DEVELOPMENTAL DYNAMICS 236:1144 –1156, 2007

WHAT’S COOL IN DEV BIO

Epigenetics in Development
Julie C. Kiefer*

It has become increasingly evident in recent years that development is under epigenetic control.
Epigenetics is the study of heritable changes in gene function that occur independently of alterations to
primary DNA sequence. The best-studied epigenetic modifications are DNA methylation, and changes in
chromatin structure by histone modifications, and histone exchange. An exciting, new chapter in the field
is the finding that long-distance chromosomal interactions also modify gene expression. Epigenetic
modifications are key regulators of important developmental events, including X-inactivation, genomic
imprinting, patterning by Hox genes and neuronal development. This primer covers these aspects of
epigenetics in brief, and features an interview with two epigenetic scientists. Developmental Dynamics 236:
1144 –1156, 2007. © 2007 Wiley-Liss, Inc.

Key words: epigenetics; genomic imprinting; X chromosome inactivation; REST; PcG; DNA methylation; chromosomal
interactions; ncRNA

Accepted 22 January 2007

INTRODUCTION
Presented here is an introduction to
epigenetic modifications, followed by
descriptions of epigenetically regu-
lated developmental events. The
primer concludes with an insightful
discussion about current topics in the
field with epigeneticists Giacomo Cav-
alli, Ph.D., and Noel Buckley, Ph.D.
EPIGENETIC
MODIFICATIONS
DNA Methylation
DNA methylation is a covalent modi-
fication that triggers heritable gene
silencing (Levenson and Sweatt,
2005). DNA methyltransferases
(Dnmts) catalyze the reaction by
transferring a methyl group to the 5
position of cytosine on CpG dinucleoti-
des. This triggers silencing in one of
two ways. First, methylation can di-
rectly interfere with transcription fac-
tor binding to recognition sites on
DNA. Second, methyl-CpG binding
domain proteins (MBPs) can reinforce
silencing by recruiting corepressor
complexes that harbor histone
deacetylases (HDACs) or histone
methyltransferases (HMTs). MBP-as-
sociated HMTs fuel a positive feed-
back loop between DNA methylation
and another epigenetic silencing
mark, methylated lysine 9 on histone
3 (H3K9; Fujita et al., 2003; Fuks et
al., 2003).
Histone Methylation
The nucleosome, consisting of 146 bp
of DNA wrapped around an octamer of
histones, H2A, H2B, H3, and H4, is
the basic structural unit of chromatin
(Margueron et al., 2005). Histones
have a central globular domain and
relatively unstructured N- and C- ter-
minal tails. The N-terminal tail is pri-
marily subjected to posttranslational
modifications, such as acetylation,
phosphorylation, ubiquitination, and
methylation, which can result in pro-
found changes in local chromatin
structure.
The histone code hypothesis states
that specific combinations of histone
modifications form a language that
specifies the structural state of chro-
matin (Strahl and Allis, 2000). Effec-
tor proteins read and carry out the
code’s instructions to specify hetero-
chromatin formation, DNA that is
tightly packed with nucleosomes, or
more loosely packed euchromatin.
Compared with euchromatin, hetero-
chromatic DNA is largely inaccessible
to transcription factors and chromatin
remodelers, making it relatively tran-
scriptionally inert.
Three histone marks encode epige-
netic processes that are described in

Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, Utah
*Correspondence to: Julie C. Kiefer, Ph.D., Department of Neurobiology and Anatomy, 20 North 1900 East, 401 MREB,
University of Utah, Salt Lake City, UT 84132. E-mail: jkiefer@neuro.utah.edu
DOI 10.1002/dvdy.21094
Published online 15 February 2007 in Wiley InterScience (www.interscience.wiley.com).

© 2007 Wiley-Liss, Inc.


the sections below. H3K9 and H3K27
methylation are marks of silent or re-
pressed chromatin (Cheung and Lau,
2005). Heterochromatin protein 1
(HP1) binds methylated H3K9 by
means of its chromodomain and in-
duces local chromatin condensation.
The chromodomain protein Polycomb
(PC) binds methylated H3K27 and in-
duces silencing in ways that are not
yet understood. Promoters and tran-
scribed regions of many active genes
are decorated with methylated H3K4,
a modification recognized by two fac-
tors associated with transcriptionally
active genes. CHD1 and BPTF, a sub-
unit of the multiprotein complex
NURF, both remodel nucleosomes in
an ATP dependent manner (Sims and
Reinberg, 2006).
Compared with other histone post-
translational modifications, lysine
methylation is a long-lasting mark
(Margueron et al., 2005). It is thermo-
dynamically very stable, persists
through mitosis, and is found on chro-
matin regions that are silenced for the
long term, such as pericentric chroma-
tin (dimethylated H3K9, H3K9me2)
and the inactive X chromosome
(H3K27me3, H3K9me2). Only re-
cently was it discovered that histone
methylation can be catalytically re-
versed by histone demethylases (Shi
et al., 2004; Cloos et al., 2006). De-
methylases are probably under tight
control, because continual histone
methylation is a hallmark of heritable
transcriptional cellular memory.
It should be noted that specific his-
tone modifications are not always pre-
dictive of gene activity. In embryonic
stem (ES) cells, lineage specific genes
that are either repressed or tran-
scribed at low levels are “bivalent,”
bearing marks of active (H3K4me,
H3K9ac) and repressive (H3K27me3)
chromatin (Azuara et al., 2006; Bern-
stein et al., 2006). These observations
have led to the hypothesis that dual
markings enable developmental genes
to be repressed in ES cells while
poised for quick activation.
It is likely that chromatin remodelers
and other histone modifications are addi-
tional sources of epigenetic changes. For
example, histone deacetylase (HDAC) is
a component of the polycomb group and
REST/NRSF corepressor complexes,
both epigenetic modifiers (see Polycomb
and REST/NRSF sections). Relative hi-
stone acetylation state affects factor re-
cruitment and/or structural changes in
chromatin. The ATPase Brahma-re-
lated gene 1 (Brg1), a member of the
epigenetic activators trithorax group, is
also a component of the SWI/SNF nu-
cleosome remodeling complex, and reg-
ulates neurogenesis, myogenesis, and
left–right asymmetry (Seo et al., 2005;
Ohkawa et al., 2006; Takeuchi et al.,
2007). SWI/SNF causes nucleosomes to
slide along DNA, either exposing or oc-
cluding regulatory elements that affect
the transcriptional status of nearby
genes. Are these epigenetic mecha-
nisms? It is partly a question of seman-
tics (see Interview). At present, modifi-
cation by HDACs and nucleosome
remodeling are considered dynamic,
whereas histone methylation is a tried
and true long-lasting epigenetic mecha-
nism. Although it is clearly not so black-
and-white, the discussion here is
limited to established epigenetic mech-
anisms.
Histone Variants
Chromatin proteins are dynamic, and
a histone can be exchanged for a vari-
ant within its own class (Cheung and
Lau, 2005). Structural differences be-
tween variants impact overall nucleo-
some structure, altering accessibility
of DNA to transcription factors and
chromatin remodelers.
H2A has the largest number of iden-
tified variants, and because of its stra-
tegic position in the nucleosome, H2A
histone variants are particularly
suited to dramatically alter histone
and DNA contacts. Two variants,
macroH2A and H2A-Barr body-defi-
cient (H2A-Bbd), are involved in X
chromosome inactivation. macroH2A
promotes gene silencing by preventing
remodeling by the SWI/SNF complex,
initiation of p300-dependent polymer-
ase II transcription, and p300-depen-
dent histone acetylation (Angelov et
al., 2003; Doyen et al., 2006a). H2A-
Bbd is associated with active chroma-
tin. The structure of its central globu-
lar domain allows only 130 bp of DNA
to be wrapped around the nucleosome,
creating a more relaxed, accessible
chromatin structure (Doyen et al.,
2006b). H2A.Z is associated with both
active and inactive chromatin
(Cheung and Lau, 2005). It is not un-
EPIGENETICS PRIMER 1145
derstood how histone variants are tar-
geted to specific chromosomal regions.
Long-Distance Chromosomal
Interactions
Another epigenetic mechanism has
been brought to the fore in recent
years: alterations in gene activity in-
duced by direct interactions between
chromosomal regions that are posi-
tioned at long-distances from one an-
other. Long-distance chromosomal
interactions can mediate gene activa-
tion or repression (Grimaud et al.,
2006; Lomvardas et al., 2006), and
both intra- and interchromosomal as-
sociations have been observed. In
2002, two papers were the first to re-
port intrachromosomal interactions at
the endogenous ␤-globin locus, be-
tween the locus control region (LCR)
and active ␤-globin genes located
some 50 kb away. Subsequently, it
was found that, in T-cells, the LCR for
cytokines Il4, Il5, and Il13 is posi-
tioned in close proximity to each of
their promoters, bridging 120 kb in
total (Spilianakis and Flavell, 2004).
The interactions may facilitate com-
munication between regulatory ele-
ments, influencing the transcriptional
state of associated genes.
Within the past year, there has
been a surge of papers documenting
nuclear colocalization of domains on
two different chromosomes (Spili-
anakis et al., 2005; Bacher et al., 2006;
Grimaud et al., 2006; Lomvardas et
al., 2006; Xu et al., 2006). In naive
T-helper cells, there is an interchro-
mosomal association between the cy-
tokine LCR mentioned above and in-
terferon-␥, a determinant of the TH1
cell fate. Upon differentiation, the in-
teraction is lost, suggesting that it co-
ordinates active gene expression
(Spilianakis et al., 2005). Interactions
between X-chromosomes, polycomb
response elements (PREs), and olfac-
tory receptor enhancer and promoters
have also been observed, and are de-
scribed in the X-chromosome inactiva-
tion, Polycomb and Olfactory receptor
sections below. The discovery of inter-
chromosomal interactions highlights
a possible role for nuclear architecture
in epigenetic regulation. Regulatory
chromosomal domains may be seques-
tered to a subcompartment of the nu-
cleus, which acts as a center for coor-
1146 KIEFER ET AL.
dinating either gene silencing or
activation (O’Brien et al., 2003).
Broad implications can be gleaned
from the long-distance chromosomal
associations that have been discov-
ered, despite that there is only a hand-
ful of examples so far. Because intra-
and interchromosomal association oc-
curs in different types of genes and in
varied cell types, the phenomenon is
likely global. Moreover, all reported
interactions are cell-type specific, and
among interactions that are transient,
the timing of chromosomal domain co-
localization tracks with changes in
cellular differentiation. Taken to-
gether, these findings suggest that
long-distance chromosomal interac-
tions are developmental regulators.
DEVELOPMENTAL GENE
REGULATION
X-chromosome Inactivation
Mammalian females have two X chro-
mosomes (XX), whereas males have
one (XY), posing the biological prob-
lem of how to equalize the dosage of
X-linked genes between the two sexes.
The mammalian subclasses euther-
ians, marsupials, and monotremes,
and other species, such as C. elegans
and Drosophila, have each taken dif-
ferent approaches to solving this prob-
lem (Lucchesi et al., 2005). Presented
here is the eutherian solution, mainly
studied in mice, which uses epigenetic
mechanisms to inactivate one of the X
chromosomes in female cells (Heard,
2005; Thorvaldsen et al., 2006).
In the preimplantation mouse em-
bryo, all cells initially undergo im-
printed inactivation of the paternal X
chromosome (Xp). In the late blasto-
cyst, inner cell mass cells reactivate
Xp, while extraembryonic cells (tro-
phoectoderm, primitive endoderm)
maintain Xp inactivation. Subse-
quently, the epiblast, or future em-
bryo, initiates random X inactivation
(Allegrucci et al., 2005). The mecha-
nisms of imprinted inactivation of Xp
and random X inactivation are largely
similar; differences are noted below.
Before random X inactivation, there
are transient interchromosomal inter-
actions between X inactivation center
(Xic) loci on the X chromosomes
(Heard, 2005; Xu et al., 2006). This
event may promote cross-talk between
the two Xics, somehow enabling its
critical functions of counting the num-
ber of X chromosomes in a cell, and
determining which X chromosome will
be inactivated in female cells.
The future inactivated X chromo-
some (Xi) is characterized by accumu-
lation of the noncoding (nc) RNA, Xist.
Within Xic are three genes encoding
ncRNAs that cross-regulate one an-
other: Xist, Tsix, and Xite. Xist is re-
quired to initiation inactivation of one
of the X chromosomes, Tsix is anti-
sense to Xist and invokes Xist silenc-
ing, and Xite positively regulates Tsix
transcription. Following Xic pairing,
Xi down-regulates Tsix transcription,
while Xist is up-regulated. On the ac-
tive X chromosome (Xa), Tsix re-
presses Xist, but counterintuitively
does not do so by destabilizing Xist
RNA (Sado et al., 2005; Sun et al.,
2006). Rather, Tsix induces H3K4me2,
a mark of euchromatin, which some-
how results in Xist repression. The
precise manner by which this occurs is
under debate (Navarro et al., 2006;
Sun et al., 2006). There are no inter-
actions between Tsix and Xist during
imprinted X inactivation, because
they are imprinted maternally and
paternally (Xi), respectively. In both
random and imprinted X inactivation,
Xist transcripts accumulate on Xi,
“coat” it, and may function as a scaf-
fold to recruit silencing histone modi-
fying enzymes.
Many epigenetic mechanisms pro-
mote heterochromatization of Xi.
First, Xi is decorated with histone
methylation marks characteristic of
chromatin silencing. The Polycomb
group (PcG) HMT, EZH2, associates
with Xi and endows it with the silenc-
ing mark H3K27me3 (Plath et al.,
2003). Xi also acquires the repressive
mark H3K9me2, and the activation
mark H3K4me2 is underrepresented
(Valley et al., 2006). Second, the his-
tone composition on Xi is altered com-
pared with Xa and autosomes. Xi is
relatively devoid of activating H2A-
Bbd, H2A.Z, and a large proportion
of H2A are replaced by repressive
macroH2A histone variants (Chad-
wick and Willard, 2001, 2003). Third,
Xi in embryonic cells is sprinkled
with hypermethylated CpG islands
(Kratzer et al., 1983). Layers upon
layers of inactivation might guarantee
Xi repression, which must be extraor-
dinarily stable to remain inactive for
the lifetime of the animal.
Genomic Imprinting
Imprinting is the process by which
epigenetic marks permit monoallelic
expression from a single parental al-
lele. There are around one hundred of
imprinted genes, many of which regu-
late placental and fetal growth. So far,
research of different imprinted loci
has shown that there is variation be-
tween the molecular processes that
regulate imprinting. Nevertheless,
there are parallels between the epige-
netics of genomic imprinting and X
chromosome inactivation (XCI; Reik
and Lewis, 2005).
Like XCI, silencing of imprinted
genes can be regulated by ncRNAs.
Global identification of imprinted
genes has shown that 30% encode for
ncRNAs, indicating that they may be
commonly used for imprinting. Im-
printed genes usually occur in clusters
of 3–11 genes. Two noncoding RNAs
that silence their respective ICRs are
Air in the Igf2r cluster and Kcnq1ot1
in the Kcnq1 cluster (Delaval and Feil,
2004). Genes in the Igf2r and Kcnq1
clusters are expressed on the mater-
nal chromosome and encode a fetal
growth regulator and cardiac potas-
sium channel, respectively. On the
maternal chromosome, DNA methyl-
ation prevents transcription of Air
and Kcnq1ot1, allowing clusters of im-
printed genes to be expressed (Stoger
et al., 1993; Engemann et al., 2000).
On the paternal chromosome, all
three coding genes in the Igf2r cluster,
Igf2r, slc22a2, and slc22a3, are si-
lenced by Air, even though it is only
antisense to Igf2r (Sleutels et al.,
2002). How Air silences Igf2r cluster
remains to be determined, but regula-
tory mechanisms behind the silencing
of Kcnq1 may yield some clues. Pater-
nal Kcnq1ot1 induces silencing by tar-
geting PcG-mediated H3K27 and
H3K9 methylation to the cluster (Um-
lauf et al., 2004). This finding suggests
that ncRNA regulates genomic im-
printing by altering chromatin states,
like ncRNAs Tsix and Xist during XCI.
Indeed, imprinting and XCI are be-
lieved to be evolutionarily linked
(Reik and Lewis, 2005).
In contrast to the Igf2r and Kcnq1

clusters, silencing of the clustered im-
printed genes Igf2 and H19 appears to
be mainly regulated by DNA methyl-
ation. Igf2 and H19 are in the same
cluster, but expression of Igf2 is pater-
nal and H19 is maternal. Their mutu-
ally exclusive expression is regulated
by an imprint control region (ICR), a
stretch of DNA that regulates expres-
sion of all genes in imprinted clusters
(Delaval and Feil, 2004). On the ma-
ternal allele, Igf2/H19 ICR is unmeth-
ylated. This permits an insulator,
CCCTC binding factor (CTCF), to bind
a boundary element within the ICR.
CTCF inhibits Igf2 expression by pre-
venting interactions between its pro-
moter and enhancer (Hark et al.,
2000). At the same time, lack of ICR
methylation is permissive for mater-
nal H19 expression. On the paternal
allele, the ICR is methylated, directly
preventing H19 expression and allow-
ing for Igf2 expression by blocking
CTCF binding (Engel et al., 2004).
H19 encodes an ncRNA, but unlike
Air and Kcnq1ot1, does not function as
a silencer.
Polycomb Group Regulates
Gene Silencing
Beyond XCI and genomic imprinting,
one might think that epigenetics and
embryonic development are incompat-
ible. Epigenetics positively or nega-
tively affect transcription over the
long term, while development is a dy-
namic process dependent on rapid and
frequent changes in gene expression.
Work with PcG genes first demon-
strated that at least some aspects of
development are under control of epi-
genetic mechanisms. PcG maintains
the spatial pattern of Hox genes, con-
served gene groups that regulate an-
terior/posterior patterning. Through
deposition of the silencing epigenetic
marks, H3K27me3 and to a lesser ex-
tent H3K9me2, PcG represses Hox
genes in regions where they are not
normally expressed. Flies and mice
mutant for PcG genes exhibit ectopic
Hox expression, sometimes eliciting
homeotic transformations (Jürgens,
1985; van der Lugt et al., 1994). Mice
mutant for the PcG gene Bmi1 also
show defects in hematopoietic and
neural stem cell self-renewal, and in
proliferation of primary fibroblasts
(Jacobs et al., 1999; Lessard and Sau-
vageau, 2003; Molofsky et al., 2003;
Park et al., 2003). As mentioned pre-
viously, trimethylation of H3K27 by
PcG also promotes silencing on Xi and
imprinted genes. These examples il-
lustrate that PcG controls many dif-
ferent cellular processes.
PcG binds to PREs in regulatory re-
gions of target genes. Thus far only
defined in Drosophila, PREs are com-
prised of different combinations of
specific binding sites. PcG targets in-
clude developmental genes such as
various transcription factors (Fox,
Sox, Pax), and signaling molecules
(Wnts, Shh, BMPs; Bracken et al.,
2006; Negre et al., 2006). Taken to-
gether with the finding that PcG tar-
get profiles differ in undifferentiated
and differentiated cells, PcG may reg-
ulate many developmental processes.
PcG is composed of two complexes,
Polycomb repressive complex (PRC) 1
and 2. Mammalian PRC2 contains
four core proteins, EED, SUZ12, and
RbAP48, and EZH2, and PRC1 is com-
posed of more than 10 subunits in-
cluding the chromodomain protein
Polycomb (PC), the oncoprotein BMI1,
and E-3 ubiquitin ligases RING1A
and B (Lund and van Lohuizen, 2004).
PcG exerts its repressive effects in
multiple ways. The catalytically
active component of PRC2, EZH2,
trimethylates H3K27, and recruits
DNMTs to PcG target genes (Vire et
al., 2006). PC binds H3K27me3 and
brings with it the rest of the PRC1
complex (Min et al., 2003). PRC1 also
imposes silencing, at least partially by
inhibiting chromatin remodeling by
SWI/SNF (Francis et al., 2001). Other
properties of PRC1 may also contrib-
ute to its ability to repress transcrip-
tion: PRC1 ubiquitination of H2A is
critical for Hox silencing by an un-
known mechanism (Cao et al., 2005),
and at least one PRC1 variant con-
tains HDAC1 (Huang and Chang,
2004). PcG has also been reported to
prevent transcription initiation (Del-
lino et al,. 2004). Multiple epigenetic
mechanisms may make repression by
PcG failsafe.
There is mounting evidence that re-
pression is at least partly mediated by
PRE-dependent chromosomal interac-
tions. PREs on the same chromosome
or on different chromosomes can pair
together, with the former resulting in
a looping out of intervening sequence
EPIGENETICS PRIMER 1147
(Comet et al., 2006). These interac-
tions induce paring-sensitive silenc-
ing: PRE pairs silence gene expression
more efficiently than a single PRE.
Paired PREs and bound PcG proteins
aggregate as tight foci in the nucleus,
forming so-called PcG nuclear bodies
(Grimaud et al., 2006). The purpose of
the nuclear bodies may be to sequester
PRE-containing genes to a nuclear
subcompartment where silencing is
propagated and/or maintained.
PcG nuclear bodies colocalize with
or lie in close proximity to RNAi nu-
clear bodies bearing components of
RNAi machinery, including Dicer-2,
ARGONAUT1, and PIWI. Genetic
data show that RNAi machinery lies
upstream of long-range PRE associa-
tion. Mutants for single RNAi proteins
experience decay in PRE association
over time. PcG-dependent cosuppres-
sion, a phenomenon whereby intro-
duction of multiple copies of a trans-
gene results in their silencing, also
requires RNAi machinery, but is ap-
parently governed by an independent
mechanism. While cosuppression is
faulty in mutants of the RNAi machin-
ery gene homeless, PRE association is
not (Pal-Bhadra et al., 2004). Cosup-
pression mediates heterochromitiza-
tion of repeat sequences, but how
RNAi machinery maintains long-
range association of PREs is unclear.
The answer may lie in the observation
that siRNAs are generated from PRE-
containing transgenes. The authors
propose that siRNAs from PRE loci act
as “molecular glue” that keeps PcG
complexes on different PREs together.
So far, siRNAs from endogenous PREs
have not yet been detected.
Trithorax Group Promotes
Active Chromatin
As the mechanistic opposite of PcG,
trithorax group (trxG) maintains gene
expression, including Hox genes, by
facilitating euchromatin (Beisel et al.,
2002). Mammalian trxG consists of
multiple proteins and includes a pan-
oply of homologs to the Drosophila
H3K4 HMT trithorax, namely Set1a,
Set1b, Mll1, Mll2, Mll3, and Mll4, and
the SWI/SNF ATPase Brg1. trxG
binds the same chromosomal element
as PcG. In the silenced state, the ele-
ment recruits PcG (PRE), and in its
active state recruits trxG (TRE). Acti-
1148 KIEFER ET AL.
vation of PRE/TRE requires tran-
scription of the TRE. Noncoding TRE
transcripts bind and recruit the Dro-
sophila trxG HMT Ash1, which depos-
its marks for euchromatin, H3K4me3
(Sanchez-Elsner et al., 2006). Recent
studies show that BPTF specifically
binds H3K4me3, and loss of Xenopus
BPTF results in aberrant Hox expres-
sion. BPTF is a subunit of NURF and
may target the chromatin remodeling
complex to histones methylated by
trxG. NURF facilitates active chroma-
tin by repositioning nucleosomes
along DNA (Sims and Reinberg,
2006). TrxG also promotes expression
of the Hox gene Ubx by facilitating
transcriptional elongation (Petruk et
al., 2006). These findings may explain
how trxG regulates gene activation.
LINEAGE RESTRICTION
REST/NRSF Activities
Govern Neuronal Fate
Decisions
As the embryo develops, multipotent
cells activate specific gene programs
that trigger differentiation into spe-
cialized cell types. Equally important,
a cell must silence expression of genes
specific to other cell types to secure its
fate. Because repression must be
maintained throughout the life of the
animal, epigenetic mechanisms are
ideal for mediating such events.
How a cell keeps from expressing
inappropriate gene programs has
been worked out for genes from just
one cell type, the neuron. Repressor
element 1 (RE-1) silencing transcrip-
tion factor/neuron restrictive silencing
factor (REST/NRSF) is a zinc finger
protein that triggers epigenetic silenc-
ing of neuronal genes (Ballas and
Mandel, 2005). In mice lacking REST/
NRSF, the neural-specific gene ␤III-
tubulin is expressed in some non-neu-
ral tissues, but many cell types are
unaffected (Chen et al., 1998). How-
ever the mice die at E11.5, precluding
insight into the roles for REST in ner-
vous system development. These data
illustrate that REST/NRSF is not a
master regulator of neuronal gene si-
lencing, but rather maintains repres-
sion.
In non-neuronal cells, REST/NRSF
binds RE-1 elements in regulatory re-
gions of target genes and blocks their
transcription. The elements reside in
genes central to neurogenesis, includ-
ing ion channels, neurotransmitter re-
ceptors, axonal guidance molecules,
and the neurogenic gene NeuroD
(Bruce et al., 2004). REST/NRSF im-
poses dynamic, impermanent repres-
sion through its association with the
corepressors CTD phosphatase, which
inhibits RNA polymerase II, and
HDACs, which limit chromatin acces-
sibility (Battaglioli et al., 2002; Yeo et
al., 2005). The corepressor Co-REST
coordinates stable, epigenetic repres-
sion by directly binding the H3K9
HMT G9a and the H3K4 demethylase
LSD1 (Roopra et al., 2004; Shi et al.,
2004). Co-REST also recruits addi-
tional epigenetic silencing factors, the
methyl DNA binding protein MeCP2,
and the H3K9 HMT SUV39H1 (Lu-
nyak et al., 2002). Finally, heterochro-
matin protein 1 (HP1) binds and con-
denses neuronal genes marked with
methylated H3K9 (Lunyak et al.,
2002).
REST/NRSF is also expressed in
cells that have the potential to become
neurons, embryonic stem cells and
neuronal progenitors. In multipotent
cells, neuronal genes must be permis-
sive for activation. Therefore, REST/
NRSF must not simply be an “off”
switch for neuronal genes. Rather, the
degree of repression mediated by
REST/NRSF is tailored to the devel-
opmental stage of the cell (Ballas et
al., 2005). In pluripotent embryonic
stem cells, REST/NRSF binds RE1
sites, but H3K9 remains unmethyl-
ated, suggesting that neuronal genes
are poised for activation. In neuronal
progenitors, posttranslational modifi-
cations lead to the down-regulation of
REST/NRSF. A cell with low levels of
REST/NRSF is permissive for tran-
scription of some neuronal genes. In
differentiated neurons, transcrip-
tional repression of REST/NRSF re-
sults in derepression of most RE1-con-
taining genes. Interestingly, in the
mature neuron, a class of stimulus-
inducible genes remains methylated
at DNA and continues to be bound by
the REST corepressors MeCP2 and
Co-REST. Upon membrane depolar-
ization, expression of one such gene,
brain-derived neurotrophic factor
(BDNF), is up-regulated and MeCP2
is released while Co-REST remains
unaffected. The authors speculate
that Co-REST persists as a platform
to facilitate dynamic gene regulation
in mature neurons. This work shows
that slight manipulations of REST
and its corepressors prepare a cell
with neuronal potential for the next
phase of development.
TERMINAL
DIFFERENTIATION
DNA and Histone
Methylation Are Required
for Differentiation of
Specific Organs
Many genes that regulate epigenetic
mechanisms are expressed ubiqui-
tously, and are essential for life. These
characteristics have made it difficult
to parse out epigenetic requirements
for the development and differentia-
tion of specific cell types. Recently, Rai
et al. circumvented this problem by
reducing, but not eliminating, expres-
sion of DMNT1 and the HMT Suv39h1
in zebrafish embryos by injecting
translation blocking antisense mor-
pholino oligos (Rai et al., 2006). Sur-
prisingly the phenotypes in mor-
phants for both genes largely
phenocopy one another, and the ob-
served defects are very specific. Ter-
minal differentiation is impaired in
the intestine, exocrine pancreas, and
retina, while other organs are unaf-
fected. Explaining the observed iden-
tical phenotypes, genetic and bio-
chemical data show that DMNT1 and
Suv29h1 function in the same path-
way. Both cytosine DNA methylation
and H3K9 methylation are reduced in
DMNT1 morphants, and overexpres-
sion of suv391 rescues DMNT1 mor-
phants. It remains to be determined
what genes are targeted by the two
regulators, and what molecular cues
trigger their silencing. The answers to
these questions may help determine
whether these epigenetic modifica-
tions regulate differentiation of other
cell types.
Long-Distance Chromosomal
Interactions Mediate
Olfactory Receptor Choice
In mice, an olfactory sensory neuron
expresses only 1 of 1,300 olfactory re-
ceptor (OR) genes. How one gene is
expressed exclusive of all others has

Fig. 1. Noel Buckley, Professor, Molecular
Neurobiology, King’s College London, London,
U.K. (Left), and Giacomo Cavalli, Group Leader,
Institute of Human Genetics-CNRS, Montpel-
lier, France (Right).
been a subject of much study. Richard
Axel and colleagues recently reported
that OR choice is mediated by inter-
chromosomal interactions (Lomvar-
das et al., 2006). The H DNA sequence
was previously found to act as an en-
hancer element in cis for a cluster of
OR genes. In this study, fluorescent in
situ hybridization (FISH) visualized
the association of H with OR genes in
trans. The authors also discovered
that one H allele is silenced by DNA
methylation, a finding that gave rise
to the idea that H might only activate
one gene at a time. In support of this,
olfactory neurons in transgenic mice
bearing more than one active H ele-
ment, and OR pseudogenes express
two OR receptors, one endogenous and
one pseudogene, in ⱕ1% of cells. An
OR pseudogene was used to circum-
vent a negative feedback loop whereby
OR gene selection prevents expression
of a second OR gene. The authors hy-
pothesize that OR choice is controlled
by random, long-distance chromo-
somal association of a single H en-
hancer element with a single OR gene.
AN INTERVIEW WITH THE
EXPERTS
Developmental Dynamics: Describe
your research.
Giacomo Cavalli (Fig. 1): We are
interested in patterning mechanisms
that involve the function of Polycomb
group (PcG) and trithorax group
(trxG) proteins. These epigenetic com-
ponents were originally discovered as
regulators of Hox gene expression.
Through this function, they play a
master role in the specification of the
anteroposterior axis of the body plan.
In addition, they regulate a variety of
other genes that play important roles
in developmental patterning and reg-
ulation of cell differentiation and pro-
liferation. One important feature of
PcG and trxG proteins is that they are
able to maintain the memory of devel-
opmental decisions through cell divi-
sion.
We are studying the molecular
mechanisms of PcG/trxG-dependent
epigenetic inheritance of chromatin
states, and we are trying to under-
stand the global regulatory logic be-
hind the function of these proteins.
We have recently found that PcG pro-
teins have the ability to induce the
association of some of their target
genes in the three-dimensional space
of the cell nucleus. Now we would like
to understand how widespread are
these contacts and what is their influ-
ence on genome regulation.
Noel Buckley (Fig. 1): We have
looked at transcriptional regulation in
neurons for several years. The idea of
“epigenetic regulation” is one that has
taken hold relatively recently and has
great resonance within neurobiology
because of the potential to provide an
understanding of the molecular un-
derpinnings of the sorts of long-term
changes that are the hallmark of neu-
ronal plasticity and memory. Having
said that, caution must be used to dis-
tinguish “epigenetic changes” from
simple readily reversible histone mod-
ifications— even though the former
may depend upon the latter. This is a
well-recognized caveat and one that
many, including Bryan Turner, have
been careful to highlight.
Our own work has focused upon
changes in histone modifications that
accompany the transition from pluri-
potent ES cells, through multipotent
neural stem cells to differentiated
neurons, particularly in relation to the
neuronal transcription factor, REST.
More recently, we have begun to ex-
amine chromatin modifications at
pan-neuronal and subtype specific
genes as neuronal differentiation un-
folds.
How did you become interested in
epigenetics?
G.C.: Toward the end of the 1980s,
it started to become clear in yeast that
chromatin was not just a dull way to
condense DNA in order to pack it into
the nucleus, but rather a functional
substrate of genomic regulatory pro-
EPIGENETICS PRIMER 1149
cesses. I had the privilege to join in
the chromatin field during these years
and to witness crucial advances show-
ing that epigenetic regulation of chro-
matin is at the heart of genome function
and contributes important information
that can be passed on to the cellular
progeny along with the DNA sequence.
This is not only true in yeast and Dro-
sophila, our model organism, but also in
plants and vertebrates.
N.B.: We took up the theme of epi-
genetics relatively recently—more a
slow embrace rather than an impas-
sioned charge. The drive largely came
in parallel with our burgeoning inter-
est in neural stem cells. First, there
was an interest in identifying tran-
scription factors that determined or
regulated neuronal phenotype. Sec-
ond was a frustration borne from the
dearth of suitable tractable, meaning-
ful cellular systems to carry out these
studies—there was no neural equiva-
lent to the myoblast cell lines and
hematopoietic stem cells that under-
pinned much of our current knowl-
edge of hematopoiesis and myogen-
esis. This barrier has now been broken,
and there are several robust neural
stem cell models that are amenable to
dissecting neurogenesis in a similar
manner. The final thread of this trin-
ity was the realization that many
long-term changes induced by tran-
scription factors were mediated by
means of changes in histone modifica-
tions and/or DNA methylation. So, a
rather indirect route brought us to
epigenetics.
What papers have influenced you the
most?
G.C.: One striking paper by Shiv
Grewal and Amar Klar (Grewal and
Klar, 1996) showed that alternative
chromatin states of the Schizosaccha-
romyces pombe mating type locus
could be passed on to the majority of
the mitotic and meiotic cellular prog-
eny. This was the first clear demon-
stration that epigenetic regulation
can affect heredity, making the point
that genes are more than their pri-
mary DNA sequence. A second semi-
nal paper in the field is that of Rea et
al., identifying the first histone meth-
yltransferase enzymes, which turned
out to be proteins previously known
for their role in heterochromatin for-
mation (Rea et al., 2000). This study
paved the way to the identification of
1150 KIEFER ET AL.
many histone methylases involved in
chromatin assembly and inheritance,
including the widely conserved En-
hancer of zeste protein of the PcG as
well as histone methylase proteins of
the trxG. More than a single paper, I
would like to mention as a third event
in epigenetics a flurry of papers pub-
lished independently by several
groups in 2006, all of them mapping
the genomic distribution of PcG pro-
teins in flies and mammals (Boyer et
al., 2006; Bracken et al., 2006; Lee et
al., 2006; Negre et al., 2006; Schwartz
et al., 2006; Squazzo et al., 2006; Tol-
huis et al., 2006). Together, these
studies have a huge impact since they
show that PcG proteins regulate in a
coordinated fashion a number of con-
served transcriptional pathways of
fundamental importance for the de-
velopment of the body plan. They also
mark the beginning of a new phase in
genome-wide location studies, where
DNA chips of the latest generation,
tiling through the large genomes of
vertebrates, are coupled to chromatin
immunoprecipitation to yield very ac-
curate protein distribution maps. This
robust technology will produce mas-
sive amounts of information in the
next few years.
N.B.: As a neurobiologist I remain
intrigued by the molecular and cellu-
lar basis of mood, perception, and cog-
nition. Some time ago, a colleague
pointed out a study that had discov-
ered a significant increase in the inci-
dence of adult schizophrenia in the
offspring of mothers who had under-
gone prolonged starvation in the Chi-
nese famine of 1959 – 61 in the prov-
ince of Anhui (St. Clair et al., 2005). It
is thankfully rare that we have access
to studies such as these on the scale
necessary to have sufficient statistical
power to allow robust conclusions to
be drawn, and although not an ounce
of mechanism can be inferred, the in-
fluence of epigenetics on something as
profound as human behavior can be
clearly inferred.
Although, not strictly epigenetics,
ChIPchip studies (first pioneered by
Rick Young in yeast) have shown on a
global scale how changes in histone
modifications can be mapped over the
whole genome (Ren et al., 2000). This
is an immensely powerful technology
and allows individual histone marks
to be overlaid on to features of the
genomic landscape, and this land-
scape, in turn, can be viewed at differ-
ent developmental time points. There
are now many of these papers, but I
would pick two papers from Eric
Lander’s and Mark Groudine’s groups
since they were the first to show large-
scale correlation between histone
methylation, and acetylation and gene
activity (Schubeler et al., 2004; Bern-
stein et al., 2005). I was also taken
with Mandy Fisher’s work (Azuara et
al., 2006) and her demonstration that
these marks can coexist in pluripotent
stem cells — an idea echoed by Eric
Lander later that same year (Bern-
stein et al., 2006). Both of these stud-
ies get away from the idea of individ-
ual histone marks as the definitive
feature of the epigenome.
Relatively few epigenetically regu-
lated developmental decisions have
been identified (e.g., X inactivation,
imprinting, maintenance of Hox si-
lencing by PcG, control of neuronal
genes by REST). Why are they so dif-
ficult to find?
N.B.: We have to first deal with the
old chestnut of what we mean by “epi-
genetics.” There is a semantic and a
scientific aspect to this and unfortu-
nately, one clouds the other, especially
in neurobiology. A textbook definition
of epigenetics usually goes along the
lines “reversible heritable changes in
gene function or other cell phenotype
that occur without a change in DNA
sequence.” Neurons are terminally
differentiated postmitotic cells, so any
change in gene function is not herita-
ble at either the cellular or organismal
level. Epigenetic theories of neural de-
velopment, therefore, embrace the im-
plicit assumption that any “epigenetic
change” must also occur in the germ
cells in order for the change in gene
function to be heritable. Changes in
chromatin that are restricted to neu-
rons cannot lead to heritable changes.
Nevertheless, I want to expand this
argument by reference to schizophre-
nia (Perkins et al., 2005; Sharma,
2005), a devastating psychosis that ef-
fects around 1% of the population.
Schizophrenia is a neurodevelopmen-
tal disorder, at least in the mind of
many neurobiologists, and since large-
scale population studies such as those
used in the Dutch and Chinese fam-
ines show a robust epigenetic compo-
nent to inheritance (see reply to third
question), then we must accept that
there is an epigenetic component to
development—in this case, the devel-
opment of behavior.
The hard bit is the last part of the
question. Except for a few studies
showing changes in DNA methylation
at schizophrenia risk genes, there is
nothing to hint at the exact nature of
these heritable changes. This I think
is the principle difficulty in identifying
epigenetic mechanisms in develop-
ment— demonstration of the phenom-
enon of epigenetic inheritance and
provision of a direct molecular corre-
late. All too often, the twin aspects are
not robustly linked.
G.C.: Noel is right, there has been a
drift in the semantic definition of what
is epigenetics, shifting from the devel-
opmentalist viewpoint of Wadding-
ton—the ensemble of the processes
that use the information of the geno-
type to bring the phenotype into be-
ing—to a sharper and DNA-centered
definition focused on mechanisms of
inheritance not relying on the primary
DNA sequence. This second definition
was going against the mainstream
view following which ALL inheritance
was dependent on DNA. Although fas-
cinating as a concept, this put the peo-
ple working in epigenetics in the
trenches. Every work they were deliv-
ering “had to” demonstrate that there
was such a thing as inheritance after
DNA. In my opinion, this preoccupa-
tion has prevented many attempts to
even identify developmental processes
regulated by epigenetic components.
Because everybody was trying to de-
crypt mechanisms, they took phenom-
ena that were overtly regulated epige-
netically. This is typically the case of
the processes you mention.
In fact, it is likely that many pro-
cesses are regulated by “classical”
transcription factors as well as epige-
netic components. Moreover, many
processes regulated by epigenetic fac-
tors may not have a strong heritable
component. Luckily the groundwork
of convincing the scientific community
that epigenetic factors do regulate
genes and can convey inheritance is
now done, and this idea starts getting
across even to the larger public. This
brings development and also cognition
and behavior at the center of interest
of people involved in epigenetics, and
also facilitates access to epigenetic re-

search to those who were not doing it
before. I am sure that, in the next
years, we will watch discoveries of ev-
ery sort of process being regulated, in
part, by known epigenetic compo-
nents.
Coming back to semantics, however,
this is not without consequences. If we
don’t need a demonstration of DNA
sequence-independent inheritance to
admit the implication of epigenetics in
a certain process, then are there really
“epigenetic” and “nonepigenetic” regu-
lators? In an age when every tran-
scriptional regulator is being found
forming chromatin regulatory com-
plexes, is there a fundamental differ-
ence between epigenetic stars like
Polycomb or heterochromatin proteins
and any other transcription factor?
The only one I can imagine is that
some epigenetic processes involve in-
heritance, while others don’t. In this
view, epigenetic regulation would be a
very general concept and epigenetic
inheritance would be restricted to a
part of these processes.
Do you think that many develop-
mental decisions are epigenetically
regulated?
N.B.: It is hard to believe otherwise.
Any developmental change is accom-
panied by wholesale changes in gene
expression with entire batteries of
genes activated or silenced. Changes
in histone acetylation and methyl-
ation accompany all these events and
appear to be stably maintained in the
differentiated state. Phenomenologi-
cally, this is epigenetics. Again, as in
my earlier comments, the missing as-
pect is often the mechanistic link. Fur-
ther proof comes from gene knockout
studies that show the impact of chro-
matin modifying activities on develop-
mental decisions. There are many
examples, including the role of chro-
matin remodeling activities such as
Brg1 in myogenesis (de la Serna et al.,
2001) and neurogenesis (Eroglu et al.,
2006). Before being hoist upon my own
petard, I accept that this does not in
itself constitute proof-positive of epi-
genetics, but it is a very strong indi-
cator.
G.C.: I fully agree with Noel. When
you look at the list of genes bound and
repressed by PcG proteins in mouse
and human cells (Boyer et al., 2006;
Lee et al., 2006), and when you fur-
ther consider that many of these genes
are derepressed upon ES cell differen-
tiation, while they actually become
permanently silenced by DNA methyl-
ation upon cancer development
(Schlesinger et al., 2006; Widsch-
wendter et al., 2006), how can one sen-
sibly doubt that epigenetics regulates
important developmental processes?
Both REST and PcG repress/silence
genes are involved in neuronal differ-
entiation. Why do neuronal genes need
their own repressor/silencer?
N.B.: This is one of the hardest out-
standing questions and usually sits
beside the equally vexed question of
what governs whether a particular
gene falls under REST (or PcG) regu-
lation? The reason for coupling these
two questions is that it unifies the
idea that we need to provide an expla-
nation for why particular genes be-
come regulated by any individual
transcription factor. In the case of
REST, many targets, but by no means
all, are neuronal genes. Equally im-
portant is the corollary that, by no
means, are all neuronal target genes
regulated by REST. Inspection of the
1,300 or so RE1 sites in the human
genome (Johnson et al., 2006) pro-
vides no insight into any linkage
among the targets, other than a bias
toward genes associated with neuro-
nal development or function.
One speculation that provides in-
sight, if not explanation, is our recent
demonstration that many REST bind-
ing sites seemed to have been depos-
ited into 5⬘UTRs by means of LINE2
retrotransposons (Johnson et al.,
2006). In other words, the “selection”
of genes may be random. Deposition of
a LINE2 element into a 5⬘UTR usu-
ally decreases the rate of transcription
so deposition of a LINE2 element that
carries a transcription factor binding
site, confers regulated repression.
Maybe this is an advantage in allow-
ing batteries of genes to be regulated
whilst in the continued presence of an
activator. Simply switching off the ac-
tivator may be more expensive in
terms of alternative molecular com-
pensatory mechanisms that would be
necessary to keep essential genes
turned on. Such fine-tuning of gene
expression may be more necessary in
the vertebrate nervous system where
cell number and cell– cell interaction
surpass any other biological system.
To address the latter part of the
EPIGENETICS PRIMER 1151
question, PcG, is normally (but not al-
ways) associated with silenced genes
(stem cells provide an interesting ex-
ception) whereas our studies (Belyaev
et al., 2004; Bruce et al., 2004; Green-
way et al., 2006) show that REST is
normally associated with active genes,
i.e., employment of REST can main-
tain repression of an otherwise active
gene, whereas in the differentiated
state, PcG normally acts to establish
and/or maintain silence.
G.C.: Noel is making a strong case
for the study of these questions from
an evolutionary perspective. We often
have a biased view of the processes we
are studying, thinking that they are
“mature,” i.e., close to their endpoint
evolution. There is a temptation to
think that if something is as we see it
today there must be a very good rea-
son (strong selective pressure) to
maintain it. If we understand how cer-
tain regulatory circuits have evolved,
we come closer to the understanding
of whether “chance” or “necessity” as-
signed certain regulators to certain
processes. This approach is going to
become more and more important as
genomics progresses and many organ-
isms, including nonmodel species are
sequenced and studied.
Coming to the specific question,
maybe that PcG proteins do regulate a
lot of neuronal genes, but the nervous
system might require its own dedi-
cated set of epigenetic factors as an
additional layer of regulation.
Do you think other tissue-specific
gene programs (e.g. muscle) have spe-
cialized repressors/silencers?
N.B.: It is interesting that they
haven’t been discovered, even in the
light of complete sequencing of several
vertebrate genomes. As alluded to ear-
lier, regulation of gene expression in
the nervous system may require finer
controls than other tissues because of
the scale of the cellular heterogeneity.
One further point to bear in mind is
that REST is an evolutionary new-
comer on the block—it is only found in
vertebrates—again coincident with a
rapid expansion in brain size and neu-
ronal diversity. Maybe not a com-
pletely compelling argument, but an
interesting coincidence.
G.C.: I am at a loss here, on the one
hand I share Noel’s feeling that the
nervous system is a very “epigenetic
object” with lots of cross-talk between
1152 KIEFER ET AL.
stable cell fates and plasticity, which
is the realm of action of epigenetic reg-
ulators. On the other hand, I would be
very cautious not to make a prediction
in this infant field; we may learn soon
that muscle development is epigeneti-
cally regulated. For instance, a recent
paper (Mal, 2006) showing a regula-
tory association between SUV39H1
and MYOD makes me feel very ex-
cited about the amount of hidden reg-
ulatory potential in tissue develop-
ment. I hope to read beautiful stories
about these subjects in the future to
help make up my mind.
Both REST and PcG can occupy ac-
tively transcribed loci. How do you ex-
plain this?
N.B.: This comes back to the idea of
whether you think REST and PcG are
repressors or silencers. In most adult
tissue and differentiated cells, we find
REST present at actively transcribed
loci, and abrogation of REST function
leads to derepression, indicating that
REST acts to regulate levels of tran-
scription rather than to simply silence
transcription. This derepression is ac-
companied by reciprocal increases in
histone H3K9 acetylation and de-
creases in H3K9 dimethylation at the
REST binding site (Belyaev et al.,
2004; Greenway et al., 2006).
The idea of singular histone modifi-
cations offering a read-out of gene ac-
tivity is losing ground, and it is likely
that no singular mark will have such
predictive or causative power. This is
especially evident in studies of stem
cells where much of the cells chroma-
tin appears to carry a “bivalent” or
mixed signature comprising both “ac-
tive” and repressed’ marks (Azuara et
al., 2006; Bernstein et al., 2006). Also,
remember that in the REST⫺/⫺ em-
bryo, very few target genes were pre-
cociously activated (Chen et al., 1998).
This of course could be due to absence
of appropriate activators. Since em-
bryonic lethality occurred before neu-
rogenesis, then in vivo testing of these
ideas awaits production of a condi-
tional REST knockout.
G.C.: Noel is reminding us of a very
important point, many regulatory pro-
cesses result from a balance between
putative activation and repression,
with activators and repressors coex-
isting on their target rather than ex-
cluding one another. For instance,
PcG proteins have to leave with their
trxG partners on their targets and the
papers cited above by Noel, as well as
a fly study by Muller and coworkers
(Papp and Muller, 2006) really sug-
gest that this is not just a mix of active
and silent cells, but rather a true pres-
ence of bivalent marks on one and the
same chromatin region.
The recent discoveries of the LSD1
and GASC1 histone demethylases sug-
gest histone methylation is more dy-
namic than previously thought. How
might this affect the interpretation
of “silencing” histone methylation marks?
G.C.: The discovery of histone dem-
ethylases was very important, although
in a way expected, since histone meth-
ylation patterns were earlier found to
vary rather rapidly following certain
treatments. Nonetheless, one should
keep in mind that methyl groups have
an average half-life of about two orders
of magnitude longer (approaching the
half-life of histones themselves) com-
pared to other histone marks like acet-
ylation. Therefore, methyl marks are
likely to be maintained stably on the
chromatin template of most genes,
making histone methylation a good can-
didate modification for transmission of
epigenetic inheritance. Histone dem-
ethylases are probably able to “reset”
this memory when needed, and it will
be important to dissect when and how is
this reset brought upon.
N.B.: I think Giacomo’s idea of “re-
setting” is key. Just because demethy-
lases exist does not mean that all
methyl marks will be erased. It simply
lends more adaptability to allow reac-
tivation of genes previously silenced.
This could be particularly important
in the recruitment, expansion, and
differentiation of tissue-specific stem
cells in response to damage in the
adult where silenced genes may need
to be reactivated (or conversely, active
genes needed for maintenance of the
multipotent state may need to be si-
lenced). Identification of the transcrip-
tion factors that target the demethy-
lases will provide insight into when
and where demethylation is em-
ployed.
Will it be important to distinguish
stably methylated genes from ones that
are subject to demethylation?
G.C.: I predict that, thanks to the
explosion of high-throughput epige-
netic mapping technology, we will
actually “see” some of these demethyl-
ation events “on the fly.” Most impor-
tantly, we will learn about the ge-
nome-wide distribution of histone
demethylases, and this might help un-
derstanding the whole issue. It is
early to say whether there will be spe-
cific signatures identifying genes that
are reversibly methylated as opposed
to genes that are not. Even the most
stable regulatory gene states are
likely to be reprogrammable under ex-
treme circumstances. However, it is
likely that different genes (or classes
of genes) can be differentially repro-
grammed during development or in
response to external stimuli.
N.B.: I certainly concur with the
power of ChIPchip studies to provide
genome-wide views of epigenetic
changes. Considering that an oocyte
nucleus can reprogram a somatic cell
to derive a whole organism then it is
clear that all marks are erasable.
Silencing of at least some PcG tar-
gets is mediated by pairing-sensitive
silencing. Do you think other epige-
netic silencers use this strategy?
G.C.: Long-distance pairing can be
driven also by heterochromatin. In-
deed, the first ever reported case of
long-distance repressory chromo-
somal associations were published by
the Henikoff and the Sedat lab 10
years ago and involved heterochro-
matic regions (Csink and Henikoff,
1996; Dernburg et al., 1996). It is a
common observation that heterochro-
matic loci cluster at the so-called chro-
mocenters in both insects and verte-
brate cells, and this clustering may
affect silencing. Other epigenetics si-
lencing systems may employ similar
strategies and, indeed, long-range
chromosomal associations are also
linked to gene activation, as the work
of several labs is recently showing
(Spilianakis et al., 2005; Lomvardas
et al., 2006).
N.B.: The notion of long distance
interactions, both in cis and in trans is
extremely important both for silenc-
ing and activation since it offers a
mechanism to allow coordinate regu-
lation across multiple genes— exactly
the sort of mechanism you would ex-
pect to see during development and
differentiation. The paper by Lomvar-
das et al. cited by Giacomo is a great
example of the power of chromosome
capture conformation (3C) to unravel
these interactions. In passing I should

point out that we have no evidence
that REST undertakes such interac-
tions, although I accept that absence
of evidence is not evidence of absence.
The one report in the literature by
Lunyak et al. (Lunyak et al., 2002)
that purports to show that REST can
silence an entire locus is unfortu-
nately based upon an incorrectly an-
notated gene order (Belyaev et al.,
2004).
What exciting ideas are emerging in
the field?
G.C.: One new idea is related to nu-
clear compartmentalization. The es-
tablished view of “genes” is that they
are regulated by cis-elements located
somewhere close to the transcription
start site or within “reasonable” (1–
100 kb) distances from it. However,
the mounting evidence for chromo-
somal contacts involving loci from dif-
ferent chromosomes or from different
parts of the same chromosome raises
the exciting possibility that gene reg-
ulation uses these contacts.
Another new concept is that noncod-
ing RNAs play important regulatory
roles in the genome, affecting all steps
of gene regulation, from chromatin
structure to stability and trafficking of
the mRNA and to regulation of RNA
translation. Many different classes of
noncoding RNAs exist, many of them
playing a role in developmental regu-
lation. It is thus important to analyze
each of them, and the initial studies
are very promising.
Finally, the flood of genome-wide
data raises the need to put a huge
effort in understanding the logic of
gene-regulatory circuits. For instance,
a glance at the PcG target genes re-
veals that they often include multiple
genes involved in the same pathways.
Frequently, these genes code for tran-
scriptional regulators. In the case of
transactivators, one may speculate
that PcG proteins secure a tight block
of certain pathways by simulta-
neously shutting down multiple genes
at different steps of the regulatory
cascade. But what is the logic of si-
lencing several transcriptional repres-
sors along multiple steps of a path-
way? Superficially, shutting down the
expression of a tanscriptional repres-
sor as well as of its own target genes
does not make much sense. Clearly,
one needs to understand how these
events are regulated both spatially
and temporally.
N.B.: Giacomo is absolutely right.
We have produced detailed linear
maps of multiple vertebrate genomes,
and the application of increasingly so-
phisticated bioinformatics and cross-
genome comparisons is overlaying de-
tailed annotations of regulatory sites.
The discoveries of long-range interac-
tions, nuclear compartmentalization
and noncoding RNAs all impose new
dimensions of complexity onto this lin-
ear map. I think, as always, those of
us working on development in higher
eukaryotes will be keeping a close eye
on the work of our colleagues in yeast
genetics particularly in relation to
building regulatory networks (Lee et
al., 2002; Wyrick and Young, 2002).
What important questions remain to
be answered?
G.C.: Concerning nuclear architec-
ture, a critical implication of the exis-
tence of long-range regulatory chro-
mosomal contacts is that a phenotype
may be induced from very remote
parts of the genome. Therefore, it will
be important to map long-range chro-
mosome contacts systematically and
to understand how frequently they af-
fect gene regulation. It will also be
crucial to study whether these con-
tacts are the cause or the effect of reg-
ulation of gene transcription.
Another unanswered question con-
cerns the mechanisms of epigenetic
inheritance through DNA replication
and mitosis (or meiosis). Indeed, chro-
mosomes undergo major reorganiza-
tion events during these two phases of
the cell cycle. How can a chromatin
state survive these events is not
known and, largely, not even studied,
because of the lack of appropriate ap-
proaches. Much of current effort in
molecular biology aims to develop
tools in order to work on the scale of
limited number of molecules (ideally
single molecules). This may allow
tackling the issue of chromatin inher-
itance at the molecular scale in next
few years.
Finally, a grand challenge for the
future is to understand how the differ-
ent gene regulatory “tools” are har-
nessed in a coordinated way during
development. Orchestration has al-
ways been an issue in developmental
biology, but certainly the new discov-
eries give a mind-boggling perspective
EPIGENETICS PRIMER 1153
of the combinatorial complexity that
can be reached. Everything, from
transcriptional output to gene prod-
uct’s location, can be dynamically reg-
ulated in a fine-tuned way by different
processes. The other side of the coin is
that there are also many risks for mo-
lecular mistakes that can potentially
damage the developmental process.
We will thus need to analyze how
these complex regulatory processes
can be coordinated in order to combine
developmental plasticity with robust-
ness.
N.B.: All of this is true. Develop-
ment has often been perceived as a
balance between external signals and
intrinsic programs, a description that
finds its reflection in some classic em-
bryological terms such as “competent”
and “determined.” The interplay be-
tween intercellular signals and whole-
sale remodeling of the genome that
occurs as differentiation and develop-
ment proceed will increasingly occupy
our experiments—whether such changes
are defined as epigenetic or otherwise.
At the moment, most researchers fo-
cus on either the chromatin or on the
signaling events, but these must con-
verge if we are to offer comprehensive
molecular correlates of “fate” and “po-
tential.” Again, I think this interplay
will be at its most complex in the ner-
vous system where the complexity of
signals and diversity of cell type is
greater than with any other tissue.
ACKNOWLEDGMENTS
Many thanks to Jacqueline Witt-
meyer, Christine Disteche, Rebecca
Oakey, Nurit Ballas, and Itys Comet
for helpful suggestions. I am also
grateful to Noel Buckley and Giacomo
Cavalli for sharing their insights.
REFERENCES
Allegrucci C, Thurston A, Lucas E, Young
L. 2005. Epigenetics and the germline.
Reproduction 129:137–149.
Angelov D, Molla A, Perche PY, Hans F,
Cote J, Khochbin S, Bouvet P, Dimitrov
S. 2003. The histone variant macroH2A
interferes with transcription factor bind-
ing and SWI/SNF nucleosome remodel-
ing. Mol Cell 11:1033–1041.
Azuara V, Perry P, Sauer S, Spivakov M,
Jorgensen HF, John RM, Gouti M,
Casanova M, Warnes G, Merkenschlager
M, Fisher AG. 2006. Chromatin signa-
tures of pluripotent cell lines. Nat Cell
Biol 8:532–538.
1154 KIEFER ET AL.
Bacher CP, Guggiari M, Brors B, Augui S,
Clerc P, Avner P, Eils R, Heard E. 2006.
Transient colocalization of X-inactiva-
tion centres accompanies the initiation
of X inactivation. Nat Cell Biol 8:293–
299.
Ballas N, Mandel G. 2005. The many faces
of REST oversee epigenetic program-
ming of neuronal genes. Curr Opin Neu-
robiol 15:500 –506.
Ballas N, Grunseich C, Lu DD, Speh JC,
Mandel G. 2005. REST and its corepres-
sors mediate plasticity of neuronal gene
chromatin throughout neurogenesis.
Cell 121:645–657.
Battaglioli E, Andres ME, Rose DW, Che-
noweth JG, Rosenfeld MG, Anderson
ME, Mandel G. 2002. REST repression of
neuronal genes requires components of
the hSWI.SNF complex. J Biol Chem 277:
41038 –41045.
Beisel C, Imhof A, Greene J, Kremmer E,
Sauer F. 2002. Histone methylation by
the Drosophila epigenetic transcrip-
tional regulator Ash1. Nature 419:857–
862.
Belyaev ND, Wood IC, Bruce AW, Street
M, Trinh JB, Buckley NJ. 2004. Distinct
RE-1 silencing transcription factor-con-
taining complexes interact with different
target genes. J Biol Chem 279:556 –561.
Bernstein BE, Kamal M, Lindblad-Toh K,
Bekiranov S, Bailey DK, Huebert DJ,
McMahon S, Karlsson EK, Kulbokas EJ
III, Gingeras TR, Schreiber SL, Lander
ES. 2005. Genomic maps and compara-
tive analysis of histone modifications in
human and mouse. Cell 120:169 –181.
Bernstein BE, Mikkelsen TS, Xie X, Kamal
M, Huebert DJ, Cuff J, Fry B, Meissner
A, Wernig M, Plath K, Jaenisch R, Wag-
schal A, Feil R, Schreiber SL, Lander ES.
2006. A bivalent chromatin structure
marks key developmental genes in em-
bryonic stem cells. Cell 125:315–326.
Boyer LA, Plath K, Zeitlinger J, Bram-
brink T, Medeiros LA, Lee TI, Levine SS,
Wernig M, Tajonar A, Ray MK, Bell GW,
Otte AP, Vidal M, Gifford DK, Young
RA, Jaenisch R. 2006. Polycomb com-
plexes repress developmental regulators
in murine embryonic stem cells. Nature
441:349 –353.
Bracken AP, Dietrich N, Pasini D, Hansen
KH, Helin K. 2006. Genome-wide map-
ping of Polycomb target genes unravels
their roles in cell fate transitions. Genes
Dev 20:1123–1136.
Bruce AW, Donaldson IJ, Wood IC, Yer-
bury SA, Sadowski MI, Chapman M,
Gottgens B, Buckley NJ. 2004. Genome-
wide analysis of repressor element 1 si-
lencing transcription factor/neuron-re-
strictive silencing factor (REST/NRSF)
target genes. Proc Natl Acad Sci U S A
101:10458 –10463.
Cao R, Tsukada Y, Zhang Y. 2005. Role of
Bmi-1 and Ring1A in H2A ubiquitylation
and Hox gene silencing. Mol Cell 20:845–
854.
Chadwick BP, Willard HF. 2001. A novel
chromatin protein, distantly related to
histone H2A, is largely excluded from
the inactive X chromosome. J Cell Biol
152:375–384.
Chadwick BP, Willard HF. 2003. Chroma-
tin of the Barr body: histone and non-
histone proteins associated with or ex-
cluded from the inactive X chromosome.
Hum Mol Genet 12:2167–2178.
Chen ZF, Paquette AJ, Anderson DJ. 1998.
NRSF/REST is required in vivo for re-
pression of multiple neuronal target
genes during embryogenesis. Nat Genet
20:136 –142.
Cheung P, Lau P. 2005. Epigenetic regula-
tion by histone methylation and histone
variants. Mol Endocrinol 19:563–573.
Cloos PA, Christensen J, Agger K, Maiolica
A, Rappsilber J, Antal T, Hansen KH,
Helin K. 2006. The putative oncogene
GASC1 demethylates tri- and dimethyl-
ated lysine 9 on histone H3. Nature 442:
307–311.
Comet I, Savitskaya E, Schuettengruber B,
Negre N, Lavrov S, Parshikov A, Juge F,
Gracheva E, Georgiev P, Cavalli G. 2006.
PRE-mediated bypass of two Su(Hw) in-
sulators targets PcG proteins to a down-
stream promoter. Dev Cell 11:117–124.
Csink AK, Henikoff S. 1996. Genetic mod-
ification of heterochromatic association
and nuclear organisation in Drosophila.
Nature 381:529 –531.
de la Serna IL, Carlson KA, Imbalzano AN.
2001. Mammalian SWI/SNF complexes
promote MyoD-mediated muscle differ-
entiation. Nat Genet 27:187–190.
Delaval K, Feil R. 2004. Epigenetic regula-
tion of mammalian genomic imprinting.
Curr Opin Genet Dev 14:188 –195.
Dellino GI, Schwartz YB, Farkas G, Mc-
Cabe D, Elgin SC, Pirrotta V. 2004. Poly-
comb silencing blocks transcription initi-
ation. Mol Cell 13:887– 893.
Dernburg AF, Broman KW, Fung JC, Mar-
shall WF, Philips J, Agard DA, Sedat
JW. 1996. Perturbation of nuclear archi-
tecture by long-distance chromosome in-
teractions. Cell 85:745–759.
Doyen CM, An W, Angelov D, Bondarenko
V, Mietton F, Studitsky VM, Hamiche A,
Roeder RG, Bouvet P, Dimitrov S. 2006a.
Mechanism of polymerase II transcrip-
tion repression by the histone variant
macroH2A. Mol Cell Biol 26:1156 –1164.
Doyen CM, Montel F, Gautier T, Menoni H,
Claudet C, Delacour-Larose M, Angelov
D, Hamiche A, Bednar J, Faivre-
Moskalenko C, Bouvet P, Dimitrov S.
2006b. Dissection of the unusual struc-
tural and functional properties of the
variant H2A.Bbd nucleosome. EMBO J
25:4234 –4244.
Engel N, West AG, Felsenfeld G, Bar-
tolomei MS. 2004. Antagonism between
DNA hypermethylation and enhancer-
blocking activity at the H19 DMD is un-
covered by CpG mutations. Nat Genet
36:883– 888.
Engemann S, Strodicke M, Paulsen M,
Franck O, Reinhardt R, Lane N, Reik W,
Walter J. 2000. Sequence and functional
comparison in the Beckwith-Wiedemann
region: implications for a novel imprint-
ing centre and extended imprinting.
Hum Mol Genet 9:2691–2706.
Eroglu B, Wang G, Tu N, Sun X, Mivechi
NF. 2006. Critical role of Brg1 member
of the SWI/SNF chromatin remodeling
complex during neurogenesis and neural
crest induction in zebrafish. Dev Dyn 235:
2722–2735.
Francis NJ, Saurin AJ, Shao Z, Kingston
RE. 2001. Reconstitution of a functional
core polycomb repressive complex. Mol
Cell 8:545–556.
Fujita N, Watanabe S, Ichimura T, Tsu-
ruzoe S, Shinkai Y, Tachibana M, Chiba
T, Nakao M. 2003. Methyl-CpG binding
domain 1 (MBD1) interacts with the
Suv39h1-HP1 heterochromatic complex
for DNA methylation-based transcrip-
tional repression. J Biol Chem 278:24132–
24138.
Fuks F, Hurd PJ, Wolf D, Nan X, Bird AP,
Kouzarides T. 2003. The methyl-CpG-
binding protein MeCP2 links DNA meth-
ylation to histone methylation. J Biol
Chem 278:4035–4040.
Greenway DJ, Street M, Jeffries A, Buck-
ley NJ. 2006. REST maintains a repres-
sive chromatin environment in embry-
onic hippocampal neural stem cells.
Stem Cells [Epub ahead of print].
Grewal SIS, Klar AJS. 1996. Chromosomal
inheritance of epigenetic states in fission
yeast during mitosis and meiosis. Cell
86:95–101.
Grimaud C, Bantignies F, Pal-Bhadra M,
Ghana P, Bhadra U, Cavalli G. 2006.
RNAi components are required for nu-
clear clustering of Polycomb group re-
sponse elements. Cell 124:957–971.
Hark AT, Schoenherr CJ, Katz DJ, Ingram
RS, Levorse JM, Tilghman SM. 2000.
CTCF mediates methylation-sensitive
enhancer-blocking activity at the H19/
Igf2 locus. Nature 405:486 –489.
Heard E. 2005. Delving into the diversity
of facultative heterochromatin: the epi-
genetics of the inactive X chromosome.
Curr Opin Genet Dev 15:482–489.
Huang DH, Chang YL. 2004. Isolation and
characterization of CHRASCH, a poly-
comb-containing silencing complex.
Methods Enzymol 377:267–282.
Jacobs JJ, Kieboom K, Marino S, DePinho
RA, van Lohuizen M. 1999. The oncogene
and Polycomb-group gene bmi-1 regu-
lates cell proliferation and senescence
through the ink4a locus. Nature 397:164 –
168.
Johnson R, Gamblin RJ, Ooi L, Bruce AW,
Donaldson IJ, Westhead DR, Wood IC,
Jackson RM, Buckley NJ. 2006. Identifi-
cation of the REST regulon reveals ex-
tensive transposable element-mediated
binding site duplication. Nucleic Acids
Res 34:3862–3877.
Jürgens G. 1985. A group of genes control-
ling the spatial expression of the bitho-
rax complex in Drosophila. Nature 316:
153–155.
Kratzer PG, Chapman VM, Lambert H,
Evans RE, Liskay RM. 1983. Differences
in the DNA of the inactive X chromo-
somes of fetal and extraembryonic tis-
sues of mice. Cell 33:37–42.
Lee TI, Rinaldi NJ, Robert F, Odom DT,
Bar-Joseph Z, Gerber GK, Hannett NM,

Harbison CT, Thompson CM, Simon I,
Zeitlinger J, Jennings EG, Murray HL,
Gordon DB, Ren B, Wyrick JJ, Tagne JB,
Volkert TL, Fraenkel E, Gifford DK,
Young RA. 2002. Transcriptional regula-
tory networks in Saccharomyces cerevi-
siae. Science 298:799 –804.
Lee TI, Jenner RG, Boyer LA, Guenther
MG, Levine SS, Kumar RM, Chevalier B,
Johnstone SE, Cole MF, Isono K, Koseki
H, Fuchikami T, Abe K, Murray HL,
Zucker JP, Yuan B, Bell GW, Herbol-
sheimer E, Hannett NM, Sun K, Odom
DT, Otte AP, Volkert TL, Bartel DP,
Melton DA, Gifford DK, Jaenisch R,
Young RA. 2006. Control of developmen-
tal regulators by polycomb in human em-
bryonic stem cells. Cell 125:301–313.
Lessard J, Sauvageau G. 2003. Bmi-1 de-
termines the proliferative capacity of
normal and leukaemic stem cells. Nature
423:255–260.
Levenson JM, Sweatt JD. 2005. Epigenetic
mechanisms in memory formation. Nat
Rev Neurosci 6:108 –118.
Lomvardas S, Barnea G, Pisapia DJ, Men-
delsohn M, Kirkland J, Axel R. 2006.
Interchromosomal interactions and ol-
factory receptor choice. Cell 126:403–
413.
Lucchesi JC, Kelly WG, Panning B. 2005.
Chromatin remodeling in dosage com-
pensation. Annu Rev Genet 39:615–651.
Lund AH, van Lohuizen M. 2004. Polycomb
complexes and silencing mechanisms.
Curr Opin Cell Biol 16:239 –246.
Lunyak VV, Burgess R, Prefontaine GG,
Nelson C, Sze SH, Chenoweth J,
Schwartz P, Pevzner PA, Glass C, Man-
del G, Rosenfeld MG. 2002. Corepressor-
dependent silencing of chromosomal re-
gions encoding neuronal genes. Science
298:1747–1752.
Mal AK. 2006. Histone methyltransferase
Suv39h1 represses MyoD-stimulated
myogenic differentiation. EMBO J 25:
3323–3334.
Margueron R, Trojer P, Reinberg D. 2005.
The key to development: interpreting the
histone code? Curr Opin Genet Dev 15:
163–176.
Min J, Zhang Y, Xu RM. 2003. Structural
basis for specific binding of Polycomb
chromodomain to histone H3 methylated
at Lys 27. Genes Dev 17:1823–1828.
Molofsky AV, Pardal R, Iwashita T, Park
IK, Clarke MF, Morrison SJ. 2003.
Bmi-1 dependence distinguishes neural
stem cell self-renewal from progenitor
proliferation. Nature 425:962–967.
Navarro P, Page DR, Avner P, Rougeulle C.
2006. Tsix-mediated epigenetic switch of
a CTCF-flanked region of the Xist pro-
moter determines the Xist transcription
program. Genes Dev 20:2787–2792.
Negre N, Hennetin J, Sun LV, Lavrov S,
Bellis M, White KP, Cavalli G. 2006.
Chromosomal distribution of PcG pro-
teins during Drosophila development.
PLoS Biol 4:e170.
O’Brien TP, Bult CJ, Cremer C, Grunze M,
Knowles BB, Langowski J, McNally J,
Pederson T, Politz JC, Pombo A,
Schmahl G, Spatz JP, van Driel R. 2003.
Genome function and nuclear architec-
ture: from gene expression to nano-
science. Genome Res 13:1029 –1041.
Ohkawa Y, Yoshimura S, Higashi C,
Marfella CG, Dacwag CS, Tachibana T,
Imbalzano A. 2006. Myogenin and the
SWI/SNF atpase BRG1 maintain myo-
genic gene expression at different stages
of skeletal myogenesis. J Biol Chem
[Epub ahead of print].
Pal-Bhadra M, Leibovitch BA, Gandhi SG,
Rao M, Bhadra U, Birchler JA, Elgin SC.
2004. Heterochromatic silencing and
HP1 localization in Drosophila are de-
pendent on the RNAi machinery. Science
303:669 –672.
Papp B, Muller J. 2006. Histone trimethy-
lation and the maintenance of transcrip-
tional ONand OFF states by trxG and
PcG proteins. Genes Dev 20:2041–2054.
Park IK, Qian D, Kiel M, Becker MW,
Pihalja M, Weissman IL, Morrison SJ,
Clarke MF. 2003. Bmi-1 is required for
maintenance of adult self-renewing
haematopoietic stem cells. Nature 423:
302–305.
Perkins DO, Jeffries C, Sullivan P. 2005.
Expanding the ‘central dogma’: the reg-
ulatory role of nonprotein coding genes
and implications for the genetic liability
to schizophrenia. Mol Psychiatry 10:69 –
78.
Petruk S, Sedkov Y, Riley KM, Hodgson J,
Schweisguth F, Hirose S, Jaynes JB,
Brock HW, Mazo A. 2006. Transcription of
bxd noncoding RNAs promoted by tritho-
rax represses Ubx in cis by trascriptional
interference. Cell 127:1209 –1221.
Plath K, Fang J, Mlynarczyk-Evans SK,
Cao R, Worringer KA, Wang H, de la
Cruz CC, Otte AP, Panning B, Zhang Y.
2003. Role of histone H3 lysine 27 meth-
ylation in X inactivation. Science 300:
131–135.
Rai K, Nadauld LD, Chidester S, Manos
EJ, James SR, Karpf AR, Cairns BR,
Jones DA. 2006. Zebra fish Dnmt1 and
Suv39h1 regulate organ-specific termi-
nal differentiation during development.
Mol Cell Biol 26:7077–7085.
Rea S, Eisenhaber F, O’Carroll D, Strahl
BD, Sun ZW, Schmid M, Opravil S,
Mechtler K, Ponting CP, Allis CD, Jenu-
wein T. 2000. Regulation of chromatin
structure by site-specific histone H3
methyltransferases. Nature 406:593–
599.
Reik W, Lewis A. 2005. Co-evolution of X-
chromosome inactivation and imprinting
in mammals. Nat Rev Genet 6:403–410.
Ren B, Robert F, Wyrick JJ, Aparicio O,
Jennings EG, Simon I, Zeitlinger J,
Schreiber J, Hannett N, Kanin E, Volk-
ert TL, Wilson CJ, Bell SP, Young RA.
2000. Genome-wide location and func-
tion of DNA binding proteins. Science
290:2306 –2309.
Roopra A, Qazi R, Schoenike B, Daley TJ,
Morrison JF. 2004. Localized domains of
G9a-mediated histone methylation are
required for silencing of neuronal genes.
Mol Cell 14:727–738.
EPIGENETICS PRIMER 1155
Sado T, Hoki Y, Sasaki H. 2005. Tsix si-
lences Xist through modification of chro-
matin structure. Dev Cell 9:159 –165.
Sanchez-Elsner T, Gou D, Kremmer E,
Sauer F. 2006. Noncoding RNAs of
trithorax response elements recruit Dro-
sophila Ash1 to Ultrabithorax. Science
311:1118 –1123.
Schlesinger Y, Straussman R, Keshet I,
Farkash S, Hecht M, Zimmerman J,
Eden E, Yakhini Z, Ben-Shushan E, Reu-
binoff BE, Bergman Y, Simon I, Cedar H.
2006. Polycomb-mediated methylation
on Lys27 of histone H3 pre-marks genes
for de novo methylation in cancer. Nat
Genet [Epub ahead of print].
Schubeler D, MacAlpine DM, Scalzo D,
Wirbelauer C, Kooperberg C, van Leeu-
wen F, Gottschling DE, O’Neill LP,
Turner BM, Delrow J, Bell SP, Groudine
M. 2004. The histone modification pat-
tern of active genes revealed through ge-
nome-wide chromatin analysis of a
higher eukaryote. Genes Dev 18:1263–
1271.
Schwartz YB, Kahn TG, Nix DA, Li XY,
Bourgon R, Biggin M, Pirrotta V. 2006.
Genome-wide analysis of Polycomb tar-
gets in Drosophila melanogaster. Nat
Genet 38:700 –705.
Seo S, Richardson GA, Kroll KL. 2005. The
SWI/SNF chromatin remodeling protein
Brg1 is required for vertebrate neuro-
genesis and mediates transactivation of
Ngn and NeuroD. Development 132:105–
115.
Sharma RP. 2005. Schizophrenia, epige-
netics and ligand-activated nuclear re-
ceptors: a framework for chromatin ther-
apeutics. Schizophr Res 72:79 –90.
Shi Y, Lan F, Matson C, Mulligan P, Whet-
stine JR, Cole PA, Casero RA, Shi Y.
2004. Histone demethylation mediated
by the nuclear amine oxidase homolog
LSD1. Cell 119:941–953.
Sims RJ, 3rd, Reinberg D. 2006. Histone
H3 Lys 4 methylation: caught in a bind?
Genes Dev 20:2779 –2786.
Sleutels F, Zwart R, Barlow DP. 2002. The
non-coding Air RNA is required for si-
lencing autosomal imprinted genes. Na-
ture 415:810 –813.
Spilianakis CG, Flavell RA. 2004. Long-
range intrachromosomal interactions in
the T helper type 2 cytokine locus. Nat
Immunol 5:1017–1027.
Spilianakis CG, Lalioti MD, Town T, Lee
GR, Flavell RA. 2005. Interchromosomal
associations between alternatively ex-
pressed loci. Nature 435:637–645.
Squazzo SL, O’Geen H, Komashko VM,
Krig SR, Jin VX, Jang S, Margueron R,
Reinberg D, Green R, Farnham PJ. 2006.
Suz12 binds to silenced regions of the
genome in a cell-type-specific manner.
Genome Res 16:890 –900.
St. Clair D, Xu M, Wang P, Yu Y, Fang Y,
Zhang F, Zheng X, Gu N, Feng G, Sham
P, He L. 2005. Rates of adult schizophre-
nia following prenatal exposure to the
Chinese famine of 1959 –1961. JAMA 294:
557–562.
Stoger R, Kubicka P, Liu CG, Kafri T, Ra-
zin A, Cedar H, Barlow DP. 1993. Mater-
1156 KIEFER ET AL.
nal-specific methylation of the imprinted
mouse Igf2r locus identifies the ex-
pressed locus as carrying the imprinting
signal. Cell 73:61–71.
Strahl BD, Allis CD. 2000. The language of
covalent histone modifications. Nature 403: printing along the Kcnq1 domain on
41–45.
Sun BK, Deaton AM, Lee JT. 2006. A tran-
sient heterochromatic state in Xist pre-
empts X inactivation choice without
RNA stabilization. Mol Cell 21:617–628.
Takeuchi JK, Lickert H, Bisgrove BW, Sun
X, Yamamoto M, Chawengsaksophak K,
Hamada H, Yost HJ, Rossant J, Bruneau
BG. 2007. Baf60c is a nuclear Notch sig-
naling component required for the estab-
lishment of left-right asymmetry. Proc van der Lugt NM, Domen J, Linders K, van
Natl Acad Sci U S A.
Thorvaldsen JL, Verona RI, Bartolomei
MS. 2006. X-tra! X-tra! News from the
mouse X chromosome. Dev Biol 298:344 –
353.
Tolhuis B, Muijrers I, de Wit E, Teunissen
H, Talhout W, van Steensel B, van Lo-
huizen M. 2006. Genome-wide profiling
of PRC1 and PRC2 Polycomb chromatin
binding in Drosophila melanogaster. Nat
Genet 38:694 –699.
Umlauf D, Goto Y, Cao R, Cerqueira F,
Wagschal A, Zhang Y, Feil R. 2004. Im-
mouse chromosome 7 involves repressive
histone methylation and recruitment of
Polycomb group complexes. Nat Genet
36:1296 –1300.
Valley CM, Pertz LM, Balakumaran BS,
Willard HF. 2006. Chromosome-wide, al-
lele-specific analysis of the histone code
on the human X chromosome. Hum Mol
Genet 15:2335–2347.
Roon M, Robanus-Maandag E, te Riele
H, van der Valk M, Deschamps J, So-
froniew M, van Lohuizen M, et al. 1994.
Posterior transformation, neurological
abnormalities, and severe hematopoietic
defects in mice with a targeted deletion
of the bmi-1 proto-oncogene. Genes Dev
8:757–769.
Vire E, Brenner C, Deplus R, Blanchon L,
Fraga M, Didelot C, Morey L, Van Eynde
A, Bernard D, Vanderwinden JM, Bollen
M, Esteller M, Di Croce L, de Launoit Y,
Fuks F. 2006. The Polycomb group pro-
tein EZH2 directly controls DNA meth-
ylation. Nature 439:871–874.
Widschwendter M, Fiegl H, Egle D, Muel-
ler-Holzner E, Spizzo G, Marth C,
Weisenberger DJ, Campan M, Young J,
Jacobs I, Laird PW. 2006. Epigenetic
stem cell signature in cancer. Nat Genet
[Epub ahead of print].
Wyrick JJ, Young RA. 2002. Deciphering
gene expression regulatory networks.
Curr Opin Genet Dev 12:130 –136.
Xu N, Tsai CL, Lee JT. 2006. Transient
homologous chromosome pairing marks
the onset of X inactivation. Science 311:
1149 –1152.
Yeo M, Lee SK, Lee B, Ruiz EC, Pfaff SL,
Gill GN. 2005. Small CTD phosphatases
function in silencing neuronal gene ex-
pression. Science 307:596 –600.