Chromatin modification and disease

Chromatin modiﬁcation and disease

Colin A Johnson

“Physicians consider that when they have discov-

ered the cause of disease, they have also discovered

the method of treating it.” Cicero, Tusculan Dis-

putations, III.x.23.

In the last few years, the exciting realisation

in the ﬁeld of gene regulation is that transcrip-

tion factors can function by recruiting large,

multiprotein complexes which mediate several

types of chromatin modiﬁcation and remodel-

ling events that alter the structure of chroma-

tin. Chromatin structure changes include post-

translational modiﬁcations of histones, DNA

methylation, remodelling of the chromatin, and

the maintenance of a heterochromatic or

euchromatic state. Most of these events are

brought about by enzymatic mechanisms. In

general, the catalytic subunits are only one

component of the complexes, with the distribu-

tion and localisation of the structural changes

dependent on targeting components. Many of

the catalytic components (sometimes called

coactivators and corepressors) interact with the

activator and repressor proteins that mediate

the actual process of transcriptional regulation.

Transcriptional dysregulation can therefore

arise from mutations that cause the loss or per-

turbation of chromatin modiﬁcation or remod-

elling, which are now known to have an impor-

tant role in the pathogenesis of cancer and

other genetic diseases. Some of the proteins

that mediate these events are therefore novel

molecular targets for future treatments.

In eukaryotes, DNA is packaged by histone

proteins into nucleosomes, the fundamental

repeating structural unit of chromatin.

The

nucleosomal core particle consists of an

octomeric complex of core histones (two each

of H2A, H2B, H3, and H4) around which 147

bp of DNA is wrapped in 1.65 turns of a left

handed superhelix.

The minor and major

grooves of adjacent turns of the DNA superhe-

lix line up and form channels through which

the histone N-termini domains protrude from

the core. These regions are in the form of

“tails” that appear to lack secondary structure

and are subject to various enzyme catalysed,

post-translational modiﬁcations which aVect

their charge and can inﬂuence the degree of

chromatin compaction. The tightness with

which DNA is packaged into chromatin will

limit the binding and function of proteins that

mediate transcriptional regulation, and this will

therefore inﬂuence the transcriptional compe-

tence of any given gene in such a chromatin

environment.

Covalent post-translational acetylation and

deacetylation of speciﬁc lysine residues in the

histone N-termini is one of the most widely

studied chromatin modiﬁcations. In the past

four years there have been rapid advances in

identifying the enzymes and multiprotein com-

plexes that bring about histone acetylation (the

family of histone acetyltransferases or HAT

coactivators) and deacetylation (the histone

deacetylases or HDAC corepressors). This

review will focus on some of the clinical aspects

of this recent work on acetylation and the inti-

mate connection that it is now known to have

with the methylation of cytosine residues in

DNA. A third type of chromatin remodelling is

the direct physical repositioning or disruption

of nucleosomes mediated by a family of DNA

dependent ATPases. The connection between

this latter type of remodelling and either

histone acetylation or DNA methylation is

complicated, but progress is being made. For

example, the NuRD multiprotein complex (see

below, ﬁg 1C) contains histone deacetylase and

chromatin remodelling activities, as well as the

methyl DNA binding protein MBD3, which

suggests that a profound interplay between

these modiﬁcations is required during gene

regulation. Therefore, it is probable that a par-

ticular pathogenesis may be caused by defects

in more than one type of chromatin modiﬁca-

tion. Relevant pathologies and syndromes are

discussed in following sections and are summa-

rised in table 1.

Histone acetylation, protein acetylation,

and gene regulation

HISTONE DEACETYLASES AND COREPRESSOR

COMPLEXES

Deacetylation of histones is, in general, associ-

ated with repression of gene transcription, pre-

sumably because the highly positively charged

N-terminal tails of the core histones can now

interact with DNA on the nucleosome surface

and in the linker DNA.

In addition, the posi-

tively charged lysines in the H4 tail may inter-

act with the negative face of an H2A-H2B

dimer from a neighbouring nucleosome,

and

hence bring about further compaction of the

chromatin. Deacetylation is brought about by

the action of the histone deacetylases

(HDACs), which would therefore enhance

histone-histone interactions by maintaining the

positively charged (unmodiﬁed) state of lysines

in the histone tails. HDACs are now known to

be corepressor components of many multipro-

tein complexes that modify and remodel chro-

matin.

Targeting of complexes containing HDAC1

and HDAC2 is achieved by the interaction of

the repressor proteins Sin3A, Sin3B,

and

other Sin3 associated proteins (SAPs)

in a

large multiprotein complex that comprises at

least seven subunits

(ﬁg 1A). The mammalian

Sin3 complex mediates repression for an

extensive and ever growing list of transcrip-

tional regulator proteins,

81011

which include

DNA binding components such as the Mad/

J Med Genet 2000;37:905–915 905

Chromatin and Gene

Expression Group,

Department of

Anatomy, University of

Birmingham,

Birmingham B15 2TT,

Correspondence to:

Dr Johnson,

[email protected]

www.jmedgenet.com

Max heterodimer and nuclear hormone recep-

tors (see below, ﬁg 1B). The members of the

Mad/Mxi1 family

are able to replace Myc in

the Myc/Max heterodimer, and can therefore

repress transcription at promoters with Myc

consensus DNA binding sites. Mutations in the

Sin3 interaction domains (SIDs) of Mad/Mxi1

can abolish binding of the corepressors to the

Sin3 proteins and hence HDACs, and this cor-

relates with the abolition of transcriptional

repression and anti-oncogenic activity.

addition, transfection studies have shown that

HDACs and Mad cooperate to repress cell

proliferation.

In a similar mechanism, the

transcriptional corepressor proteins N-CoR

(nuclear hormone receptor corepressor) and

SMRT (silencing mediator of retinoid and thy-

roid hormone receptor) target deacetylase

activity to non-liganded thyroid hormone and

retinoic acid nuclear receptors

15–17

and to

antagonist bound oestrogen and progesterone

receptors.

As discussed below, the presence of

receptor ligands, for example, retinoic acid,

appears to induce an exchange of the corepres-

sor complexes containing HDACs for those

with coactivator functions that contain histone

acetyltransferase (HAT) activities

18 19

(compare

ﬁgs 1B and 2A).

Histone deacetylases are also recruited by

the retinoblastoma protein pRb

(ﬁg 1A), the

product of a tumour suppressor gene, and an

inhibitor of cell proliferation.

The inhibitory

action of pRb is the result, in part, of its ability

to bind to the E2F family of DNA binding

transcription factors, which results in the

sequestration of E2F and repression of E2F

target genes during the G1 phase of the cell

cycle.

The interaction is mediated by the A/B

pocket domain in pRb, and it is no coincidence

that the great majority of Rb mutations in

human tumours are located in this domain.

The pocket domain can also interact with a

variety of other cellular proteins, including viral

transforming oncoproteins (such as E1A from

adenovirus and SV40 large T

) and histone

deacetylases (HDAC1 and HDAC2) that share

the common LXCXE motif which allows

interactions with pocket proteins. However, the

interaction between Rb and either HDACs or

viral oncoproteins appears to be competitive,

and the Rb-HDAC1 interaction may be one of

the intracellular targets for these transforming

proteins. The Rb-HDAC interaction has been

analysed by transient transfection experiments,

which show that Rb and the HDACs cooperate

in repressing an E2F1-driven promoter,

24–26

and the repression exerted by Rb and other

pocket proteins during the G1 phase of the cell

cycle

can be reversed by treatment with

chemical inhibitors of HDACs (see below).

The HDAC inhibitors can also upregulate

some of the E2F target genes.

It is probable

that aberrant targeting of deacetylase activity

and incorrect chromatin remodelling are one

step in the process of transformation and

implies that these processes have a fundamen-

tal role in the suppression of carcinogenesis. In

Figure 1 Schematic representation of multiprotein complexes that mediate chromatin modiﬁcations. Some of the known

components of the complexes are shown on the left, with arrows indicating additional interactions with other proteins on the

right (refer to main text for details). Histone deacetylases (HDAC) are shown in purple, components of the DNA

methylation system in yellow, and ATPase/helicase that mediates chromatin remodelling in green. Other corepressor

accessory proteins in the complexes (panels A-C) are shown in grey. (A) Components of the HDAC/Sin complex and

known interacting proteins. (B) Additional interactions of the HDAC/Sin3 complex, mediated by the corepressors

NcoR/SMRT, with unliganded nuclear receptors and leukaemogenic fusion proteins. Note that these interactions occur at

low concentrations, or in the absence of the receptor ligand. (C) Components and interactions of the NuRD complex.

Figure 2 Schematic representation of interactions mediated by histone acetyltransferases

(HATs). Unbroken arrows indicate known interactions of HATs with the proteins listed on

the right (refer to main text for details). Histone acetyltransferases are shown in red, the

SWI/SNF complex that mediates chromatin remodelling in green, and other accessory

proteins in grey. (A) Interactions of three human histone acetyltransferases. The p300/CBP

coactivator can interact with nuclear receptors at physiological concentrations of the receptor

ligand, with other transcription factors and with the histone acetyltransferase PCAF. PCAF

can also interact with the coactivator ACTR and transcription factors. (B) Putative

interactions of the human and/or yeast Hat1 protein with both RbAp48 and, through the

bromodomain, with histones H3 and H4. Histone H4 can also interact directly with both

RbAp46 and RbAp48. The bromodomain may also mediate an interaction with the yeast

SWI/SNF complex (broken arrow).

906 Johnson

www.jmedgenet.com

the case of pRb, histone modiﬁcation is impli-

cated in the induction of cell cycle arrest which

may explain why the Rb gene is mutated in

almost all cancer cells.

In addition to E2F and HDACs, Rb has also

been shown to interact with other proteins that

regulate chromatin modiﬁcations: TAFII 250

is a transcription factor that has intrinsic

histone acetyltransferase (HAT) activity

;

BRG1 is a transcriptional activator and

ATPase/helicase that is a component of the

mammalian SWI/SNF chromatin remodelling

complex

; and Rb associated protein 48

(RbAp48) is a component of the chromatin

assembly factor CAF-1,

interacts with

HDAC1,

and is a component of the human

Hat1 acetyltransferase

(ﬁg 2B). RbAp48, and

the related protein RbAp46, can bind directly

to an alpha helix in histone H4,

so it is likely

that these proteins mediate core histone

binding for the Sin3-HDAC complex, CAF-1

and Hat1.

A similar network of interactions that

regulate cell proliferation is also seen with the

HATs (see below) and for histone deacetylase

complexes other than the Sin3-HDAC com-

plex. For example, a novel multiprotein com-

plex has been isolated recently that contains

both nucleosome remodelling and histone

deacetylase activities (hence the NuRD com-

plex,

ﬁg 1C). The NuRD complex contains,

in addition to HDAC1 and HDAC2, the

dermatomyositis speciﬁc autoantigen Mi-2â

that contains an ATPase/helicase domain of the

SWI/SNF type (see below). In addition, the

NuRD complex contains a protein, MTA2,

that is homologous to the metastasis associated

protein MTA1,

which is expressed at high

levels in several human cancer cell lines and

tissues. The NuRD complex can also be

recruited to DNA by speciﬁc DNA binding

factors, in a similar mechanism to the targeting

of the Sin3-HDAC complex. For example, two

determinants of the lymphoid lineage in T

cells, Ikaros and Aiolos, have been shown to

associate with the NuRD complex.

However,

Ikaros and Aiolos can also function as tran-

scriptional repressors during lymphocyte de-

velopment by recruiting the Sin3-HDAC com-

plex.

HISTONE DEACETYLASES AND LEUKAEMOGENIC

FUSION PROTEINS

The interaction of HDACs with chimeric

mutant proteins of the retinoic acid receptor is

one of the mechanisms that underlies the

molecular pathogenesis of acute promyelocytic

leukaemia (APL),

39 40

one of the best character-

ised forms of acute myeloid leukaemia

(AML).

In this disease, chromosomal translo-

cations create fusion proteins of retinoic acid

receptor-á and either PML (for promyelocytic

leukaemia) or, in rare cases, PLZF (for promye-

locytic leukaemia zinc ﬁnger).

42 43

In the case of

PML/RAR-á forms of APL, the fusion protein

retains the ability of the wild type nuclear

receptor to recruit the N-CoR/HDAC complex

(ﬁg 1B) and to block haematopoietic diVeren-

tiation. However, physiological concentrations

of retinoic acid dissociate the corepressor com-

plex from wild type RAR-á (see above and ﬁg

2A), so that it can therefore function as a tran-

scriptional activator. In contrast, the fusion

protein retains the ability to bind to the

corepressor complex under these conditions,

thereby constitutively repressing RAR-á target

genes. Treatment with higher, pharmacological

concentrations of the hormone overcomes this

interaction and converts the PML/RAR-á

fusion protein back into an activator. As a con-

sequence, cell proliferation is inhibited and

neutrophilic diVerentiation of neoplastic cells

is induced, which is the basis of diVerentiation

therapy of APL.

One of the target genes of

PML/RAR-á during RA induced diVerentia-

tion of APL cells is that encoding the

cyclin-CDK inhibitor p21

(also known as

WAF1 and CIP1), which exerts a G1 cell cycle

arrest in response to a variety of stimuli.

In contrast, cells expressing the PLZF/

RAR-á fusion are not sensitive to RA induced

diVerentiation, and patients with this type of

Table 1 Summary of human diseases in which a defect in chromatin modiﬁcation and remodelling is believed to contribute to a clinical pathology. The

defects can arise from mutations in gene products, or by the aberrant recruitment of other proteins that are components of multiprotein complexes. Refer to

the main text for details

Type of chromatin

modiﬁcation

Enzymes and other proteins that

mediate the chromatin modiﬁcation

Mutated genes

implicated as a

cause of a

pathogenesis

Relevant proteins that

interact with the

enzymes etc

Clinical conditions and pathologies that

correlate with a listed mutation or

protein-protein interaction References

Histone acetylation Histone deacetylases

HDAC1/2 Rb pRb Neoplasia 24, 25, 26

p53 p53 Neoplasia 92

— Mi-2â Dermatomyositis and neoplasia 33, 34, 122

— ÌÔÁ2 Metastasis 35

RARá Fusion proteins Acute promyelocytic leukaemia 39, 40, 47, 48

Histone acetyltransferases

p300/CBP — E1A Transformation 82

MLL and MOZ Fusion proteins Acute myeloid leukaemia 94, 95

CBP — Rubinstein-Taybi syndrome 96

TAF II 250 — pRb Loss of tumour suppression? 28

Histone phosphorylation Rsk-2 kinase RSK2 Not known CoYn-Lowry syndrome 103

DNA methylation DNA methyltransferase DNMT3B Not known ICF syndrome 113, 114

MethylCpG binding proteins MECP2 HDAC/Sin3 Rett syndrome 125

MBD4 Not known Microsatellite unstable colon cancers 124

Chromatin remodelling ATPase/helicases

Mi-2â — NuRD complex Dermatomyositis and neoplasia 130, 131

ATRX ATRX Not known ATRX syndrome 134

BRG-1/hBRM — pRb Neoplasia 28

hSWI5/INI1 subunit of

SWI2/SNF2 complex

hSWI5/INI1 SWI2/SNF2 complex Malignant rhabdoid tumours,

chronic myeloid leukaemias

141, 142

Chromatin modiﬁcation and disease 907

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APL respond poorly, if at all, to pharmacologi-

cal doses of the hormone.

The interaction

between PLZF/RAR-á and the corepressor

complex is resistant to retinoic acid because the

wild type PLZF protein can itself interact

directly with corepressors such as SMRT and

N-CoR.

39 40 47

This interaction is mediated by

the broad complex/tramtrack/bric a brac/

poxviruses and zinc ﬁnger (BTB/POZ) repres-

sion domain from PLZF,

which also allows

SMRT to interact with another BTB/POZ

oncoprotein, LAZ3/BCL6.

In contrast, other

transcriptional repressors that also contain the

BTB/POZ domain, such as the product of the

putative tumour suppressor gene HIC-1 (for

hypermethylated in cancer), do not recruit the

SMRT/N-CoR-histone deacetylase complex as

a general mechanism to repress transcription.

However, since HDAC inhibitors restore the

retinoid responses of RA resistant APL cell

lines,

clinical and cytogenetic remission of a

PLZF/RAR-á type of APL has been achieved

with a combination therapy of retinoic acid and

phenylbutyrate, an HDAC inhibitor.

Such

combination therapies may therefore be appli-

cable to other types of neoplastic diseases that

are associated with oncogenic repression of

gene transcription by histone deacetylases.

HISTONE DEACETYLASE INHIBITORS AND CANCER

CHEMOTHERAPY

Inhibitors of HDACs have also received

considerable attention as possible therapeutic

agents to induce growth arrest and terminal

diVerentiation in malignant cells and therefore

prevent the progression of cancers. HDAC

inhibitors can be classiﬁed on the basis of

structure and mode of inhibition. Reversible

inhibitors include n-butyric acid and other

related short chain fatty acids,

52 53

the microbial

antibiotic trichostatin A (TSA

), and hybrid

polar compounds such as suberoylanilide

hydroxamic acid (SAHA

). The carboxylic

and hydroxamic acid groups in this set of com-

pounds are likely to be speciﬁc ligands of a

catalytic zinc ion at the active site of HDAC

55 56

because a similar zinc binding site exists in a

prokaryotic homologue of the enzyme.

Irre-

versible inhibitors include trapoxin

58 59

and

trapoxin related natural products, such as

chlamydocin and HC toxin.

These com-

pounds are tetrapartite cyclic peptides with

2-amino-9,10-epoxy-8-oxodecanoic acid

(Aeo) as one residue. Aeo contains an epoxyke-

tone group that is isosteric with N-acetyl lysine

and is presumed to inhibit HDACs by binding

covalently and irreversibly to nucleophilic

groups in the active site of the enzymes. TSA,

trapoxin, and depudicin (a fungal metabolite

that resembles Aeo

) have all been shown to

revert the morphologies of oncogene trans-

formed cells and cells derived from tumours to

those with a normal cytoskeletal architec-

ture.

58 61 62

TSA and SAHA are also potent

inducers of transformed cell diVerentiation and

apoptosis.

55 63

A novel antibiotic and HDAC

inhibitor, FR901228,

can strongly inhibit

proliferation of tumour cells in vitro by arrest-

ing cell cycle transition at G1 and G2/M

phases, a property that is common between the

unrelated types of HDAC inhibitors.

For this

reason, HDAC inhibitors are being assessed as

therapeutic agents for cancer chemotherapy

and as adjuncts to established agents such as

retinoic acid (see above). In view of this, it is

interesting to note that n-butyric acid causes

growth inhibition, diVerentiation, and apopto-

sis in colon cancer derived cell lines.

Butyrate

is a natural fermentation product of certain

dietary ﬁbres by anaerobic bacteria in the

lumen of the colon. The production of this

short chain fatty acid may be the key factor that

allows dietary ﬁbre, for example ﬁbre from

wheat, to protect against colon carcinogenesis.

The molecular mechanism that underlies this

protection is presumed to be inhibition of his-

tone deacetylase activity and induction of

histone hyperacetylation by butyrate.

Archer

et al

showed that butyrate induces expression

of the G1 cell cycle inhibitor p21 gene (see

above), presumably in response to hyper-

acetylation of the p21 promoter, which induces

growth arrest in colonic cancer cells. Trapoxin

has also been shown to increase p21 expression

in human tumour cells, with an increase of his-

tone H3 acetylation at the p21 promoter and

the induction of apoptosis in a cell line that

contained wild type p53.

HISTONE ACETYLTRANSFERASES AND

COACTIVATOR COMPLEXES

In parallel with the developments in our

understanding of HDACs, there have also been

the recent identiﬁcation and functional analysis

of several human histone acetyltransferases

(HATs

) and the mechanisms by which

histone acetylation can activate transcription.

Histone acetylation, at lysines within the

N-terminal tails of H3 and H4, appears to be a

prerequisite for the process of transcriptional

activation in vivo.

The acetylation appears to

mediate chromatin remodelling (which makes

the chromatin more accessible to transcription

factors) by the speciﬁc, targeted interaction of

coactivator proteins that contain a bromodo-

main with acetyl lysines.

In particular, the

bromodomain of a yeast nuclear HAT is able to

bind in vitro to acetylated peptides of the H3

and H4 N-terminal tails

and the bromodo-

main is required for the subsequent recruit-

ment and coordination of in vivo remodelling

activity by the yeast SWI/SNF complex

(ﬁg

2B).

Recent work has shown the intimate rela-

tionship between histone modiﬁcations and the

processes of cell proliferation, cell diVerentia-

tion, and oncogenesis. As discussed in previous

sections, nuclear receptors have a key role in

determining the balance of cell proliferation

and cell diVerentiation in response to extracel-

lular signals, such as the hormone retinoic acid.

In the absence of ligand, the receptors remain

constitutively bound to target promoters and

recruit a transcriptional repressor complex (see

above), which maintains the cell in a proliferat-

ing state. However, once the nuclear receptors

bind the ligand, the repressor complex is

replaced by an activator complex that induces

cell diVerentiation (ﬁg 2A). Components of the

activator complexes include CREB binding

908 Johnson

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protein (CBP)

75 76

and the highly homologous

protein p300,

77 78

both of which are now known

to be histone acetyltransferases (HATs).

79 80

addition to CREB and nuclear hormone

receptors, the p300/CBP protein can also

interact with other DNA binding transcription

factors, such as c-Jun, c-Myb, c-Fos, and

MyoD

(ﬁg 2A), as well as p300/CBP

associated factor (PCAF) that is itself a histone

acetyltransferase.

The studies by Yang et al

also show that the E1A oncoprotein stimulates

proliferation by disrupting the interaction of

p300/CBP and PCAF, which would normally

suppress cell growth. PCAF is also implicated

in nuclear receptor coactivation.

83 84

Two addi-

tional nuclear hormone receptor coactivators,

SRC-1 and ACTR, which are members of the

p160 family, are also HATs

85 86

(see below).

The human transcription factor TAF II 250, a

component of the TFIID complex that recog-

nises the TATA element at promoters, also

contains HAT activity.

A further level of complexity in the regula-

tion of gene expression by HATs is that the

enzymes can acetylate lysine groups of non-

histone proteins. For example, p300/CBP and

PCAF are able to acetylate transcription

factors such as p53 and components of the

general transcription machinery such as the

TFIIEâ subunit.

87 88

Acetylation of both p53

and the haematopoietic transcription factor

GATA-1 increased their DNA binding activi-

ties.

87 89

In the latter study, the acetylation of

GATA-1 in vivo was implicated in the activa-

tion of target genes. A similar mechanism of

regulation has been shown for the nuclear hor-

mone receptor coactivator and acetyltrans-

ferase ACTR, which can itself be acetylated by

p300/CBP.

As expected, hormone treatment

of cells caused an increase in histone acetyla-

tion at receptor target genes. However, this

eVect was transient because the subsequent

acetylation of ACTR led to dissociation of the

receptor-coactivator complex and down regu-

lation of transcription, and suggests that

non-histone protein acetylation is the key

element that autoregulates hormone induction.

In a separate study, the acetylation of E2F1 by

both PCAF and p300/CBP appears to enhance

the function of this transcription factor,

which would presumably stimulate the tran-

scription of target genes during S phase of the

cell cycle. As discussed above, the E2F

transcription factors repress transcription of

target genes during G1 phase by interacting

with the Rb tumour suppressor gene product

pRb and histone deacetylases (HDACs). In

addition, the p53 tumour suppressor gene

product has also been shown to repress

transcription during apoptosis by recruiting

the Sin3-HDAC complex,

and itself to

become acetylated at deﬁned lysine residues in

vivo.

These observations suggest that tran-

scriptional regulation involves the addition and

removal of acetyl groups not only from histones

but also from other nuclear proteins, which has

important implications for the understanding

of cell growth and oncogenesis.

HISTONE ACETYLTRANSFERASE AND

LEUKAEMOGENIC FUSION PROTEINS

Transcriptional dysregulation by fusion pro-

teins that contain histone acetyltransferases

(HATs) has been implicated in leukaemogen-

esis

94 95

and in Rubinstein-Taybi syndrome

(RTS). RTS is a developmental disorder in

which patients have an increased incidence of

malignancies.

Microdeletions, translocations,

inversions, and point mutations in the CREB

binding protein (CBP) gene have been identi-

ﬁed in patients with RTS.

CBP translocations

that correlate with acute myeloid leukaemia

(AML) include fusions to the MLL (also

known as ALL-1, see below) gene

and to the

MOZ gene.

MOZ is itself a histone acetyl-

transferase

and a chromosomal translocation

in AML can also fuse it to the nuclear receptor

coactivator TIF2.

There are, as yet, no known

chemical inhibitors of HATs that could there-

fore be assessed for AML therapy, in contrast

to the retinoic acid and HDAC inhibitor treat-

ments discussed above. It is likely that a nuclear

HAT inhibitor will be isosteric for the acetate

group, which would allow it to inactivate either

the catalytic site or the bromodomain of the

enzyme.

OTHER HISTONE MODIFICATIONS

The N-terminal tails of the core histones are

subject to other post-translational modiﬁca-

tions, which include phosphorylation, methyla-

tion, ADP ribosylation, and ubiquitination.

Phosphorylation of histone H3 at serine 10 has

been the most extensively studied modiﬁcation

to date. It occurs during mitosis in many cells

as a transient and rapid response after mitogen

stimulation, which correlates with the expres-

sion of c-fos and c-jun.

100

H3 phosphorylation

in response to epidermal growth factor (EGF)

appears to be mediated by the Rsk-2 kinase,

101

which is a member of the pp90

rsk

(ribosomal S6

kinase) family that is implicated in cell

proliferation and diVerentiation.

102

It is inter-

esting to note that mutations in Rsk-2 (but not

in any other kinase in this family) are associated

with CoYn-Lowry syndrome,

103

which is char-

acterised by severe psychomotor retardation,

facial and digital dysmorphism, and skeletal

deformations. However, it remains unclear

what role H3 phosphorylation has during

cellular response to mitogens and if it has a

direct involvement in gene regulation.

Methylation of histone H3 has been linked to

gene activation by the p160 family of coactiva-

tors, which include the histone acetyltrans-

ferases SRC-1 and ACTR (see above). Both of

these proteins, and a third member of this fam-

ily called GRIP1, have been shown to interact

with the coactivator associated arginine (R)

methyltransferase (CARM1), which has exten-

sive homology to other arginine methyltrans-

ferases.

104

CARM1 can also methylate histone

H3 in vitro, and enhances the expression of a

reporter gene in a transient transfection

experiment. Methylation of histones, or other

proteins in the transcription initiation com-

plex, may therefore be a gene regulatory mech-

anism that cooperates with histone and protein

acetylation.

Chromatin modiﬁcation and disease 909

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DNA methylation and gene silencing

At the level of DNA modiﬁcations, methylation

of the C5 atom of cytosine residues is a power-

ful and prevalent mechanism for the repression

and inactivation of genes. Methylation of

promoter regions, on opposite DNA strands of

the dinucleotide sequence CpG, correlate, in

general, with transcriptional inhibition.

105

Hy-

drolytic deamination of 5'-methylcytosine to

thymine residues generates T-G mispairs,

which contribute to many germline point

mutations associated with human genetic

disease and other somatic mutations that lead

to cancer.

106 107

For this reason, CpG dinucle-

otides tend not to be found in coding regions,

but are clustered near the promoters of widely

expressed housekeeping genes, but remain

unmethylated at all levels of expression of the

gene. CpG islands can become de novo meth-

ylated during normal development (to silence

imprinted genes and the genes on the inactive

X chromosome of female mammals), but the

CpG islands of autosomal genes can also

become methylated as a result of in vitro cell

culture or neoplasia. It is possible that the

silencing of certain tumour suppressor genes

may, in part, be the consequence of de novo

methylation of an adjacent CpG island. For

example, the frequency with which promoter

methylation contributes to the gene inactiva-

tion is 33% for VHL and 84% for MLH1,in

von Hippel-Lindau (VHL) disease and micro-

satellite unstable colorectal tumours, respec-

tively.

108 109

A causal link between promoter

methylation and carcinogenesis is implied from

studies of transgenic mice with targeted

deletions of the DNA methyltransferase 1

(Dnmt1) gene, in which the formation of intes-

tinal polyps was suppressed.

110

Dnmt1 can also

recruit histone deacetylase activity and can

interact with HDAC1 in vitro.

111

Dnmt1 meth-

ylates DNA containing hemimethylated CpG

dinucleotides more eYciently than unmethyl-

ated DNA, and is therefore presumed to be the

major maintenance methyltransferase in

vivo.

112

Since Dnmt1 protein colocalises with

replication foci,

112

it will be interesting to see if

HDACs have a particular role at the replication

fork. Other mammalian DNA methyltrans-

ferases (Dnmt3a and b

113

) are likely to de novo

methylate promoters, with Dnmt3b speciﬁcally

required to methylate centromeric minor satel-

lite (in mouse embryonic stem cells).

114

Muta-

tions in one of the conserved catalytic domains

of DNMT3B, which presumably cause a partial

loss of function of the enzyme, are associated

with ICF syndrome (for immunodeﬁciency,

centromeric instability, facial anomalies).

114 115

ICF syndrome is characterised by immuno-

logical defects, hypomethylation, and instabil-

ity of centromeric heterochromatin, and facial

anomalies such as hypertelorism, epicanthic

folds, and macroglossia.

The mechanism by which methylated pro-

moters are inactivated has also become clearer

and appears to involve a long term remodelling

of the chromatin at the promoter. DNA meth-

ylation is able to reduce the binding aYnity of

sequence speciﬁc transcription factors or

recruit sequence speciﬁc DNA binding pro-

teins, such as methylated DNA binding protein

(MDBP),

116 117

which may act as transcriptional

repressors. However, a more general process

recruits sequence non-speciﬁc methyl-CpG

binding proteins (MeCPs) that exclude tran-

scription factors from the methylated pro-

moter.

10 118

It is now clear that the key molecu-

lar mechanism that underlies this repression

involves the recruitment of histone deacety-

lases (HDACs, see above and ﬁg 1A). The

MeCP2 protein contains a methyl binding

domain (MBD), which allows it to bind to a

single, symmetrically methylated CpG site and

a transcriptional repression domain (TRD)

which recruits the Sin3-HDAC corepressor

complex.

119

This mechanism is now known to

be implicated in repression by members of the

MBD protein family, which were identiﬁed on

the basis of homology with the MBD domain

of MeCP2.

120

MBD2 forms the so called

MeCP1 complex, together with HDAC1,

HDAC2, and RbAp46 and 48,

10 121

and MBD3

is a component of the Mi2-NuRD deacetylase

complex

35 122

(see above). It is interesting to

note that MBD4 contains the canonical methyl

CpG binding domain in addition to a thymine

DNA glycosylase catalytic domain, which

binds preferentially to methyl CpG-TpG

mismatches in DNA.

123

It is therefore probable

that the function of the MBD4 enzyme is to

suppress mutation at methyl CpG, rather than

to act as a transcriptional repressor. Frameshift

mutations in MBD4 that would cause trunca-

tion of the protein between the MBD and gly-

cosylase domains, and hence cause a defect in

mismatch repair, correlate with over 40% of

microsatellite unstable sporadic colon can-

cers.

124

Mutations in the MECP2 gene, which

encodes the X linked methyl CpG binding

protein 2 (MeCP2, see above), are of particular

clinical signiﬁcance. Genetic linkage analysis

has established that de novo missense muta-

tions in the methyl CpG binding domain

(MBD), and other mutations that disrupt the

transcriptional repression domain (TRD), are

a cause of Rett syndrome in about a quarter of

the sporadic patients studied.

125

Rett syndrome

is a progressive neurodevelopmental disorder

that occurs almost exclusively in females, and is

typiﬁed by the onset of autism, dementia,

ataxia, and loss of purposeful hand movements

from the ages of 6 to 18 months.

126

additional study has made the suggestion that

the eVect of MECP2 mutations may not be

limited to Rett syndrome, since heterozygote

females with skewed X inactivation patterns

may have a mild disease phenotype.

127

How-

ever, it is clear that the mechanism that under-

lies the pathogenesis of Rett implicates a

dysregulation in chromatin remodelling, al-

though it is not yet known if a similar

pathogenesis is seen for mutations in the MBD

family, or their partners in multiprotein

complexes.

Chromatin remodelling and

carcinogenesis

A further causal link between epigenetic

dysregulation and carcinogenesis is provided

910 Johnson

www.jmedgenet.com

by defects in ATP utilising chromatin remodel-

ling complexes. The role of these complexes is

to physically reposition or disrupt nucleosomes

by altering histone-DNA contacts.

128 129

The

ATPase/helicase subunits of the complexes are

members of three related families: homologues

of yeast SWI2/SNF2 (for mating type switch/

sucrose non-fermenting 2), the Mi-2 family

(also known as the CHD family), and the ISWI

(imitation SWI) family.

As discussed above, the dermatomyositis

speciﬁc autoantigen and ATPase/helicase

Mi-2â (also called CHD4) is a component of

the NuRD complex

that contains the histone

deacetylases HDAC1 and HDAC2 (ﬁg 1C).

This observation suggests that chromatin

remodelling by the ATPase activity makes the

histone tails more accessible to the NuRD

deacetylases, with the subsequent formation of

a repressive chromatin structure. However,

neither the eVect of NuRD mediated histone

deacetylation on gene expression nor the

targets of this type of chromatin remodelling

are known, although the MBD3 component of

the complex may recruit it to extensive, meth-

ylated regions of the genome such as hetero-

chromatin. Since 15% to 30% of patients with

dermatomyositis develop cancer,

130 131

it is

therefore probable that changes in chromatin

modiﬁcation and remodelling can aVect cell

proliferation.

It is interesting to note that the Mi-2â

protein contains two cysteine rich PHD (plant

homeodomain)/zinc ﬁnger regions and two

chromodomains, in addition to the helicase/

ATPase domain. Homeodomains and chromo-

domains are common features of other tran-

scriptional regulators that bind to

chromatin,

72 132

which include the DNMT3

family of DNA methyltransferases

112

and

ALL-1 (also known as MLL, HRX, or HTRX,

see below).

133

Another such protein is ATRX

(for á thalassaemia/retardation on the X chro-

mosome) which, like Mi2â, also contains

PHD-like ﬁngers and other domains that clas-

sify it as a member of the ATPase/helicase

superfamily. The functional importance of the

PHD-like ﬁngers is shown by the observation

that two thirds of all mutations that cause

ATRX syndrome lie in this region.

134

ATRX

syndrome comprises a severe form of mental

retardation, characterised by the presence of á

thalassaemia, urogenital abnormalities, and

facial dysmorphism.

135 136

In view of the interac-

tion of Mi2â protein with HDAC1,

it is prob-

able that PHD-like domains in other transcrip-

tional regulators also mediate the same

interaction. In addition, mutations in ATRX

are associated with changes in DNA methyla-

tion patterns at highly repeated sequences,

such that rDNA repeat arrays are hypomethyl-

ated whereas others (the Y speciﬁc repeat

DYZ2) are hypermethylated.

137

This observa-

tion implies a link between chromatin remodel-

ling, mediated by the ATRX protein, and the

DNA methylation system. However, it is

unclear if ATRX functions as a transcriptional

coactivator (similar to the SWI2/SNF2 pro-

teins) to increase the expression of a compo-

nent of the DNA methylation system, or if it

increases the accessibility of chromatin to a

DNA methyltransferase.

The human SWI/SNF complex has been

found to act as a coactivator for several nuclear

receptors

138

and appears to be both recruited to

speciﬁc regions of chromatin by DNA binding

proteins and to remodel promoter regions to

facilitate the binding of other factors.

129 139

The

two human homologues of SWI/SNF2, BRG-1

and hBRM, are also implicated in transcrip-

tional repression, since both can interact with

the tumour suppressor gene product Rb.

20 140

As discussed above, Rb regulates cell cycle

progression by inhibiting the activity of the

transcriptional activator E2F and recruiting

histone deacetylases. Transient transfection of

BRG-1 or hBRM into cell lines that do not

express these proteins but that do express Rb

leads to growth arrest

and, furthermore,

ﬁbroblasts transformed with ras and lacking

endogenous expression of BRM revert to a

ﬂattened, growth arrested phenotype after

reintroduction of hBRM.

140

These observations

establish the link between chromatin remodel-

ling by SWI/SNF and control of the cell cycle.

A further connection is provided by another

subunit of the SWI/SNF complex, called

hSNF5/INI1. Genetic linkage has identiﬁed

biallelic deletions or mutations in the hSWF5/

INI1 gene to be responsible for malignant

rhabdoid tumours, which are very aggressive

cancers of early childhood that tend to occur in

the kidney, brain, and soft tissues.

141

Another

study has shown that deletions of hSNF5/INI1

can be aquired during leukaemogenesis in

patients with chronic myeloid leukaemia.

142

Maintenance of chromatin states and

carcinogenesis

Recent work has shown that the maintenance

of active and inactive chromatin states is an

important determinant of gene expression. In

Drosophila, the coordinated expression of the

homeotic genes determines segmentation and

body plan along the anterior-posterior axis.

The maintenance of this expression through-

out development is mediated by the Polycomb-

group (Pc-G) repressor proteins and the

trithorax-group (trx-G) activator proteins.

143

Many of the polycomb- and trithorax-group

proteins contain the SET domain (for

Suvar3-9, Enhancer-of-zeste, Trithorax), that

is conserved in a number of mammalian

homologues. ALL-1, for example, is the human

homologue of Drosophila Trithorax protein,

which is a positive regulator of homeotic gene

expression. A similar role is implied for the

mammalian MLL/ALL-1 because hetero-

zygous mll

+/-

mice have defects in axial skeletal

development and haematopoiesis, which arise

from alterations in the pattern of Hox gene

expression.

144

The ALL-1 gene, at the 11q23

locus, is one of the most common targets of

chromosomal translocations in acute lym-

phocytic leukaemia and other acute leukae-

mias,

145

and there are at least 30 partner genes

that produce in frame leukogenic fusion

proteins with ALL-1/MLL, including the

histone acetyltransferase CBP

(see above). In

general, the C-terminal SET domain of

Chromatin modiﬁcation and disease 911

www.jmedgenet.com

ALL-1/MLL is lost during chromosomal

translocation. It is interesting to note that the

hSNF5/INI1 component of the SWI/SNF

complex (see above) has been shown to interact

with the SET domain of ALL-1.

146

One of the

consequences of ALL-1/MLL translocations

would be the inability to recruit SWI/SNF, and

it is therefore possible that the transformation

of haematopoietic cells involves the dysregula-

tion of chromatin remodelling, as well as the

aberrant expression of MLL/ALL-1 target

genes.

Mammalian polycomb-group homologues

are thought to be negative regulators of home-

otic gene expression. EED (for embryonic

ectoderm development) is required at a very

early stage in embryonic development and is

the only known homologue of the Drosophila

extra sex combs (esc) Pc-G protein. Disruption

of the eed gene in mice causes defects in

anterior mesoderm production, followed by

death at day 8.5 of gestation.

147

In addition, the

histone deacetylases HDAC1 and HDAC2

have been shown to interact with EED, but not

with other vertebrate Pc-G proteins.

148

This

result again underlines the complex and subtle

interplay between the separate mechanisms of

gene regulation.

Conclusion

In the past few years, the exciting progress in

the ﬁeld of gene regulation has made it clear

that chromatin is not just a static structure, but

that it has a pivotal role in regulating transcrip-

tion. Chromatin has a dynamic structure that

can be modulated during cell diVerentiation

and transformation, and the nucleosome is a

substrate that can both receive and transmit

intracellular signals. In addition, chromatin has

the capacity to encode epigenetic information

on levels of gene expression, which is inde-

pendent of the genetic information encoded by

the sequence of genes.

149

A parallel develop-

ment is the realisation that many transcription

factors can function by recruiting large, multi-

protein complexes that mediate several types of

chromatin modiﬁcation and remodelling

events. It is therefore not surprising that muta-

tions that cause loss or perturbation of

chromatin modiﬁcation and remodelling ac-

tivities will cause changes in gene expression

levels. This points to abnormal epigenetic

regulation as a general mechanism that under-

lies human carcinogenesis and the pathogen-

esis of other genetic diseases (see table 1 for a

summary). For example, the types of mutations

that are associated with most classes of acute

myeloid leukaemia are typiﬁed by balanced

chromosome translocations that result in the

expression of chimaeric proteins. Most of these

chimaeric proteins involve the in frame fusion

of transcriptional regulator that results in tran-

scriptional dysregulation. Some of the proteins

that mediate chromatin modiﬁcation and

remodelling are therefore novel molecular

targets for future treatments of cancer and

other genetic diseases.

One of the most intriguing aspects of this

recent work is the probable interplay between

modiﬁcation and remodelling events. Future

work will undoubtedly uncover further mecha-

nistic links between distinct gene regulation

systems. But a hint of this interplay is seen with

the possible interactions of histone deacetylase.

As discussed above, HDAC1 and HDAC2 can

interact with the DNA methyltransferase

Dnmt1, the polycomb-like protein EED, and

the ATPase/helicase Mi-2â. To add to this

complexity, it is not known if other histone and

protein post-translational modiﬁcations (nota-

bly histone phosphorylation and methylation)

can modulate the eVects of chromatin modiﬁ-

cation and remodelling, or if they participate in

other gene regulation systems of their own.

Another important aspect is a description and

understanding of the signal transduction path-

ways that use chromatin as a target, and how

these signals can mediate changes in gene

expression. For example, a very recent develop-

ment has shown that recombinant yeast and

mouse Sir2 proteins, and other yeast Sir2

homologues, are novel, NAD dependent his-

tone deacetylases.

150 151

In yeast, Sir2 is a

mediator of transcriptional silencing at the het-

erochromatic regions associated with silent

mating loci, telomeres, and ribosomal DNA.

152

However, the absolute requirement of NAD for

the deacetylation reaction suggests that this

form of chromatin modiﬁcation can be regu-

lated by the metabolic levels of NAD and

NADH, and hence by caloriﬁc intake.

153

The author would like to thank Doug Higgs, Institute of

Molecular Medicine, Oxford and members of the Chromatin

and Gene Expression Group for suggestions and critical evalu-

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