological boundaries such that caudal Hox genes leave the repressed domain and become subject to transcriptional activation. Hence, CTCF is required to insulate facultative heterochromatin from impinging euchromatin to produce discrete positional identities. Copyright © 2015, American Association for the Advancement of Science.

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