Cell, Vol.
108, 489–500, February 22, 2002, Copyright 2002 by Cell Press
Epigenetic Codes for
Heterochromatin Formation and
Silencing: Rounding up the Usual Suspects
Eric J. Richards and Sarah C.R. Elgin1
Department of Biology
Washington University
One Brookings Drive
St. Louis, Missouri 63130
Recent results from diverse organisms point to a self-
reinforcing network of interactions among the three
best-characterized covalent modifications that mark
heterochromatin: histone hypoacetylation, histone H3-
Lys9 methylation, and cytosine methylation. These
modification systems suggest a mechanistic basis for
spreading of heterochromatin over large domains and
for stable epigenetic inheritance of the silent state. All
three modifications used in packaging heterochroma-
tin are also used in stable silencing of euchromatic
genes.
Introduction
Significant increases in genome size can be correlated
with the acquisition of epigenetic mechanisms for estab-
lishing and maintaining transcriptionally silent “off”
states via chromatin packaging (Bird, 1995), and this
may have been a necessary adaptation for coping with
repetitive DNA. The ability to package large genomes
is associated with a fundamental shift in the logic of
gene regulation between prokaryotes and eukaryotes.
Whereas in prokaryotes the ground state is nonrestric-
tive, in eukaryotes transcriptional activity is generally
impeded by nucleosomal packaging (see Struhl, 1999,
for development of this contrast). Activators and repres-
sors appear to influence gene expression in eukaryotes
by recruiting chromatin-modifying activities to promot-
ers (see review by Narlikar et al., 2002 [this issue of
Cell]). Large segments of the genome, primarily repeti-
tive sequences, are packaged in a permanently inactive
form, constitutive heterochromatin. This requires spe-
cific modifications of the histones, recruitment of partic-
ular protein complexes, and/or specific modification of
the underlying DNA (see Figure 1). The silenced state
can persist through mitotic and meiotic cell divisions,
indicating that the particular chromatin structure is itself
replicated during the process of DNA replication and
chromosome duplication.
Structural Characteristics of Heterochromatin
Early cytological studies distinguished two types of
chromatin: euchromatin and heterochromatin. Hetero-
chromatin was originally defined as that portion of the
genome that remains condensed and deeply staining
(heteropycnotic) as the cell makes the transition from
metaphase to interphase; such material is generally as-
sociated with the telomeres and pericentric regions of
chromosomes. Subsequent work has identified a cluster
of structural features that characterizes heterochroma-
tin (Table 1; reviewed by Henikoff, 2000). While hetero-
1
Correspondence: selgin@biology.wustl.edu
Review
chromatic regions are rich in repetitive sequences and
have a low gene density, they are not devoid of genes;
it is estimated that there are ⵑ40–50 genes within the
pericentric heterochromatin of Drosophila (Weiler and
Wakimoto, 1995). An altered packaging of heterochro-
matin, to a less-accessible form, has been demon-
strated by probing with nucleases and other reagents
such as prokaryotic DNA methyltransferases. The data
suggest that while nucleosome arrays in euchromatin
are irregular, punctuated by the nucleosome-free hyper-
sensitive sites (HS sites) characteristic of active genes,
the nucleosomes in heterochromatin have a regular
spacing over large arrays, with a higher proportion of
the DNA associated with the histone core rather than in
the linker (Grewal and Elgin, 2002; Sun et al., 2001, and
references cited therein).
Genes normally active in a euchromatic domain will
typically be silenced, often showing a variegating phe-
notype, when placed adjacent to or within a heterochro-
matic domain by chromosome rearrangement or trans-
position (i.e., position effect variegation, PEV). The
variegation is thought to reflect heterochromatin assem-
bly of formerly euchromatic regions at the euchromatin/
heterochromatin boundary in a stochastic process. That
euchromatic regions can be silenced by packaging in a
heterochromatic form was initially recognized by identi-
fication of “facultative heterochromatin,” defined as ge-
nomic regions that exhibit such packaging in only a
subset of the cells or for only one homolog. A prominent
example is the inactive X chromosome in female mam-
mals, where either the paternal or maternal X chromo-
some is selected for such packaging. The silent state
of “facultative heterochromatin” persists through mi-
totic cell divisions, providing epigenetic regulation.
Here we will refer to euchromatic regions silenced by
such packaging as “silent chromatin,” reserving the term
“heterochromatin” for constitutive heterochromatin ex-
hibiting the cluster of properties shown in Table 1. While
we will focus on the extreme states, in reality there are
no doubt a number of intermediates in which a subset
of silencing mechanisms is used to regulate gene ex-
pression.
Biochemical Characteristics of Heterochromatin
In the last few years, we have learned a great deal about
the biochemistry of heterochromatin. In the process it
has become clear that some of the biochemical modifi-
cations important for packaging heterochromatic do-
mains are used to silence genes in euchromatin. These
modifications include covalent modification of the DNA,
association of particular nonhistone proteins, and par-
ticular patterns of covalent modification of the histones.
It is an interesting paradox that while the histones are
among the most conserved proteins known in evolution,
they are also among the most variable in posttransla-
tional modification. The pattern of modifications has
been suggested to act as an information code (the his-
tone code), dictating both nucleosomal interactions and
the association of nonhistone chromosomal proteins
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490

Figure 1. Heterochromatin Epigenetic Mark-

ers: Interconnections
Three different biochemical marks important
in distinguishing the heterochromatic state
from the euchromatic state are shown. Re-
cent evidence suggests that each of the epi-
genetic markers can be directly propagated
and may influence acquisition of the other
two. The hypothesized signaling from the
5mC mark to the histone H3-mLys9 mark
(dashed line) has not yet been reported. Ab-
breviations: H3-mLys9, histone H3 methyl-
ated on lysine 9; H3-mLys4, H3 methylated
on lysine 4; H3/4 aLys, acetylated isoforms
of H3 and H4; H3/4 Lys⫹, deacetylated iso-
forms of H3 and H4. The H3-mLys4 and H3-
mLys9 modifications are antagonistic (Noma
et al., 2001; Wang et al., 2001).

that collectively influence packaging and gene regula-
tion (Rice and Allis, 2001; Strahl and Allis, 2000; Turner,
2000). Modifications include acetylation, methylation,
phosphorylation, ubiquitination, and ADP-ribosylation.
Given the number of sites of posttranslational modifica-
tion for each of the four core histones, an imposing
number of differently modified nucleosomes is possible.
The modification states of the N-terminal tails of his-
tones H3 and H4 appear to play a major role in hetero-
chromatin formation (Figure 2).
Hypoacetylation of lysine residues is associated with
both formation of heterochromatin and gene silencing.
Early attempts to fractionate chromatin and characterize
the components led to the suggestion that heterochro-
matic domains were associated with hypoacetylated
histones, while euchromatic domains were associated
with hyperacetylated histones. This distinction is ob-
served not only between constitutive heterochromatin
and euchromatin, but also in mapping studies compar-
ing an active or inducible gene to flanking regions (Heb-
bes et al., 1994; Litt et al., 2001).
An interesting recent finding has been the identifica-
tion of histone H3 methylated at lysine 9 (H3-mLys9) as
characteristic of the heterochromatic state. Immunoflu-
orescent staining of Drosophila polytene chromosomes
shows that the bulk of the H3-mLys9 is present in the
pericentric heterochromatin and in a banded pattern on
the fourth chromosome, known sites of repetitive DNA
(Jacobs et al., 2001). Similarly, chromatin immunopre-
cipitation (ChIP) experiments demonstrate that H3-
mLys9 is a prominent component of the silent mating
type locus in fission yeast (Schizosaccharomyces
pombe), while essentially absent from flanking regions
containing inducible genes (Noma et al., 2001). Methyla-
tion of histone H3-Lys9 has also been associated with
the silencing of euchromatic genes (Hwang et al., 2001;
Nielsen et al., 2001).
A third biochemical marker of heterochromatin is the
presence of cytosine methylation, the most common
form of DNA modification in eukaryotes. Although ab-
sent in some eukaryotes, this DNA modification is widely
distributed in the eukaryotic kingdom. It is particularly
prevalent in plants and mammals where it is an important
epigenetic mark that contributes to the stability of peri-
centromeric heterochromatin (Bachman et al., 2001;
Okano et al., 1999; Xu et al., 1999) and plays a central
role in cementing and maintaining epigenetic expression
states, not only in heterochromatin but in silenced eu-
chromatic domains (Jones and Takai, 2001; Martienssen
and Colot, 2001).

Table 1. Distinctions between Euchromatic and Heterochromatic Domains

Feature Euchromatin Constitutive Heterochromatin

Staining/packaging in interphase Dispersed Appears condensed, heteropycnotic

DNA sequence Predominantly unique Predominantly repetitive (satellites;
derivatives of viruses, transposons, etc.)
Presence of genes High/variable density Low density
Meiotic (reciprocal) recombination Normal frequency Low frequency
Replication timing Throughout S phase Late S phase
Chromatin structure HS sites, irregular nucleosomes; Loss of HS sites, regular nucleosome array;
accessible to nucleases less accessible to nucleases
Activity state
Euchromatic genes Genes inducible Genes silenced (variegated)
Heterochromatic genes Genes silenced (variegated) Genes inducible
Characteristic modifications Histone hyperacetylation Histone hypoacetylation
Histone H3-mLys4 present Histone H3-mLys9 present
Cytosine hypomethylation Cytosine hypermethylation
Review
491
A General Framework for Information Flow
in Epigenetic Codes?
Although our understanding of epigenetic codes is in its
infancy, a synthesis of recent results from experimental
organisms as diverse as fungi, mammals, and plants
points toward the operation of a self-reinforcing network
of interactions among the three best-understood cova-
lent modifications that mark heterochromatin. The
emerging framework, illustrated in Figure 1 and elabo-
rated below, is composed of self-reinforcing loops and
feedback mechanisms driving formation, maintenance,
and spreading of heterochromatin. Evidence for these
connections comes from a wide range of experimental
organisms. We will examine here data indicating the
commonality of these relationships but pointing out
some instances where a particular relationship does not
appear to hold for a given organism. The framework
suggests experiments to identify connections that have
not heretofore been recognized.
Histone Hypoacetylation as a Mark for
Heterochromatin and Silent Domains
Hypoacetylation, particularly of histones H3 and H4, as-
sociated with heterochromatic domains from a range of
organisms, has been studied in greatest detail in Sac-
charomyces cerevisiae. Histone acetylation occurs at a
low level ubiquitously throughout much of the genome,
resulting from a balance between the several histone
acetyltransferase (HAT) activities and histone deacety-
lase (HDAC) activities (Vogelauer et al., 2000). Specific
local alterations are then superimposed upon this base-
line. While the chromosomes of S. cerevisiae are too
small to allow cytological detection of heterochromatin,
the silent mating type loci (HML and HMR), the regions
adjacent to telomeres, and the rRNA gene repeats show
many heterochromatic features. Maintenance of this si-
lencing is important for the biology of the organism;
e.g., relaxation of silencing at HML and HMR leads to
expression of the developmental programs for both mat-
ing types, and the haploid cells acquire many properties
Figure 2. Modification of Histones H3 and H4
The amino acid sequences of the N-terminal
tails of histones H3 and H4 from mammals
(top) and S. cerevisiae (bottom), with some
of the covalent modifications characterized
in different systems. Modification reactions
are influenced by the prior modification state,
as indicated for some H3 cases. Specific
functions have been mapped to various sub-
domains as shown, either by a genetic ap-
proach in yeast or by X-ray crystallography
(internucleosomal contact; see Turner, 2000,
for review and original citations).
of nonmating diploids. Reporter genes inserted within
these regions are silenced in a stochastic manner, giving
rise to populations in which the gene is stably main-
tained in an “on” or “off” state (Freeman-Cook et al.,
2000).
Many of the cis- and trans-acting factors necessary
to establish and maintain the silent state at the telomeres
and HML/HMR loci have been identified. These studies
have demonstrated the need for hypoacetylated his-
tones. Silencing is mediated by the multiprotein, nucleo-
some binding SIR(1-4) complex, recruited by interaction
with specific DNA binding proteins (Figure 3). Sir3 and
Sir4 interact specifically with the N-terminal tails of his-
tones H3 and H4 in the hypoacetylated state. While the
N-terminal tails of the histones are not required individu-
ally for growth in yeast, they do play an essential role
in silencing, amino acids 4–20 of H3 and 16–29 of H4
being required (Figure 2). Certain sir3 alleles can sup-
press the silencing defect of histone H4 tail mutations,
and Sir3 and Sir4 can bind to the amino termini of his-
tones H3 and H4 in vitro, suggesting direct interaction
(Hecht et al., 1995, and references therein). Recent stud-
ies using antibodies against different histone acetylated
isoforms indicate that histones in the telomeric and
HML/HMR heterochromatin are hypoacetylated at all
modification sites (Suka et al., 2001).
What is the mechanism for histone hypoacetylation
specifically at the heterochromatic domains? This func-
tion is apparently provided, at least in part, by Sir2,
shown to have a NAD-dependent protein deacetylase
activity. Sir2 can efficiently deacetylate histones in vitro,
preferentially deacetylating histone H4 at Lys16, al-
though direct action in vivo has not yet been reported.
Enzymatic activity of Sir2 is required for silencing in the
heterochromatic domains (see Moazed, 2001, for review
and original citations). The acetylation status of H4-
Lys16 may be of particular importance. Lys16 is the
preferred site of acetylation in monoacetylated H4 of
euchromatin in yeast (Clarke et al., 1993), and this is
the only acetylatable H4 site whose mutation strongly
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492
Figure 3. Heterochromatin Assembly in S. cerevisiae
A model for assembly of heterochromatin at yeast telomeres. Telo-
meric repeats bind to Rap1, a sequence-specific DNA binding pro-
tein, which together with telomeric proteins yKu70 and yKu80 re-
cruits the SIR complex. (Note that Sir1 is required for nucleation
only at the mating type loci.) Step-wise assembly of the heterochro-
matin has been suggested, with binding of the Sir2-Sir4 complex
(1) followed by association with Sir3. Following deacetylation of the
H3/H4 tails by Sir2 (and possibly other histone deacetylases), Sir3
is recruited through interactions with Rap1, Sir4, and the hypoace-
tylated histone tails, binding to the nucleosomes (2). Multimerization
of Sir3 and Sir4, with recruitment of Sir2, can result in continuing
rounds of histone modification and complex assembly, leading to
spreading of the heterochromatic state (3). (Reproduced with per-
mission from Moazed, 2001.)
affects Sir3 binding in heterochromatin (Hecht et al.,
1996). Deletion of sir3 results in increased histone acet-
ylation in heterochromatic domains, as well as a loss
in silencing (Suka et al., 2001). The results suggest an
assembly model in which interaction of the Sir2-Sir4
complex with specific DNA binding proteins leads to
local histone deacetylation, permitting binding of Sir3.
It appears that binding of Sir3 to the hypoacetylated
histone blocks reacetylation. Given the interactions be-
tween Sir2, Sir4, and Sir3, once initiated, such a complex
could spread along the nucleosome array, generating
and maintaining the altered modification state (Fig-
ure 3).
In addition to the above, studies in S. pombe, Dro-
sophila, and other organisms suggest that the histone
acetylation level is used as a heritable mark of the chro-
matin state. Mutations in HDACs or treatment with tri-
chostatin A (TSA), an inhibitor of some HDACs, fre-
quently results in a loss of function in heterochromatic
domains and a relaxation of silencing. For example,
treatment with TSA results in functionally deficient cen-
tromeres and chromosome loss in S. pombe, concomi-
tant with a loss of silencing for test genes within the
centromeric heterochromatin. The hyperacetylated state
is heritable following removal of TSA, is linked in cis
to the treated centromere locus, and correlates with
inheritance of functionally defective centromeres (Ek-
wall et al., 1997), demonstrating an epigenetic phenome-
non based on the chromatin structure. In contrast, acet-
ylation is used as an inherited mark of activity. Histone
H4-aLys16 is prominently associated with the dosage-
compensated, 2-fold active X chromosome in males of
Drosophila (Bone et al., 1994). This specific modification
is due to MOF, an essential acetyltransferase of the
dosage compensation complex that coats the male X
chromosome (Smith et al., 2000). The complex remains
associated with its target DNA throughout the cell cycle,
providing the means to replicate the modification state.
Recruitment of HATs to chromosome regions showing
histone acetylation patterns corresponding to their own
catalytic specificity has been observed, e.g., the histone
acetyltransferase P/CAF binds preferentially to acet-
ylated H4 and H3 peptides via a bromo domain (Dhalluin
et al., 1999; Rice and Allis, 2001). These observations
provide evidence for use of the histone acetylation state
as an epigenetic mark (Table 2).
How does hypoacetylation impact chromatin struc-
ture? In the case described above, the hypoacetylated
histone tails interact specifically with the SIR complex.
While Sir2 orthologs have been identified, few proteins
with similarity to the other Sir proteins have been found
in multicellular eukaryotes. Nonetheless, there may be
an equivalent of the SIR complex that makes similar
use of the histone hypoacetylation signal. However, a
significant effect might be realized through the interac-
tion of the histone H3/H4 tails with the DNA and/or other
nucleosomes in the chromatin fiber (see Annunziato and
Hansen, 2000, for review). The regions of the histone
H3 and H4 tails that contribute to DNA binding, as ob-
served in the crystal structure, are necessary for silenc-
ing of basal transcription in vivo. However, these regions
are distinct from those critical for repression at the HM
loci and telomeres (Figure 2). It appears unlikely that
simply weakening intranucleosomal histone-DNA inter-
actions by histone acetylation could alleviate the inhibi-
tory effect of heterochromatin structure on transcription.
An alternative possibility was suggested by the original
crystals of the nucleosome (using histones from Xeno-
pus), where histone H4 amino acids 16–24 were ob-
served to interact with the acidic region formed by his-
tones H2A and H2B on the surface of the adjacent
histone octamer (Luger et al., 1997). The eight H2A/H2B
amino acids involved in forming this negatively charged
patch are highly conserved. Acetylation of the H4 tail
might disrupt this interaction, leading to a loss of com-
paction along the chromatin fiber. However, this disposi-
tion of the histone H4 tail is not seen in crystals of the
nucleosome made using yeast histones (White et al.,
2001), and additional studies are needed to resolve this
interesting question.
Methylation of Histone H3 on Lysine 9
and Association of HP1 as a Mark
for Heterochromatic Domains
A key role for a second histone modification in the speci-
fication of heterochromatin is shown by the recent dem-
onstration that mammalian homologs of Drosophila
Su(var)3-9, including human SUV39H1 and murine
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493
Table 2. Presence of Silencing Components in Different Systems
Hypoacetylated Histone H3-Lys9
Organism H3/H4 Methyltransferase
S. cerevisiae Yes ? No
S. pombe Yes Clr4
N. crassa ? DIM-5
D. melanogaster Yes Su(var)3-9
mouse Yes Suv39h1, Suv39h2
human Yes SUV39H1
A. thaliana Yes ? LHP1a
a
The A. thaliana HP1-like protein, LHP1, was reported in Gaudin et al., 2001.
Suv39h1, encode enzymes that specifically methylate
histone H3 on lysine 9 (Rea et al., 2000). Su(var)3-9 was
originally identified as a suppressor of PEV in Drosoph-
ila, indicating that the wild-type gene product is involved
in heterochromatin formation (Tschiersch et al., 1994).
A homolog in S. pombe, Clr4, is also a specific histone
H3-Lys9 methyltransferase (Nakayama et al., 2001), sug-
gesting that this activity is widely distributed and well
conserved. clr4 mutants exhibit reduced heterochroma-
tin formation at centromeres, with elevated mitotic chro-
mosome loss and reduced silencing within both pericen-
tromeric heterochromatin and the silent mating type
locus (Allshire et al., 1995; Ekwall et al., 1996; Ivanova et
al., 1998). Similarly, mammalian Su(var)3-9-like proteins
have been implicated in both centromere activity and
gene silencing. Disruption of the murine Suv39h1 and
Suv39h2 paralogs causes genome instability, chromo-
some missegregation, and male meiotic defects (Peters
et al., 2001b).
Further, the Suv39h1/SUV39H1 proteins are found in
association with M31 (Aagaard et al., 1999), a mouse
heterochromatin protein 1 (HP1) homolog. HP1, perhaps
the best-characterized protein found in heterochroma-
tin, was identified in Drosophila melanogaster in a
screen of monoclonal antibodies prepared against pro-
teins tightly bound in the nucleus. Immunofluorescent
staining of the polytene chromosomes shows HP1 con-
centrated in the pericentric heterochromatin, the telo-
meres, and a banded pattern across the small fourth
chromosome, known sites of repetitive DNA with char-
acteristics of heterochromatin (James et al., 1989). A
few prominent HP1 sites are observed within the euchro-
matic arms (e.g., region 31). Homologs of HP1 are asso-
ciated with pericentric heterochromatin in organisms
from S. pombe to humans (Table 2). The protein (206
amino acids in Drosophila) has a conserved N-terminal
chromo domain (CD) followed by a variable hinge region
and a conserved C-terminal chromo shadow domain
(CSD; Figure 4). The chromo domain was first recog-
nized by similarity with a domain in Polycomb, a protein
associated with silencing of the homeotic genes during
development; this domain has now been identified in
many other chromosomal proteins. Both point muta-
tions in the chromo domain and presumed null muta-
tions (early truncation of the translation product) in the
gene encoding HP1 [Su(var)2-5] result in a loss of silenc-
ing, while an additional dose will increase silencing of
a variegating euchromatic gene, i.e., one placed in a
heterochromatic environment. Interestingly, the con-
verse is true for those few genes normally resident within
HP1 5mC
No
Swi6 No
? Yes
HP1 Little
MHP1a, M31/MOD1, M32/MOD2 Yes
HP1hs␣, HP1hs␤, HP1hs␥ Yes
Yes
the pericentric heterochromatin (e.g., light), which ap-
pear to be dependent on HP1 for normal activity (re-
viewed by Eissenberg and Elgin, 2000). The conserved
structure of HP1 suggests that it might serve as a bifunc-
tional reagent, helping to organize and maintain hetero-
chromatin structure. HP1 interacts with a number of
other chromosomal proteins, including several involved
in nuclear assembly, replication, and gene regulation
(Eissenberg and Elgin, 2000). These interactions have
generally been mapped to the chromo shadow domain.
The chromo shadow domain can homodimerize, and the
dimer has been suggested to be the interactive species
(Brasher et al., 2000).
The HP1 chromo domain specifically binds histone
H3 N-terminal tails methylated on lysine 9 (Bannister et
al., 2001; Lachner et al., 2001), and a variety of data
suggest that this interaction is essential for maintenance
of heterochromatin. The interaction appears quite spe-
cific; neither the chromo domain of Polycomb nor the
chromo shadow domain of HP1 shows this interaction
(Lachner et al., 2001). The H3 tail fits within a groove
established by conserved chromo domain residues;
Su(var) mutation V26M results in an alteration of the
structure and loss of H3-mLys9 binding (Jacobs et al.,
2001). Studies in mammalian cells suggest that localiza-
tion of HP1 in heterochromatin is dependent on the
presence of histone H3-mLys9 (Bannister et al., 2001;
Figure 4. Heterochromatin Protein 1 (HP1) from Drosophila melano-
gaster
The chromo domain of HP1 binds specifically to histone H3-mLys9;
the chromo shadow domain interacts with several proteins. While
it is not known whether the interaction is direct or indirect, M31 (an
HP1 from mouse) has been found in a complex with SUVAR39, the
mammalian homolog of Su(var)3-9. This suggests that the bifunc-
tional nature of HP1 provides a mechanism for perpetuating and
spreading heterochromatin structure (see text). Y24F and V26M indi-
cate mutations that result in suppression of PEV.
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494
Figure 5. Heterochromatin Formation in S. pombe
A flow diagram of the steps required to assemble heterochromatin
at the silent mating type locus (see text). Adapted from Nakayama
et al. (2001).
Rea et al., 2000). However, HP1 association with hetero-
chromatin in Drosophila can be driven either by the
N-terminal portion (with the chromo domain) or the
C-terminal portion (with the shadow domain), emphasiz-
ing the bifunctional nature of the protein (Eissenberg and
Elgin, 2000). The above results argue that an interaction
between the specifically modified histone H3 and HP1
is essential for maintaining a stable heterochromatin
structure.
Histone Modification/Modifier Complexes:
Maintenance and Spreading
Histone H3-Lys9 methylation is influenced by preex-
isting modifications of histone H3 and affects other his-
tone modifications, implying a set of functional interac-
tions (Figure 2; reviewed in Jenuwein and Allis, 2001).
The relationship between hypoacetylation of H3/H4 and
methylation of H3 has been clarified by studies of hetero-
chromatin formation in S. pombe (Figure 5). clr1-clr4,
clr6, swi6, and rik1 mutations all identify trans-acting
factors necessary for silencing at the S. pombe mating
type locus (reviewed in Grewal, 2000). Swi6 is a homolog
of HP1, while clr1 and rik1 code for putative DNA binding
proteins. The products of clr3 and clr6 are homologs
of HDACs. As discussed above, Clr4 is the H3-Lys9
methyltransferase. These genes work together, acting
on the entire silent mating type domain to maintain it in
the repressed state. Clr3, an H3-specific deacetylase,
and Rik1 are required for histone H3-Lys9 methylation
by Clr4, and Swi6 localization is dependent on Clr4 and
Rik1 (Ekwall et al., 1997; Nakayama et al., 2001).
These observations suggest a progression of events
leading to establishment of a distinctive heterochro-
matic structure based on the histone modification pat-
tern (Figure 5). Deacetylation of histone H3 by Clr6
and/or Clr3 creates conditions favoring methylation at
H3 Lys9 by the Clr4/Rik1 complex; methylation leads to
binding of Swi6, establishing a chromatin configuration
that is refractory to transcription and stably maintained.
Mapping studies using chromatin immunoprecipitation
show H3-mLys9 and Swi6 found throughout, and limited
to, the 20 kb silent mating type domain. This 20 kb region
is flanked by inverted repeats IR-L and IR-R, which ap-
pear to serve as barriers to the spread of silencing;
removal of these repeats results in the appearance of
H3-mLys9 and Swi6 on neighboring sequences (Noma
et al., 2001). Silencing is dependent on the dosage of
Swi6, which remains bound to the mating type region
throughout the cell cycle and may itself be a marker for
heterochromatin formation (Nakayama et al., 2000).
The findings suggest a mechanism for maintaining
heterochromatin structure following replication and for
driving the spread of heterochromatin. During replica-
tion, the DNA must be “unpackaged” and the daughter
DNA molecules repackaged into nucleosomes. Parental
histones are efficiently reutilized, distributed randomly
to the two daughter DNA molecules; an equal amount
of newly synthesized histone is required to complete
assembly (Krude and Keller, 2001). Assuming that the
histone H3-mLys9 in a heterochromatic domain is stable
(no histone demethylases have been identified as yet),
it will associate with HP1 through the chromo domain.
The presence of HP1 will result in assembly of a modi-
fying complex, presumably through the chromo shadow
domain, that will deacetylate and specifically methylate
the newly arrived histone, perpetuating the pattern of
modification and HP1 binding to establish a heterochro-
matic structure. Recovery of a SUV39H1-HDAC1 com-
plex from Drosophila embryo extracts that can methyl-
ate preacetylated histones supports such a model
(Czermin et al., 2001; see also Vaute et al., 2002). As
suggested above, formation of complexes that both rec-
ognize a particular pattern of histone modification and
have the ability to achieve that pattern provides a mech-
anism for epigenetic inheritance of chromatin structure.
The same machinery could account for spreading of
heterochromatin, requiring that boundaries to such
spread be established (Bell et al., 2001; Sun et al., 2000).
Genetic analyses in S. pombe and Drosophila indicate
that while the H3-mLys9/HP1 system is critical for het-
erochromatin formation and silencing in pericentric het-
erochromatin, it is of less importance at the telomeres,
suggesting that an additional mechanism is used in
those domains (Cryderman et al., 1999; Ekwall et al.,
1999). Association of HP1 and a dependence on Su(var)3-9
activity have also been identified as critical in silencing
particular euchromatic genes, both in mammalian sys-
tems (Firestein et al., 2000; Nielsen et al., 2001) and in
Review
495
Drosophila (Hwang et al., 2001). Interestingly, it appears
that histone H3-mLys9 at Rb-associated genes is quite
limited; one nucleosome at the promoter is so modified,
while an immediately upstream nucleosome is not (Niel-
sen et al., 2001), suggesting a difference in the capacity
of the modified structure to spread. Histone H3-mLys9
is also associated with the inactive X chromosome in
human cells (Boggs et al., 2001; Heard et al., 2001; Pe-
ters et al., 2001a), but no HP1 homologs have been
identified preferentially associated with this domain.
Whether differences in the degree of histone methylation
or other modifications of histone H3 are important in
determining any partner of H3-mLys9 in this case re-
mains to be seen.
Cytosine Methylation
The third silent chromatin mark we will consider, 5-meth-
ylcytosine (5mC), affects the DNA itself. Postreplicative
methylation of cytosine is carried out by a diverse group
of cytosine DNA methyltransferases (Dnmt’s; Colot and
Rossignol, 1999). Beyond this, little is known about the
mechanisms that establish, maintain, and modify cyto-
sine methylation patterns. At the whole genome level,
it is clear that cytosine methylation patterns can be quite
dynamic. The best example is the erasure and resetting
of cytosine methylation in early mammalian develop-
ment (Reik et al., 2001). However, large swings in cyto-
sine methylation levels have not been detected during
zebrafish development (Macleod et al., 1999), and the
evidence in plants is contradictory (Oakeley et al., 1997;
Richards, 1997). Regardless of whether de novo methyl-
ation occurs every generation or in rare initiating events,
certain DNA sequences must be targeted for cytosine
methylation. At present, little is understood about the
primary DNA sequence determinants for targeting, if
any. Analysis of a Neurospora sequence prone to de
novo methylation indicated the presence of redundant
elements promoting methylation and suggested that
TpA-rich sequences may be important (Miao et al.,
2000). Unfortunately, similar detailed studies are not
available in other organisms. Certain cytosine methyl-
transferases, such as mouse Dnmt3a and Dnmt3b, are
specialized to carry out de novo methylation (Okano et
al., 1999). However, these enzymes do not appear to
have the intrinsic capacity for discrimination among pri-
mary nucleotide sequences, nor among higher-order
structures. These considerations suggest that de novo
cytosine methyltransferases might be taking cues from
another epigenetic mark.
Establishing Cytosine Methylation Patterns:
The Histone Methylation Connection
Communication between the histone code and cytosine
methylation may provide at least a partial answer to
the long-standing question of how cytosine methylation
patterns are established. The most direct evidence for
a connection with histone methylation comes from ge-
netic screens for cytosine hypomethylation mutants in
Neurospora. The genome of this filamentous fungus
contains 5-methylcytosine (ⵑ1.5 % of total C) concen-
trated in repetitive DNA (e.g., rRNA genes) and remnants
from RIP activity (repeat induced point mutation, a hy-
permutation surveillance system that detects sequence
duplications; Selker, 1997). Two Neurospora mutations
completely abolish cytosine methylation in vegetative
cells. One of these, dim-2, disrupts a gene encoding
a cytosine methyltransferase (Kouzminova and Selker,
2001). The other, dim-5, maps to a gene encoding a
histone H3 methyltransferase (Tamaru and Selker, 2001).
The predicted DIM-5 gene product contains a SET do-
main flanked by cysteine-rich elements and has se-
quence similarity to the histone methyltransferases Clr4
and Su(var)3-9, although it lacks a chromo domain. Re-
combinant DIM-5 protein exhibits histone methyltrans-
ferase activity in vitro. Strikingly, transformation of Neu-
rospora with modified histone H3 genes with a
substituted amino acid at Lys9 (the probable site of
methylation by DIM-5) reduces cytosine methylation and
relieves 5mC mediated gene silencing (Tamaru and
Selker, 2001).
Given that the dim-5 mutation appears to abolish all
cytosine methylation, the results suggest that all DNA
methylation in Neurospora takes its cue from histone
H3-mLys9. It will be important to determine whether the
histone methylation-DNA methylation connection is also
found in other organisms, and if so, whether all cytosine
methylation lies downstream of histone methylation. The
dim-5 mutation causes more phenotypic defects than
the dim-2 cytosine methyltransferase mutation, sug-
gesting that a histone methylation deficiency has effects
beyond those that result from loss of cytosine methyla-
tion (Tamaru and Selker, 2001).
A connection between the histone code and the 5mC
code is supported by other findings. The presence in
flowering plants of cytosine methyltransferases that
contains a chromo domain (Bartee et al., 2001; Henikoff
and Comai, 1998; Lindroth et al., 2001; Papa et al., 2001)
is particularly intriguing. Such “chromo methyltransfer-
ases” (CMTs) might be recruited to a genomic region by
nucleosomes containing histone H3-mLys9; thus, histone
modification would provide a foundation for establishing
DNA methylation patterns. However, the CMTs have not
yet been demonstrated to bind methylated histone H3,
nor is it clear that these methyltransferases possess de
novo methyltransferase activity. Moreover, chromo meth-
yltransferases have not been documented outside of plant
species. Consequently, chromo methyltransferases are
unlikely to be solely responsible for translating the his-
tone methylation code into the 5mC epigenetic mark.
Indirect models for the flow of information from his-
tone H3-mLys9 to 5mC also need to be considered. The
H3-mLys9 mark creates a foundation for HP1 interaction
and subsequent heterochromatin formation. Cytosine
methylation may be targeted to heterochromatin due
to any number of characteristics, including nonhistone
chromosomal protein content, subnuclear localization,
or DNA replication timing. Disruption of heterochromatin
by loss of the H3-mLys9 mark may lead to loss of 5mC
through a number of intermediary steps. A “chromatin
first/cytosine methylation second” model is consistent
with the demonstration that loss or alteration of cytosine
methylation can be caused by mutations in SWI2/SNF2-
like proteins (see Narlikar et al., 2002 [this issue of Cell])
in Arabidopsis, mice, and humans (Dennis et al., 2001;
Gibbons et al., 2000; Jeddeloh et al., 1999).
Cell
496
Establishment of Cytosine Methylation Patterns:
The RNA Connection
Another potential targeting mechanism has emerged
from work on posttranscriptional gene silencing in
plants, which resembles RNA interference (RNAi). Es-
tablishment of posttranscriptional gene silencing is cor-
related with cytosine methylation of sequences with
similarity to the RNA targeted for turnover (Pelissier et
al., 1999). Furthermore, an engineered hairpin RNA spe-
cies has been used to target cytosine methylation to
promoter sequences in tobacco and Arabidopsis (re-
viewed in Bender, 2001; Matzke et al., 2001).
These examples of RNA-dependent DNA methylation
(RdDM) suggest a number of models (Bender, 2001;
Matzke et al., 2001), including generation of unusual
nucleic acid structures that can be targeted by cytosine
methyltransferases. Alternatively, the RNA molecules
may serve as cofactors for cytosine methyltransferases,
directing de novo methylation to particular sequences,
or the pathway may be indirect. RdDM has only been
documented in plants, and its importance as a general
strategy to establish cytosine methylation patterns re-
mains to be demonstrated. However, RNA-mediated
posttranscriptional silencing systems exploiting similar
machinery appear to be conserved in eukaryotes (Mat-
zke et al., 2001).
Inheritance of Cytosine Methylation
Once 5mC patterns have been established, they must be
maintained in order to serve as an inherited epigenetic
code. The potential of cytosine methylation as a mitotic
memory device was first described in the “maintenance
methylation” model (Holliday and Pugh, 1975; Riggs,
1975). The essential feature of the model is clonal inheri-
tance of the 5mC patterns through mitotic, and possibly
meiotic, divisions based on the symmetrical nature of
the sequences modified (e.g., CpG) and the specificity
of “maintenance” DNA methyltransferases for hemi-
methylated DNA. The basic tenets of the maintenance
methylation model have been supported by a wealth of
evidence. The bulk of cytosine methylation occurs very
shortly after DNA replication, catalyzed by methyltrans-
ferases that have hemimethylated substrate prefer-
ences, recruited to the vicinity of the replication fork by
interaction with PCNA (Chuang et al., 1997). However,
the classic maintenance methylation model is inade-
quate to explain the variability of 5mC patterns within
individuals and omits some of the known components
of the cytosine methylation system. Not all cytosine
methylation occurs at short symmetrical sequences, so
a simple maintenance methyltransferase, making refer-
ence solely to cytosine methylation on the template
strand, cannot perpetuate methylation patterns. Mainte-
nance of 5mC patterns at nonsymmetrical sites might
involve reiterated de novo methylation (Kouzminova and
Selker, 2001) and may represent an additional tier of
DNA methylation superimposed on the pattern of 5mC at
symmetrical sites. The machinery necessary to maintain
5mC at asymmetric sites has not been firmly estab-
lished, but clues are emerging. Dnmt3a has been impli-
cated in the synthesis of 5mC at asymmetric sites in
mice (Ramsahoye et al., 2000). In Neurospora, a single
cytosine methyltransferase, DIM-2, is responsible for all
vegetative 5mC, including both symmetrical and asym-
metrical sites (Kouzminova and Selker, 2001). In plants,
5mC in asymmetrical sequences has been associated with
chromo methyltransferases (Bartee et al., 2001; Lindroth
et al., 2001) and RNA-dependent DNA methylation (Matzke
et al., 2001; Pelissier et al., 1999).
The classical methylation maintenance model ac-
counts for loss of 5mC through a passive mechanism:
DNA replication in the absence of maintenance methyla-
tion. Cytological data using immuno-detection of 5mC
argues that passive demethylation causes the dramatic
erasure of DNA methylation patterns in early mammalian
development (Rougier et al., 1998). However, observa-
tion of 5mC loss in the absence of DNA replication has
suggested an active demethylation mechanism as well
(Oakeley et al., 1997; Santos et al., 2002). A 5mC-DNA
glycosylase might also contribute to the dramatic
swings in cytosine methylation seen in mammalian de-
velopment (Jost et al., 2001).
The DNA Methylation-Histone
Deacetylation Connection
The execution of gene silencing from the 5mC mark
involves modulation of another epigenetic mark, hypo-
acetylation of histones. Two independent pathways
have been discovered in vertebrates connecting 5mC
to histone deacetylation. The first uses methyl cytosine
binding proteins, MeCP, or MBD (methyl binding do-
main) proteins as adaptors connecting 5mC to histone
deacetylase complexes (Jones et al., 1998; Ng et al.,
1999). Several MBD/MeCP protein-HDAC complexes
have been identified in mammalian cells (reviewed by
Dobosy and Selker, 2001). These complexes act to re-
duce local histone acetylation levels using the 5mC
marks on the DNA as a guide.
A second pathway, also uncovered in mammals, oper-
ates through a physical interaction between the mainte-
nance cytosine methyltransferase DNMT1 and HDACs
(Robertson et al., 2000; Rountree et al., 2000). The cata-
lytic domain of DNMT1 is not necessary for this interac-
tion, suggesting that this cytosine methyltransferase is
actually a transcriptional corepressor independent of its
ability to methylate DNA. This interaction could act to
reinforce inheritance of silent chromatin by facilitating
histone deacetylation at the replication forks, where
DNMT1 acts to maintain the 5mC epigenetic mark on
methylated DNA sequences.
Epigenetic information may also flow from the histone
acetylation state back to cytosine methylation. The
HDAC inhibitor TSA leads to cytosine hypomethylation
at specific sequences in Neurospora, and a similar effect
has been noted in mammalian cells (Cervoni and Szyf,
2001; Selker, 1998). The loss of DNA methylation may
be related to transcriptional activation, but other mecha-
nisms have been proposed, including activation of cyto-
sine demethylases. Inhibition of histone deacetylation
does not lead to global loss of DNA methylation, how-
ever. For example, disruption of a histone deacetylase
gene in plants did not lead to a generalized loss of 5mC
despite a 10-fold elevation in histone H4 acetylation
(Tian and Chen, 2001). Regardless of the significance
of the retrograde signaling, the well-established flow of
information from 5mC to histone deacetylation closes
Review
497

the loop of the self-reinforcing cycle shown in Figure 1 fication pathways operating on each covalent mark also
for those organisms that utilize cytosine methylation. interact and reinforce each other.
In organisms lacking 5mC, a histone modification
code appears to be sufficient to mark and perpetuate
Targeting and Establishment of Silent
silent chromatin domains. The feedback loop between
Chromatin Domains
histone methylation and histone deacetylation, coupled
The cycle of epigenetic marks discussed here suggests
with mechanisms to maintain these modifications, ap-
that initiation of heterochromatin formation, or similar
parently provides stable silencing. In fact, S. cerevisiae
silencing of euchromatic domains, requires acquisition
appears to utilize neither DNA modification nor the HP1/
of at least one epigenetic mark. What do we know about
histone H3-mLys9 complex, relying solely on deacetyla-
entry into the cycle? In S. cerevisiae, protein interactions
tion of histones H3/H4 as an epigenetic mark to maintain
with specific cis-acting DNA sequences, such as E and
silencing. The transmission of chromatin states requires
I at the HM loci, or telomeric repeats, provide the founda-
that at least one of the covalent marks be inherited
tion to recruit the SIR silencing complexes (Figure 3).
through mitotic, and possibly meiotic, cell divisions. Evi-
The EF2-Rb-SUV39H1-HP1 interaction in mammals also
dence discussed above argues that all three of these
implicates specific DNA sequences (binding sites for
marks meet the criteria of persistence through mitosis.
EF2) as initiation sites for silencing (Nielsen et al., 2001).
While self-reinforcing mechanisms may be advanta-
Silencing within the mating type locus of S. pombe
geous to ensure maintenance of silencing on genomic
appears to be controlled both by local elements (REII
sequences to be archived for the long-term in a nonex-
and mat3 silencer) operating similarly to E and I in
pressed state (e.g., transposons, pericentromeric re-
S. cerevisiae and by packaging of the domain as a whole,
peats), there may be a need to reconfigure silenced
dependent on a block of repetitive DNA (Grewal, 2000).
chromatin as a prerequisite to expression of specific
In other organisms, the repetitive nature of the locus,
genes (e.g., mating type switching). In this case, what
rather than the primary DNA sequence, may be a trigger
general mechanisms can be used to break the hetero-
(Henikoff, 2000). The mechanisms at work are not clear,
chromatin reinforcing cycle? Removal of the histone H3-
but hints can be derived from the repeat sensing/silenc-
mLys9 mark may require turnover of the entire protein,
ing phenomena in filamentous fungi, MIP (methylation
as no histone demethylase has yet been identified. His-
induced premeiotically) in Ascobolus and RIP in Neuros-
tones, however, are generally very stable. In compari-
pora (Selker, 1997). In these systems, repeats appear
son, the 5mC mark is more easily erased by passive or
to be recognized by a DNA-DNA pairing mechanism.
active demethylation mechanisms. The most malleable
In Ascobolus, cytosine methylation can be transferred
mark is the deacetylation of histones, the levels of which
between alleles, accompanying meiotic pairing and re-
are set by the competing activities of histone acetylases
combination events (Colot et al., 1996). RNA signals
and histone deacetylases.
may provide another entrée into the cycle of epigenetic
silencing. Two noncoding RNA species, Xist and Tsix,
Concluding Remarks
are pivotal for initiation and choice in X chromosome
The goal of this review is to explore the evidence for a
inactivation in mice (Mlynarczyk and Panning, 2000),
simple signaling framework among three covalent epi-
where H3-Lys9 methylation is an early event (Heard et
genetic marks, two modifications of histones and one
al., 2001). RNA may also have a role in initiating silent
modification of DNA. Even at this early stage, the circular
chromatin formation by directing the acquisition of cyto-
nature of the signaling pathway and the involvement of
sine methylation marks (Martienssen and Colot, 2001;
covalent marks at both the protein and DNA level sug-
Matzke et al., 2001). Resolution of this question will be
gest important features of chromatin-based epigenetic
one of the major goals of future research.
inheritance. The self-reinforcing nature of the signaling
suggests that heterochromatin can be maintained and
Propagation of Silencing propagated, provided that at least one of the epigenetic
Once a genomic region has been targeted for silencing marks is inherited with the chromatin. As stressed
by acquisition of one or more covalent epigenetic marks, above, this framework is an assemblage of independent
a silent chromatin identity can be propagated through interactions teased out in different experimental sys-
the interactions illustrated in Figure 1. The general fea- tems. One of the challenges for the future will be to
tures of the system include (1) positive signaling be- fill in the gaps to determine how well the framework
tween the different covalent epigenetic marks (shown describes the operation of the epigenetic signaling path-
by arrows) and (2) enzymatic complexes/pathways that way in different eukaryotes. Other covalent epigenetic
recognize each mark and catalyze the formation of the marks, in addition to the three emphasized here, will
same mark (represented by loops at the vertices of the likely be important. The interactions between histone
triangle). For example, in yeast, the histone H3/H4 modifications already documented promises that this
deacetylation mark is recognized by Sir3, leading to simple framework will become much more complex in
recruitment of the Sir2 histone deacetylase (see Figure the near future.
3). The histone H3-mLys9 mark is recognized by HP1,
which can apparently recruit the histone methyltransfer- Acknowledgments
ase activity of Su(var)3-9 homologs. The third self-rein-
We thank our colleagues Joel Eissenberg, Boris Leibovitch, Craig
forcing loop is carried out by maintenance cytosine Pikaard, Danesh Moazed, Eric Selker, Christopher Shoenherr, and
methyltransferases, which have a substrate preference anonymous reviewers for providing helpful comments on the manu-
for hemimethylated DNA. As discussed above, the modi- script. We apologize to those whose work was not cited directly
Cell
498
due to lack of space. The authors gratefully acknowledge support
from the National Science Foundation (MCB9985348; E.J.R.) and
the National Institutes of Health (HD 23844; S.C.R.E.).
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