Erasing the methyl mark: histone
demethylases at the center of cellular
differentiation and disease
Paul A.C. Cloos,2Jesper Christensen, Karl Agger, and Kristian Helin1
Biotech Research and Innovation Centre (BRIC) and Centre for Epigenetics, University of Copenhagen,
DK-2200 Copenhagen, Denmark
The enzymes catalyzing lysine and arginine methylation
of histones are essential for maintaining transcriptional
programs and determining cell fate and identity. Until
recently, histone methylation was regarded irreversible.
However, within the last few years, several families of
histone demethylases erasing methyl marks associated
with gene repression or activation have been identified,
underscoring the plasticity and dynamic nature of his-
tone methylation. Recent discoveries have revealed that
histone demethylases take part in large multiprotein
complexes synergizing with histone deacetylases, histone
methyltransferases, and nuclear receptors to control de-
velopmental and transcriptional programs. Here we re-
view the emerging biochemical and biological functions
of the histone demethylases and discuss their potential
involvement in human diseases, including cancer.
Histones constitute the basic scaffold proteins around
which DNA is wound to form the highly ordered struc-
ture of chromatin. Histones, and in particular their tails,
are subjected to a plethora of post-translational modifi-
cations that have been implicated in chromatin remod-
eling and closely linked to transcriptional regulation,
DNA replication, and DNA repair (for recent reviews,
see Berger 2007; Kouzarides 2007). Histone acetylation
and methylation represent the most common modifica-
tions of the histone tails. These modifications differ in
two ways: Histone acetylation results in a negative charge
of the modified lysine residue, causing a decreased inter-
action between the histone and DNA that is generally
associated with active transcription. In contrast, meth-
ylation of histones occurs at both arginine and lysine
residues, and does not influence the net charge of the af-
fected residues, and hence, has no effect on DNA–histone
interactions. Rather, the effect of histone methylation
impacts on the transcriptional activity of the underlying
DNA by acting as a recognition template for effector
proteins modifying the chromatin environment and lead-
ing to either repression or activation. Thus, histone
methylation can be associated with either activation or
repression of transcription depending on which effector
protein is being recruited. It should be noted that the
unmodified residues can also serve as a binding template
for effector proteins leading to specific chromatin states
(Lan et al. 2007b).
Arginine residues can be modified by one or two meth-
yl groups; the latter form in either a symmetric or asym-
metric conformation (Rme1, Rme2s, and Rme2a), per-
mitting a total of four states: one unmethylated and
three methylated forms.
Similarly, lysine residues can be unmethylated,
mono-, di-, or trimethylated (Kme1, Kme2, and Kme3),
and the extent of methylation at a specific residue is
important for the recognition of effector proteins and has
therefore impact on chromatin and the transcriptional
Histone methylation is involved in the regulation of a
variety of nuclear processes essential for cellular regula-
tion, homeostasis, and fate. Previously, methylation has
been considered to constitute a permanent and irrevers-
ible histone modification that defined epigenetic pro-
grams in concert with DNA methylation. Recently,
however, a large number of enzymes have been discov-
ered with the ability to demethylate methylated histone
lysine residues as well as methylated arginines via amine
oxidation, hydroxylation or deimination. Here we re-
view the current knowledge on histone demethylases,
with a special focus on the Jumonji (JmjC) proteins, their
role in chromatin regulation, cellular differentiation, and
involvement in human diseases.
The identification of histone demethylases
Methylated lysine and arginine residues on histone tails
are believed to constitute important regulatory marks
delineating transcriptionally active and inactive chroma-
tin. For instance, tri- and dimethylated Lys 9 on histone
H3 (H3K9) is associated with silenced chromatin, whereas
[Keywords: Cancer; differentiation; demethylases; epigenetic; histone;
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Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1652908.
GENES & DEVELOPMENT 22:1115–1140 © 2008 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/08; www.genesdev.org1115
tri- and dimethylated Lys 4 on the same histone tail
(H3K4) is often connected to euchromatin and active
In contrast to other histone modifications such as
acetylation and phosphorylation, methylation, and in
particular trimethylation, has been regarded as irrevers-
ible because of the high thermodynamic stability of the
N–CH3bond. Hence, for several years the consensus in
the epigenetic field was that the only way to revert his-
tone methylation was by histone exchange or by cleav-
age of the methylated histone tail. The identification of
the amine oxidase LSD1 (KDM1 according to the newly
suggested nomenclature) (Allis et al. 2007) as a histone
demethylase changed this perception (Shi et al. 2004).
This enzyme demethylates the histone substrate
through a flavin adenine dinucleotide (FAD)-dependent
amine oxidase reaction. However, due to the require-
ment of a protonated methyl ?-ammonium group for
LSD1-catalyzed oxidation, and thus, for LSD1-mediated
demethylation, this and other related (and as-yet-undis-
covered) enzymes are unable to catalyze the demethyl-
ation of trimethylated lysine residues. However, re-
cently we and others have identified and characterized
histone demethylases containing the Jumonji (JmjC)
catalytic domain, which can demethylate trimethylated
lysines (Cloos et al. 2006; Klose et al. 2006b; Tsukada et
al. 2006; Whetstine et al. 2006; Yamane et al. 2006).
The JmjC-driven demethylase reaction is compatible
with demethylation of mono-, di-, and trimethylated ly-
sines and, indeed, most of the JmjC histone demethyl-
ases characterized so far are capable of demethylating
trimethylated lysines, and in most cases favor a trimeth-
ylated substrate (e.g., see Couture et al. 2007; Ng et al.
2007). JmjC proteins can also demethylate arginine resi-
dues (Chang et al. 2007), and, at least in theory, other
protein substrates or nucleotides. Certainly, the fact that
JmjC domain-containing proteins are also found in bac-
teria that do not contain histones suggests that these
proteins also serve other purposes in addition to being
The amine oxidase LSD1 (KDM1)
The idea that oxidases could function as histone demeth-
ylases was originally proposed by Kouzarides and cowork-
ers (Bannister et al. 2002). Subsequently, in a groundbreak-
ing study, the Shi lab showed (Shi et al. 2004) that the
amine oxidase LSD1 (synonyms: BHC110, KIAA0601,
p110b, and KDM1), which had been identified previously
as a component of a number of corepressor complexes
(You et al. 2001), could demethylate di- and monometh-
ylated H3K4 (H3K4me2/me1) (Shi et al. 2004). LSD1 be-
longs to the class of FAD-dependent monoamine oxi-
dases that typically catalyze the oxidation of amine-con-
taining substrates utilizing molecular oxygen as electron
acceptor. In LSD1-catalyzed histone demethylation, the
amino group of the methylated lysine in question is oxi-
dized, presumably to generate an imine intermediate
that will spontaneously hydrolyze to produce formalde-
hyde and the corresponding amine residue (Fig. 1A). Sub-
strate oxidation leads to the two-electron reduction of
the cofactor FAD, which is reoxidized by molecular oxy-
gen to produce hydrogen peroxide (Fig. 1A). The forma-
tion of the imine intermediate, and hence the LSD1-me-
diated demethylation, is as mentioned above, critically
dependent on the protonation of the nitrogen. Thus, this
enzyme can only catalyze demethylation of mono- and
LSD1 has been identified as a part of both repressive
and activating complexes. Intriguingly, the demethylase
activity and specificity of LSD1 appear to be determined
by its binding partners, including CoREST, the androgen
receptor (AR), and BHC80 (Lee et al. 2005; Metzger et al.
2005; Shi et al. 2005), as well as by the neighboring his-
tone marks surrounding the substrate (Forneris et al.
2005, 2006). Thus, when associated with the repressive
CoREST complex, it acts as an H3K4me2/me1 demeth-
ylase (Shi et al. 2004). In contrast, when in a complex
with the AR it acts as a transcriptional coactivator, de-
methylating H3K9me2/me1 (Metzger et al. 2005). Here,
inhibition of LSD1 expression resulted in an increase of
H3K9 methylation of AR target genes and a concomitant
decreased transcription of these, directly linking LSD1 to
AR-dependent transcriptional activation.
In another study, LSD1 has been linked to estrogen
receptor (ER) signaling (Garcia-Bassets et al. 2007). Gar-
cia-Bassets et al. (2007) investigated the occupancy of
estrogen-responsive promoters by chromatin modifiers
in estradiol-stimulated MCF-7 breast cancer cells. Sur-
prisingly, of 580 promoters found to be occupied by ER?,
58% were co-occupied by LSD1. RNAi-mediated inhibi-
tion of LSD1 expression led to decreased expression of ER?
target genes co-occupied by LSD1, but not by those not
occupied by LSD1. Moreover, LSD1, dependent on its
catalytically activity, was able to restore ER?-induced
expression of LSD1-positive genes (Garcia-Bassets et al.
2007). Taken together, these findings suggest that LSD1,
by demethylating H3K9me2/me1, acts as an essential
coactivator of a significant number of ER?-responsive
Studies performed in Schizosacchaomyces pombe and
Drosophilia melanogaster have implicated LSD1 or-
thologs in the organization of higher-order structure of
chromatin (Nicolas et al. 2006; Lan et al. 2007c; Rudolph
et al. 2007). These studies have corroborated the notion
that LSD1 can act as a H3K9 demethylase associated
with heterochromatin boundaries and euchromatic pro-
moters (Nicolas et al. 2006; Lan et al. 2007c) or as a H3K4
demethylase (Rudolph et al. 2007). Thus, in S. pombe the
LSD1 ortholog SWIRM1/2 (also referred to as spLsd1/2)
has been demonstrated to demethylate H3K9 and deple-
tion of SWIRM1/2 induces heterochromatin spreading
beyond normal heterochromatin regions (Lan et al.
2007c). Consistent with a role as a transcriptional core-
pressor, transcription is decreased at adjacent genomic
sites, correlating with an increase in H3K9 methylation.
In contrast, the D. melanogaster LSD1 ortholog Su-
H3K4me2 and to be important for subsequent H3K9
methylation and heterochromatin formation (Rudolph et
Cloos et al.
1116 GENES & DEVELOPMENT
al. 2007). Deletion of dLsd1 strongly reduces viability of
mutant flies and leads to sterility and defects in ovary
development (Di Stefano et al. 2007). Interestingly, mu-
tant alleles of dLsd1 suppress positional effect variega-
tion, indicating the involvement of dLsd1 in balancing
euchromatin and heterochromatin (Di Stefano et al. 2007).
In concert, these studies suggest an important role for
LSD1 in the regulation of chromatin boundaries in these
organisms (for review, see Chosed and Dent 2007). Mouse
knockout studies have demonstrated that deletion of
LSD1 leads to embryonic lethality, and that LSD1 is re-
quired for late cell lineage determination and differentia-
tion (Wang et al. 2007b). Moreover, these studies suggest
that LSD1 primarily affects gene activation or repression
programs by recruiting specific coactivator or corepres-
sor complexes (Wang et al. 2007b). In addition to the
roles as a histone demethylase, a recent study reported
that LSD1 demethylates a methylated form of Lys 370 of
the transcription factor p53 (J. Huang et al. 2007).
Whereas in vitro studies demonstrate that LSD1 can de-
methylate both mono- and dimethylated K370 on p53,
the enzyme shows a strong preference for dimethylated
K370 in vivo, repressing p53 function by inhibiting its
interaction with 53BP1 (J. Huang et al. 2007).
In summary, LSD1 can function as both a histone de-
methylase specific for H3K4me2/me1 and H3K9me2/
me1 and for nonhistone substrates, such as p53. The spe-
cific activity of the enzyme is determined partially by its
association with different complexes, thereby allowing it
to participate in the regulation of transcriptional programs,
heterochromatin spreading and stress-induced responses.
The Jumonji (JmjC) domain protein family
Similar to the discovery of LSD1, a JmJC domain-driven
demethylase reaction was proposed in a review before
the actual experimental characterization of the demeth-
ylases (Trewick et al. 2005). The Jumonji protein family
contains the conserved JmjC domain that was first iden-
tified in the Jumonji protein (JARID2). Jumonji means
cruciform in Japanese, and the gene was so named be-
cause mice with a genetrap inserted in the Jumonji locus
develop an abnormal cross-like neural tube (Takeuchi et
al. 1995). There are 27 different JmjC domain proteins
within the human genome, of which 15 have been pub-
lished to demethylate specific lysines or arginines in the
H3 tail (Fig. 2). The first JmjC domain demethylase de-
scribed, was FBXL11 (synonyms: JHDM1a and KDM2A),
which was shown to specifically demethylate mono- and
dimethylated H3K36 in a Fe(II) and ?-ketoglutarate
(?KG)-dependent manner (Tsukada et al. 2006).
oxidation reaction using FAD as a cofactor. The imine intermediate is hydrolyzed to an unstable carbinolamine that subsequently
degrades to release formaldehyde. (B) The JMJC proteins use ?KG and iron (Fe) as cofactors to hydroxylate the methylated histone
substrate. Fe(II) in the active site activates a molecule of dioxygen to form a highly reactive oxoferryl [Fe(IV) = O] species to react with
the methyl group. The resulting carbinolamine intermediate spontaneously degrades to release formaldehyde. Throughout the figure,
the wavy line indicates attachment to the peptide backbone.
Mechanisms of lysine demethylation by LSD1 and JMJC proteins. (A) LSD1 demethylates H3K4me2/me1 via an amine
Erasing the methyl mark
GENES & DEVELOPMENT1117
Although the reaction mechanism for the FBXL11-
mediated demethylation, at least in theory, could utilize
trimethyl H3K36 as substrate, no such activity could be
demonstrated. Thus, even though the identification of
LSD1 and FBXL11 as histone demethylases represent im-
portant milestones for epigenetic research, by demon-
strating the dynamic regulation of methyl marks, they
did not resolve the question of the reversibility of tri-
methylated lysine marks.
However, only a few months after publication of the
FBXL11 enzyme, a flurry of papers showed that the
JMJD2 protein can demethylate H3K9me3/me2 and
H3K36me3/2 (Cloos et al. 2006; Fodor et al. 2006; Klose
et al. 2006b; Whetstine et al. 2006), formally demonstrat-
ing the reversibility of trimethylated lysine marks. To-
day, these discoveries have been followed by the identi-
fication of the JMJD1 protein group that demethylates
H3K9me2/me1 (Yamane et al. 2006), the JARID1 group
of H3K4me3/me2-specific demethylases (Christensen et
al. 2007; Iwase et al. 2007; Klose et al. 2007; Lee et al.
2007a; Tahiliani et al. 2007), and lately, the UTX/JMJD3
group, capable of demethylating H3K27me3/me2/me1
(Agger et al. 2007; De Santa et al. 2007; Jepsen et al. 2007;
Lan et al. 2007a; Lee et al. 2007b).
The JmjC proteins belong to the ?KG-dependent oxy-
genase super family. A widespread feature of these pro-
teins is the ability to bind Fe(II) ions. In addition, many
of these proteins have the capability to hydroxylate pro-
tein substrates utilizing oxo-ferryl(IV) and ?KG as cofac-
tors. Moreover, some cupin dioxygenases, including
members of the JmjC demethylase family, have the ad-
ditional requirement of ascorbate for full catalytic activ-
ity. Various studies of ?KG-dependent oxygenases in-
cluding the Factor-Inhibiting HIF1 (FIH) suggest the fol-
lowing reaction mechanism: First, the enzyme binds
iron (Fe) through its metal-binding motif HXD/EXnH,
the so-called facial triad. Then, the Fe(II)–enzyme com-
plex binds the cofactor ?KG, and subsequently the sub-
strate and oxygen. The binding of oxygen is followed by
the oxidative decarboxylation of ?KG to produce succi-
nate, carbon dioxide, and ferryl. The latter is a highly
reactive group and can potentially oxidize a C–N bond in
a lysine ?-methyl group, forming an unstable carbinol-
amine that will rapidly break down, leading to the re-
lease of formaldehyde and loss of a methyl group from
lysine (Fig. 1B).
The mode of action of ascorbate is presently unclear
but has been suggested to reduce Fe(III) to its active state
Fe(II) or to function as a “surrogate reducing substrate”
to “rescue” the dioxygenase enzyme in the event of the
uncoupled production of a ferryl [Fe(IV) = O] intermediate.
When constructing a phylogenetic tree of the Jumonji
proteins based on an alignment of their respective JmjC
domains (Fig. 2), these proteins segregate in distinct clus-
ters. In general, it appears that each cluster has specific-
ity for demethylating a certain histone mark. So far,
most subfamilies conform to this rule; however, there
are exceptions—for instance, the FBXL cluster appar-
ently comprises both H3K36me2/me1 and H3K4me3 de-
methylase activities (Tsukada et al. 2006; Frescas et al.
2007). Below we provide an overview of the features of
the different JmjC subfamilies.
The FBXL (KDM2) cluster
Two proteins FBXL11 (synonyms: JHDM1a and KDM2A)
and FBXL10 (synonyms: JHDM1b and KDM2B) consti-
tute the FBXL cluster. These were the first JmjC histone
demethylases published (Tsukada et al. 2006). Both pro-
teins contain an F-box domain in addition to two leu-
cine-rich repeat (LRR) domains (Fig. 2). FBXL11 was
demonstrated to demethylate di- and monomethylated
H3K36 (Tsukada et al. 2006). The di- and trimethylated
family of demethylases. The names, syn-
onyms, substrate specificities, and domain
structures of the proteins are provided.
The lists of synonyms may be longer, but
due to space limitations only the most rel-
evant are provided. Putative oncoproteins
are in red and putative tumor suppressors
are in green. (JmjC) Jumonji C domain;
(JmjN) Jumonji N domain; (PHD) plant ho-
meodomain; (Tdr) Tudor domain; (Arid)
AT-rich interacting domain; (Fbox) F-box
domain; (C5HC2) C5CHC2 zinc-finger do-
main; (CXXC) CXXC zinc-finger domain;
(TPR) tetratricopeptide domain; (LRR) leu-
cine-rich repeat domain; (TCZ) treble-clef
zinc-finger domain; (PLAc) cytoplasmic
phospholipase A2 catalytic subunit.
Phylogenetic tree of the JmjC
Cloos et al.
1118GENES & DEVELOPMENT
variants of the H3K36 mark are enriched at the 3?-end of
actively transcribed genes and are involved in regulating
transcriptional elongation (Joshi and Struhl 2005). Thus,
Set 2, the histone methyl transferase responsible for the
methylation of H3K36 in Saccharmyces cerevisiae, binds
the elongating form of RNA polymerase II (RNAPII). The
Rpd3 histone deacetylase (HDAC) complex is recruited
to H3K36me2 thereby preventing transcription from
cryptic promoters (Li et al. 2007). These findings suggest
that the H3K36 demethylases could have a role resetting
this histone modification when active transcription is
turned of. Apart from its role as an H3K36me2/me1 de-
methylase, little is currently known about the biological
role of this enzyme.
As FBXL11, FBXL10 was also originally reported to
demethylate H3K36me2/me1 (Tsukada et al. 2006).
However, it was recently published that FBXL10 has
H3K4me3-specific demethylase activity (Frescas et al.
2007). Frescas et al. (2007) showed that FBXL10 acts in
the nucleolus, and is involved in the repression of ribo-
somal RNA genes. Consistent with a role for FBXL10 in
repressing ribosomal RNA genes and thus RNA synthe-
sis, FBXL10 negatively regulates cell proliferation (Fres-
cas et al. 2007). FBXL10 features a DNA-binding CXXC
zinc finger and the protein localizes together with the
RNA polymerase I transcription factor UBF at nucleolar
organizing regions, indicating a stable association with
ribosomal DNA (rDNA). Indeed, genome localization
analysis demonstrated that FBXL10 bound various re-
gions of the human rDNA repeat and was especially en-
riched on transcribed CpG-rich areas (Frescas et al.
In agreement with a role of FBLX10 in transcriptional
repression, the protein has been found in a complex com-
prising the Bcl6 corepressor (BcoR), CK2?, SKP1, YAF2,
HP1?, RING1, CBX8, BMI, and Nspc1/Pcgf1 (Gearhart et
al. 2006; Sanchez et al. 2007). Moreover, in addition to
repressing the transcription of ribosomal genes, recent
studies also showed that FBXL10 functions as a transcrip-
tional repressor of c-JUN target genes (Koyama-Nasu et
al. 2007). Thus, FBXL10 is a candidate tumor suppressor
gene. This notion has been supported by several studies.
First, retroviral insertional mutagenesis within the
FBXL10 gene has been shown to cause lymphoma in
BLM (Bloom syndrome RecQ protein-like-3 DNA heli-
case)-deficient mice (Suzuki et al. 2006). Second, inhibi-
tion of FBXL10 expression increases cell proliferation
(Koyama-Nasu et al. 2007), and third, FBXL10 expression
is significantly decreased in various primary brain tu-
mors, including glioblastoma multiforme (Frescas et al.
F-box domain proteins have foremost been character-
ized as the components of the SCF ubiquitin–ligase com-
plexes, which recognize the substrate being targeted for
ubiquitin-mediated proteolysis. The presence of an F-box
domain in the FBXL family proteins could suggest that
these proteins regulate transcription through the com-
bined action of demethylation and ubiquitylation of
transcription factors or other proteins associated with
transcription. In agreement with this, a possible function
of the FBXL10–Ring1B complex in H2A ubiquitylation,
has been demonstrated (Sanchez et al. 2007).
The JMJD1 (KDM3) cluster
JMJD1A (synonyms: TSGA, JDHM2A, and KDM3A) was
originally isolated as a male germ-specific transcript
(Hoog et al. 1991). JMJD1A was later shown to be an
H3K9me2/me1-specific demethylase (Yamane et al.
2006). The protein features an LXXLL motif that is a
signature involved in nuclear receptor interactions
(Heery et al. 1997). The expression of JMJD1A is most
prominent in testes, and has been implicated in demeth-
ylation of H3K9me2 of AR target genes (Yamane et al.
2006). Thus, JMJD1A was found to interact with the AR
in a ligand-dependent manner. Inhibition of JMJD1A ex-
pression in the prostate cancer cell line LnCaP led to an
increase in H3K9me2 in a subset of AR target genes in-
cluding PSA, NKX3.1, and TMPRSS22, and a decrease in
their expression (Yamane et al. 2006). These results
show that JMJD1A acts as a coactivator of AR-mediated
More recently, Jmjd1a genetrap mice were described
(Okada et al. 2007); these mice apparently develop nor-
mally but are infertile (Okada et al. 2007). A careful
analysis of the infertility phenotype revealed that sperm
cells from Jmjd1a genetrap mice exhibited post-meiotic
chromatin condensation defects, leading to a low num-
ber of mature sperm cells, of which all had abnormally
shaped heads and the vast majority were immotile
(Okada et al. 2007). Moreover, Jmjd1a was found to bind
to, and positively regulate, the expression of the two genes,
transition nuclear protein (Tnp1) and protamine 1 (Prm1),
by removing the repressive H3K9 marks from their pro-
moters. The gene products of Tnp1 and Prm1 are indis-
pensable for the histone replacement process that takes
place during the final stages of sperm chromatin conden-
sation and maturation, supporting the notion that
JMJD1A contributes to spermiogenesis by controlling
the expression of these genes (Okada et al. 2007). In con-
clusion, JMJD1A appears to fulfill essential roles for
spermiogenesis, and its disruption causes male infertil-
ity phenotypes reminiscent of human syndromes as azo-
ospermia and globospermia, advancing JMJD1A as a can-
didate gene for these infertility conditions.
In another study, JMJD1A was found to associate with
the cardiac and smooth muscle cell (SMC)-specific tran-
scription factor myocardin and the related proteins
MRTF-A and MRTF-B (Lockman et al. 2007). Moreover,
genomic localization analysis and gene expression analy-
sis showed that JMJD1A binds to SMC-specific promot-
ers and is regulating TGF?-mediated activation of these
genes. These results suggest that JMJD1A is involved in
regulating SMC differentiation.
Human JMJD1B (synonyms: JHDM2B, 5qCNA and
KDM3B) has also been identified as an H3K9me2/me1
demethylase (Klose et al. 2006a). The human JMJD1B
gene is located at 5q31,a chromosomal area that is often
deleted in malignant myeloid disorders, including acute
myeloid leukemia and myelodysplasia (Hu et al. 2001).
Erasing the methyl mark
GENES & DEVELOPMENT1119
Hu et al. (2001) showed that the enforced expression of
JMJD1B in a cell line carrying a 5q deletion inhibits clo-
nogenic growth, indicating that loss of JMJD1B may be
involved in the pathogenesis of these malignancies, and
that the protein may have tumor suppressor activities.
The protein JMJD1C (also known as TRIP8 or
JHDM2C) was originally described a thyroid-hormone
receptor (TR)-interacting protein and is closely related to
JMJD1A and JMJD1B, but so far, no enzymatic activity
has been reported. The protein features a conserved iron-
binding HXD/EXnH motif, as well as a ?KG-binding site,
indicating that this protein might be an H3K9me2/me1-
specific histone demethylase. Consistent with such a
role, a short variant s-JMJD1C was reported recently to
be a transcriptional coactivator interacting with the AR
(Wolf et al. 2007). In the brain, s-JMJD1C is foremost
present in AR-expressing neuronal population, and its
concentration appears to vary according to the hormonal
status in a region-specific manner. Of note, Castermans
et al. (2007) reported the identification of a de novo bal-
anced paracentric inversion 46,XY,inv(10) with a break-
point in chromosome 10q21.3 located within the first
intron of the JMJD1C gene in a boy with autism. This
chromosomal aberration caused a twofold decrease in
expression of two of three JMJD1C transcripts, implicat-
ing this JmjC family member as a candidate gene for
autism. Finally, JMJD1C expression is reduced in breast
cancer tissues compared with normal breast tissues, sug-
gesting a possible role in tumor suppression (Wolf et al.
Hairless (HR) is one of the best-studied members of the
JmjC protein family, but has so far not been ascribed
histone demethylase activity. The protein has high ho-
mology with JMJD1A and JMJD1B, but its potential Fe-
binding HXDXnH motif appears to align differently com-
pared with catalytically active JmjC members, and the
protein could therefore be devoid of demethylase activity.
Various studies point to a role of HR in hair formation;
thus, the protein appears to be crucial for the normal
regulation of several key cellular functions for hair
growth including (1) the maintenance of the dermal pa-
pilla, (2) the disintegration of the inner root sheet, (3)
club hair formation, and (4) apoptosis of keratinocytes in
the hair follicle (Panteleyev et al. 1999). Numerous stud-
ies have underscored the importance of this JmjC mem-
ber for normal hair growth, a few of which are mentioned
HR-null mice are born with fur, but loose their hairs
within their first 3 wk after birth, indicating that HR is
required for regenerating hair follicles (Beaudoin et al.
2005). The biological mechanisms underlying the reini-
tiation of hair growth are currently not fully elucidated,
but probably require the regulation of several genes by
HR. Candidate target genes, most likely repressed by
HR, include (1) Soggy, encoding a protein related to
Dickkopf-3, a member of the Dickkopf family of Wnt
inhibitors; and (2) Wnt modulator in surface ectoderm
(Wise), which is involved in the regulation of Wnt sig-
naling in the hair follicle and in hair follicle regeneration
(Beaudoin et al. 2005). Several other reports have also
suggested HR as a transcriptional corepressor for various
nuclear receptors including the vitamin D receptor
(VDR), the thyroid hormone receptor (THR), as well as
the retinoic acid receptor-related orphan receptors (ROR)
?, ?, ? (Potter et al. 2001, 2002).
The importance of HR for normal regulation of hair
growth in human beings is underlined by the fact that
several nonsense, missense, insertion, and deletion mu-
tations of the human HR gene have been identified. All
these mutations result in hair loss disorders, including
alopecia universalis congenita (AUC; OMIM 203,655)
and arthricia with papular lesions (APL; OMIM 209,500)
(Table 1). Thompson et al. (2006) demonstrated that sev-
eral mutations in rat HR corresponding to missense mu-
tations in human APL reduced or abrogated the ability of
HR to function as a corepressor activity together with
the THR nuclear receptor. Similarly, a large number of
HR mutants, previously described as the molecular
cause of APL, and of which five resulted in amino acid
changes within the JmjC domain of HR, caused loss of
corepressor activity and defective interactions with
HDAC1 (Wang et al. 2007a). In summary, HR is a tran-
scriptional corepressor essential for hair growth, whose
biochemical action may involve the removal of an “ac-
tivatory” methylation mark.
The JMJD2 (KDM4) cluster
The JMJD2 cluster consists of four genes, JMJD2A (syn-
onym: KDM4A), JMJD2B (synonym: KDM4B), JMJD2C
(synonyms: GASC1 and KDM4C), and JMJD2D (syn-
onym: KDM4D) (Fig. 2). They direct the expression of
histone lysine demethylases capable of demethylating
both the H3K9me3/me2 and H3K36me3/me2 marks
(Cloos et al. 2006; Fodor et al. 2006; Klose et al. 2006b;
Whetstine et al. 2006).
Whereas the H3K36me3/me2 mark is associated with
transcriptional elongation (Joshi and Struhl 2005; Keogh
et al. 2005; Lee and Shilatifard 2007) and possibly fulfills
important roles in suppression of inappropriate tran-
scription within the body of genes (Carrozza et al. 2005),
the H3K9me3/me2 mark has generally been associated
with transcriptional repression and formation of hetero-
chromatin involving the recruitment of factors such as
the HP1 proteins (Bannister et al. 2001; Lachner et al.
2001). Moreover, increased H3K9me3/me2 levels, pres-
ent in so-called senescence-associated heterochromatic
foci (SAHFs) together with HP1, have been associated
with stress signals inducing senescence (Narita et al.
2003). Some E2F target genes have been found associated
with SAHFs, resulting in permanent shutdown of tran-
scription from these promoters during senescence
(Narita et al. 2003). Thus, the idea is that the recruit-
ment of specific H3K9me3 methyltransferases (KMTs) to
active promoters or chromosomal regions can result in
the generation of a heterochromatic environment and
repression of transcription. Due to the apparent impor-
tant role of the H3K9me3 mark in regulating cellular
senescence and chromatin structure, Cloos et al. (2006)
searched for proteins binding to this mark in the hope of
Cloos et al.
1120GENES & DEVELOPMENT
JMJC proteins: specificity, complex partners, and association to disease
Involvement in disease
Bcl6, CK2?, Skp1, YAF2, HP1?,
CBX8, BMI1,Nspc1, Ring1B,
Candidate tumor suppressor
KIAA1196, Smad3, Smad7, Skp1
ER71, MRTFA, MRTFB,
Candidate gene for azoospermia
Candidate tumor suppressorCandidate gene for acute
myeloid leukemia andmyelodysplasia
VDRTHR ROR ?, ? ?
AUC (OMIM203655) APL (OMIM209500)
N-CoR, SMRT, pRb
Candidate gene for prostate
Candidate gene for prostate
Candidate gene for squamous
cell carcinoma and prostatecancer
pRB, p107, Myc, E2F1, E2F2,
EID-1, PLZF, BAA90908,
HDAC1HDAC4 HDAC5 HDAC7
pRB, Myc, BF1, PAX9
pRB, REST, RING1, RING2,
Candidate gene for CHD and
continued on next page
Erasing the methyl mark
GENES & DEVELOPMENT1121
Involvement in disease
Candidate gene for HSN1 and ESS1
ASH2L, RBBP5, WDR5, ASC-2,
RBQ3, Matrin, PTIP, hDPY30,
NCOA6, PA1, ZNF281,NEDD4, KIAA2032,
Candidate tumor suppressor
ASH2L, RBBP5, WDR5
Candidate tumor suppressor
Candidate tumor suppressor
Candidate gene for IE
(APL) Arthricia with popular lesion; (AUC) Alopecia universalis congenital; (CHD) congenital heart disease; (ESS1) multiple self-healing squamosus epitheloma; (HSN1) hereditary
neuropathy-1; (IE) intractable epilepsy; (XLMR) X-linked mental retardation.
Cloos et al.
1122 GENES & DEVELOPMENT
finding proteins mediating its function. Among the iden-
tified proteins was JMJD2C, which originally was named
gene amplified in squamous cell carcinoma 1 (GASC1),
because of its amplification in esophagus cell lines (Yang et
Enforced expression of JMJD2A, JMJD2B, and JMJD2C
significantly decreases H3K9me3 and H3K9me2 levels,
and delocalizes HP1, which is consistent with an antago-
nistic role of the JMJD2 family to heterochromatin
(Cloos et al. 2006; Fodor et al. 2006). The possible onco-
genic potential of the JMJD2 family could be the result of
H3K9me3/me2 demethylation, leading to the dissocia-
tion of the SAHFs and re-expression of E2F target genes.
Additionally, Whetstine et al. (2006) demonstrated that
depletion of the Caenorhabditis elegans JMJD2 homo-
log, CeJMJD2, resulted in a global increase of H3K9me3
levels, localized H3K36me3 to meiotic chromosomes
and activation of p53-dependent germline apoptosis. In
this organism, DNA damage-induced apoptosis, as op-
posed to physiological apoptosis, is dependent on the p53
ortholog CEP-1. Interestingly JMJD2-induced apoptosis
was lost in CEP-1 deletion mutants indicating a func-
tional link between JMJD2 loss-of-function and DNA
damage-induced germline apoptosis. Moreover, deple-
tion of CeJMJD2 increased RAD51-positive foci in the
mid-pachytene nucleus, suggesting an increase in the
levels of DNA double-strand breaks (DSB), or a delay in
the progression of meiotic DSB repair.
Previous studies have established that disruption of
Suv39h1/h2 leads to loss of H3K9 trimethylation at peri-
centric chromatin, impairment of heterochromatin
structures, and genomic stability (Peters et al. 2001). The
double-knockout mice also develop B cell lymphomas,
suggesting that Suv39h1/h2 act as tumor suppressors.
JMJD2A-C proteins are overexpressed in cancer, and in-
hibition of JMJD2A and JMJD2C affects cellular growth
(Cloos et al. 2006). The enzymatic activity of JMJD2 pro-
teins toward demethylation of the repressive marks
H3K9me3/me2 indicates that these proteins work as
transcriptional coactivators. Taken together, these data
suggest that the JMJD2 proteins are oncogenes, which
contribute to tumor formation by (1) inducing genomic
instability, (2) suppressing of cellular senescence, or (3)
The involvement of the JMJD2 proteins in tumorigen-
esis has been supported further by a recent report dem-
onstrating the functional interaction between JMJD2C
and the AR in prostate carcinomas (Wissmann et al.
2007). Wissmann et al. (2007) showed that JMJD2C can
bind to the AR and work as an essential coactivator of
AR-induced transcription and cellular growth.
The JMJD2 proteins have also been proposed to work
as transcriptional repressors: JMJD2A has been shown
previously to be associated with the transcriptional re-
pressor complex, N-CoR (Gray et al. 2005), and the in-
hibition of JMJD2A expression has been shown to trigger
an increase in H3K9me3 levels correlating with expres-
sion of the N-CoR target gene ASCL (Klose et al. 2006b).
The fact that JMJD2 proteins can both catalyze the
demethylation of H3K9 and H3K36 is surprising, and
suggests multiple roles for these enzymes. The crystal-
lization of the catalytical JmjC domain of JMJD2A to-
gether with its peptide substrates has provided insights
into the specificity of substrate binding as well as the
structural requirements for catalysis (Chen et al. 2007;
Couture et al. 2007; Ng et al. 2007). The studies show
that despite significant differences in the adjacent se-
quence motifs of lysine K9 and K36, the two substrates
have a very similar, but not identical, binding mode. More-
over, two of the studies found the K9 lysine to be the fa-
vored substrate (Couture et al. 2007; Ng et al. 2007),
whereas the last could not detect significant differences
in affinity toward K9 and K36 (Chen et al. 2007).
In addition to the catalytic domain the JMJD2 proteins
also contain PHD and Tudor domains (Fig. 2), which are
expected to make significant contributions to the activ-
ity and specificity of these enzymes by guiding them to
specific areas of chromatin. Thus, the tandem Tudor do-
mains of JMJD2A has been found to bind to H3K4me3,
H3K9me3, and H4K20me3, thereby potentially recruit-
ing this demethylase to areas of active or inactive tran-
scription (Huang et al. 2006; Kim et al. 2006; Lee et al. 2008).
Although we only have limited knowledge regarding
the biological role of the JMJD2 proteins, recent studies
have implicated JMJD1 and JMJD2 family members in
the regulation of self-renewal capacity of mouse embry-
onic stem cells (ES): The stem cell transcription factor
Oct4 was shown to partially control the expression of
JMJD1A and JMJD2C, and depletion of the two demeth-
ylases by shRNA decreased the expression of stem cell
markers and induced genes involved in differentiation
(Loh et al. 2007). It is noteworthy that depletion of the
two demethylases only modestly affected the expression
of stem cell markers. This finding suggests that these
demethylases are involved in fine-tuning expression lev-
els during differentiation, rather than being instrumental
for cell fate decisions and/or determining the balance
between pluripotency and differentiation, such as, for
example, the transcription factors Klf4, Sox2, and Oct4.
In summary, current data suggest that the JMJD2 pro-
teins can act as oncogenes, and are essential for normal
embryogenesis. They can function as both transcrip-
tional corepressors and coactivators. There are still sub-
stantial gaps in our understanding of how the activity
and specificity of these proteins is regulated and of the
dual role of these proteins in transcriptional regulation.
The activity of the JMJD2 proteins may be dictated, as
demonstrated for LSD1, by their complex partners. Fur-
ther studies are required to attain a deeper understanding
of these questions.
The JARID1 (KDM5) cluster
The JARID1 subfamily of JmjC proteins encompasses
four members: JARID1A, JARID1B, JARID1C, and
JARID1D (also called KDM5A-D according to the novel
nomenclature), which all can demethylate tri- and di-
methylated H3K4 (Christensen et al. 2007; Iwase et al.
2007; Klose et al. 2007; Lee et al. 2007a; Tahiliani et al.
2007; Yamane et al. 2007).
Erasing the methyl mark
GENES & DEVELOPMENT 1123
Tri- and dimethylation on histone H3 at Lys 4
(H3K4me3/me2) is often found at transcribed genes.
While H3K4me3 is highly enriched around transcrip-
tional start sites, H3K4me2 seems to be present through-
out the coding region of transcribed genes (Noma et al.
2001; Santos-Rosa et al. 2002; Liang et al. 2004; R.
Schneider et al. 2004; Bernstein et al. 2005). Evidence
indicates that trimethylation of K4 first appears after the
assembly of the preinitiation complex (PIC), leading to
the suggestion that this modification is involved in
maintenance of transcription rather than initiation of
transcription (Pavri et al. 2006). However, another study
has shown that the basal transcription factor TFIID
binds to the H3K4me3 mark via the PHD finger of TAF3,
indicating that the interaction between TFIID and
H3K4me3 may be important for PIC assembly and sub-
sequent activation of genes in mammalian cells (Ver-
meulen et al. 2007).
Consistent with a contribution of H3K4 methylation
to transcription, the bulk of data currently indicates that
JARID1 proteins function as transcriptional repressors
through the demethylation of H3K4me3/me2, and through
the recruitment of other repressive chromatin modifiers
(Christensen et al. 2007; Iwase et al. 2007; Klose et al.
2007; Lee et al. 2007a; Tahiliani et al. 2007).
The JARID1 proteins are highly homologous, and con-
tain a similar domain structure comprising a JmjN, Arid/
Bright, and C5HC2 zinc-finger domain, in addition to
two or three plant homeodomains (PHD) and the cata-
lytic JmjC domain (Fig. 2). Moreover, this group features
various conserved motifs, which are probably involved
in pRB modulation—among those a leukemia-associated
protein (LAP), a rhombotin-2 (RBTN2, LMO2)-binding
domain, and a pRB-binding motif (LXCXE). Indeed,
JARID1A (synonyms: RBP2 and KDM5A) was originally
identified in a screen for proteins binding to the retino-
blastoma protein, pRB (Defeo-Jones et al. 1991; Fattaey
et al. 1993). It has been reported that the interaction with
pRB converts JARID1A from a transcriptional repressor
to a transcriptional activator (Benevolenskaya et al.
In a recent study, Christensen et al. (2007) identified
JARID1A and its related proteins JARID1B and JARID1C
as demethylases specific for H3K4me3/me2. Consistent
with previous reports linking JARID1A to the control of
cellular differentiation (Benevolenskaya et al. 2005)
JARID1A was found to be an important regulator of
HOX transcription (Christensen et al. 2007). Hence,
JARID1A occupies several homeotic gene promoters in-
cluding Hoxa1, Hoxa5, and Hoxa7, and is displaced from
Hox genes during differentiation of mouse ES cells in
response to retinoic acid, correlating with an increase of
their H3K4 trimethylation and expression.
C. elegans and D. melanogaster both encode a single
ortholog of the JARID1 demethylases that share signifi-
cant homology and domain structure with the mamma-
lian JARID1 proteins. The C. elegans JARID1 ortholog
rbr-2 is a bona fide H3K4me3 demethylase, which is es-
sential for development. Thus, the C. elegans mutant
strain (tm1231) expressing a mutant RBR-2 protein lack-
ing the entire catalytic JmjC domain, displayed a highly
penetrant vulval phenotype with 80% of the animals be-
ing either multivulval or vulvaless (Christensen et al.
2007). In addition, mutant animals displayed a signifi-
cant increase in global H3K4 methylation in all larval
stages as well as in the adult worm, showing that ongo-
ing H3K4 demethylation by RBR-2 is essential for the
correct regulation of H3K4 methylation and for normal
development in this organism.
The D. melanogaster JARID1 ortholog, Lid (short for
little imaginal disc), is also an H3K4 demethylase, and
mutation of Lid results in a global increase of this mark
(Secombe et al. 2007). This was rather surprising, be-
cause Lid was originally classified as a Trithorax (Trx)
gene (Gildea et al. 2000) and the protein was therefore
anticipated to maintain H3K4 methylation, rather than
removing it. Two proposals have been put forward to
rationalize these findings. One model holds that a global
increase of H3K4me3 levels in Lid mutants may displace
Trx proteins from their appropriate HOX loci, resulting
in decreased expression of these genes and a Trx pheno-
type (Secombe et al. 2007). Another proposition is based
on the observation that the transcription factor Myc
binds to the JmjC domain of Lid and inhibits its demeth-
ylation activity. According to this, Myc could sequester
Lid preventing its demethylase activity triggering a local
increase in H3K4 trimethylation at Myc target genes. In
turn, this could lead to recruitment of activating com-
plexes promoting transcriptional activation at these
sites, resulting in a Trx phenotype (Secombe and Eisen-
Because of the high conservation of the JmjC domain
among JARID1 proteins between species, one would pre-
dict that the binding between the JARID1 proteins and
MYC is conserved during evolution. That this indeed
might be the case is supported by data showing that en-
dogenous JARID1A and JARID1B can bind to MYC in ES
cells (Secombe et al. 2007).
Whereas mutation of Lid is fatal and mutation of RBR-2
leads to severe developmental phenotypes, Jarid1a-null
mice only display minor phenotypes, probably reflecting
functional redundancy among mammalian JARID1 pro-
teins (Klose et al. 2007). Thus, apart from neutrophillia
and behavioral abnormalities when held upside-down by
the tail, these mice appear to be grossly normal. How-
ever, a detailed analysis documented decreased apoptosis
and increased entry into the G1 phase of the cell cycle in
hematopoietic stem cells and myeloid progenitor com-
partments (Klose et al. 2007). In addition, expression ar-
rays indicated that the levels of several genes were af-
fected in Jarid1a-null mice: The mRNA levels of genes
such as Sdf1, Cxlc5, and Foxp2 were increased compared
with wild-type littermates, whereas Kcnd3, Sema4a, and
Sorbs1 expression was decreased. Genomic location
analysis suggests that Sdf1 is a direct target gene for
Jarid1a, and that increased Jarid1a binding to the Sdf1
promoter decreased H3K4 trimethylation at this site.
JARID1B (synonyms: PLU1 or KDM5B) was originally
identified in a screen for genes regulated by the oncogene
c-ErbB2. JARID1B is highly expressed in 90% of ductal
Cloos et al.
1124 GENES & DEVELOPMENT
breast carcinomas, and the gene has been associated with
the malignant phenotype of breast (Lu et al. 1999; Barrett
et al. 2002) and prostate cancer (Xiang et al. 2007b). Based
on this link to cancer and to its rather restricted expres-
sion pattern in normal adult tissues, where only ovary,
testes, and the mammary gland of the pregnant female
display high levels of the protein, JARID1B was sug-
gested to belong to the family of testis cancer antigens
(Chen and Old 1999; Barrett et al. 2002). Consistent with
a putative role in promoting cancer, JARID1B is required
for the proliferation of the breast cancer cell line MCF-7
and for the tumor growth of mammary carcinoma cells
in nude mice (Yamane et al. 2007).
JARID1B target genes have been identified by genomic
location analysis, and several of these have been impli-
cated in breast cancer proliferation including 14–3–3?,
BRCA1, CAV1, and HOXA5 (Yamane et al. 2007). In
agreement with a role of JARID1B as a transcriptional
repressor, inhibition of its expression leads to increased
H3K4 trimethylation at these target genes and increased
transcription of these genes (Yamane et al. 2007).
Previously, other studies have linked JARID1B to re-
pression of developmental genes. Here, JARID1B was
found to bind the developmental transcription factors
such as brain factor-1 (BF-1) and paired box 9 (PAX9) via
a conserved sequence motif (AXAAXVPX4VPX4VPX8P),
termed the VP motif (Tan et al. 2003). Mutation of the
VP motif in BF-1 and PAX9 abrogated JARID1B corepres-
sion activity (Tan et al. 2003). Both BF-1 and PAX are
known to interact with members of the Groucho core-
pressor family potentially supporting a role for JARID1B
in Groucho-mediated transcriptional repression.
JARID1C (synonyms: SMCX and KDM5C) is tran-
scribed from the X chromosome, and it is one of few
genes that are not transcriptionally silenced during the
X-inactivation process (Wu et al. 1994a). Interestingly, a
large number of point mutations have been identified
within the JARID1C gene in patients affected by
X-linked mental retardation (XLMR) (Wu et al. 1994b;
Brown et al. 1995; Jensen et al. 2005; Santos et al. 2006;
Tzschach et al. 2006). One of these mutations (A388P) is
located in the vicinity of the proteins N-terminal PHD
domain. Importantly, in the context of the wild-type pro-
tein this PHD domain, but not the A388P mutant, can
bind to H3K9me3 (Iwase et al. 2007). Moreover, intro-
duction of this or other XLMR-linked mutations into
wild-type JARID1C was found to reduce the in vitro de-
methylation activity of the protein (Iwase et al. 2007).
Collectively, these findings suggest that XLMR-associ-
ated mutations in JARID1C may affect both chromatin
association and the demethylating activity of the pro-
tein, and could be important for the pathophysiology of
some forms of XLMR. Consistent with such a role,
RNAi-mediated depletion of JARID1C in rat cerebellar
granule neurons led to a significant decrease in the den-
dritic lengths of neurons that could be rescued by rein-
troduction of the wild-type protein, but not by XLMR-
linked mutants (Iwase et al. 2007). The importance of
JARID1C for brain development was further underscored
by studies in zebrafish. Here, the expression of the
JARID1C homolog is largely restricted to the brain dur-
ing development, and its depletion suggests a role for the
protein in neuronal survival (Iwase et al. 2007).
Another report has suggested a direct role for JARID1C
in chromatin dynamics and REST-mediated repression
(Tahiliani et al. 2007). In this study, JARID1C was copu-
rified with HDAC1 and HDAC2, as well as the histone
H3K9 methyltransferase G9a (synonym: KMT1C), and
the transcriptional repressor REST. Chromatin immuno-
precipitation (ChIP) analysis revealed that JARID1C and
REST co-occupy the neuron restrictive silencing ele-
ments in the promoters of a subgroup of REST target
genes. Moreover, depletion of JARID1C by RNAi led to
the derepression of several of these target genes and a
simultaneous increase in H3K4 trimethylation at the so-
dium channel type 2A (SCN2A) and synapsin I (SYN1)
promoters (Tahiliani et al. 2007). This observation led to
the interesting suggestion that loss of JARID1C activity
could abrogate REST-mediated neuronal gene regulation,
thus contributing to the pathogenesis of JARID1C-asso-
ciated XLMR (Tahiliani et al. 2007).
Analogously, JARID1D (synonym: SMCY and KDM5D)
was found to associate with the Polycomb-like ring fin-
ger-containing protein Ring6a/MBLR (Lee et al. 2007a).
Ring6a/MBLR has been identified previously as a com-
ponent of an E2F6-containing complex with the ability
to ubiquitylate Lys 119 on histone H2A (Akasaka et al.
2002; Ogawa et al. 2002; H. Wang et al. 2004). JARID1D
was reported to occupy the promoters of the Synapsin,
Engrailed 1, and Engrailed 2 genes, and to be implicated
in the regulation of the latter (Lee et al. 2007a). Consis-
tent with such a role, depletion of JARID1D by RNAi led
to an increase in H3K4me3/me2 levels at the Engrailed 2
promoter and a concurrent increase in binding of the
RNAPII machinery and an increased transcription of En-
grailed 2 (Lee et al. 2007a). In vitro reconstitution experi-
ments indicated that Ring6a/MBLR could enhance the
demethylase activity of JARID1D. Taken together, these
findings support a model where recruitment of Ring6a-
JARID1D to the transcription start sites of specific pro-
moters leads to loss of K4 methylation, as well as NURF
and RNAPII complex members leading to the repression
of the affected gene.
In summary, the four JARID1 proteins are specific his-
tone demethylases for H3K4me3 and H3K4me2, whose
main function is to work as transcriptional corepressors.
Results from C. elegans and D. melanogaster have
shown that the proteins are required for normal devel-
opment, and studies of human malignancies have, in par-
ticular, pointed to a role of JARID1B in the development
of cancer and JARID1C in XLMR.
The UTX/JMJD3 (KDM6) cluster
This cluster consists of three proteins, UTX (synonym:
KDM6A), UTY, and JMJD3 (synonym: KDM6B) (Fig. 2).
UTX and JMJD3 are histone demethylases specific for
H3K27me3/me2 (Agger et al. 2007; De Santa et al. 2007;
Hong et al. 2007; Lan et al. 2007a,b; Xiang et al. 2007a),
whereas no activity has been reported for UTY so far.
Erasing the methyl mark
GENES & DEVELOPMENT1125
UTX and UTY are highly homologous and character-
ized by the presence of 6 tetratricopeptide repeat (TPR)
domains in addition to the catalytic JmjC domain and a
treble clef zinc-finger domain (Fig. 2). The TPR domain,
is a structural motif present in a wide range of proteins in
prokaryotes and eukaryotes, and is supposed to constitute
an ancient protein–protein interaction module (Blatch and
Lassle 1999; D’Andrea and Regan 2003). TPR proteins
contain multiple copies of a degenerate 34-amino-acid
motif and self-associate via a “knobs and holes” mecha-
nism mediating the assembly of multiprotein com-
plexes. Indeed, several recent studies have demonstrated
that UTX forms part of different multiprotein com-
plexes. One study identified UTX as part of an H3K4–
methyltransferase complex containing MLL2 (ALR or
KMT2B), PTIP, ASC2, ASH2, RBQ3, WDR5, and matrin
(Issaeva et al. 2007). Three other studies, found UTX in
association with two H3K4–methyltransferase Set1-like
complexes containing MLL3 and MLL2, respectively
(Cho et al. 2007; Lee et al. 2007b; Patel et al. 2007). In
addition to ASH2L, RBBP5, and WDR5, three subunits
shared by all human Set1-like complexes this complex
also contained hDPY-30, the human homolog of the
Sdc1 subunit of the yeast COMPASS/Set1 complex as
well as NCOA6, PA1, and PTIP. Of interest, both UTX
complexes contained the protein PTIP. PTIP is known to
interact with 53BP1, a key regulator of the DNA damage
response that translocates to DNA DSB foci upon ioniz-
ing radiation. This finding indicates that these UTX-con-
taining complexes and their associated demethylase and
methyltransferase activities may be involved in DNA
damage response and/or repair. Analogously, De Santa et
al. (2007) reported that JMJD3 can coimmunoprecipitate
with all three K4-methyltransferase core subunits
(ASH2L, RBBP5, and WDR5), providing further support
for a general association of H3K27 demethylases with
Interestingly, in vitro demethylation studies using re-
combinant UTX have demonstrated the ability of this
enzyme to convert trimethylated H3K27 to its unmeth-
ylated state (Agger et al. 2007; Lan et al. 2007a). Con-
versely, several parallel studies have independently
found UTY to be enzymatically inactive (Hong et al.
2007; Lan et al. 2007a). This is surprising, given the high
homology between UTX and UTY, and may indicate
that the latter protein fulfills other functions than de-
methylation, or that additional factors are required for
its activity. Moreover, the fact that UTX escapes X in-
activation (Greenfield et al. 1998) would mean that
males only have a half dose of UTX-like H3K27 demeth-
In contrast to some of the other demethylases charac-
terized so far, the UTX/JMJD3 group has no or little ac-
tivity on the physiologically relevant substrate, the nucleo-
some in vitro (Lan et al. 2007a; J Christensen, K. Agger, P.
Cloos, and K. Helin, unpubl.). This finding suggests that
these enzymes require additional cofactors for their
function; indeed, ectopic expression of UTX alone ap-
pears to have little effect on K27 methylation levels in
most cell types.
UTX is recruited to the promoters of the anterior genes
of the HOXA and HOXB clusters upon differentiation of
NT2/D1 cells with retinoic acid (Agger et al. 2007; Lee et
al. 2007b). The recruitment correlates with the loss of
H3K27me3, SUZ12, and EZH2 from these promoters,
and with activation of the genes. Moreover, depletion of
UTX prevents H3K27 demethylation of the HOXB1 pro-
moter and its induction in response to retinoic acid treat-
ment (Agger et al. 2007). These results suggest that UTX
is required for the activation of the HOX cluster during
Similarly, De Santa et al. (2007) demonstrated that
JMJD3 is transiently associated with the HOXA7 and
HOXA11 promoters; however, the functional impor-
tance of this association was not addressed.
De Santa et al. (2007) identified JMJD3 as a gene highly
induced in activated macrophages. Bone morphogenetic
protein 2 (BMP2) was identified as a JMJD3 target gene
by expression studies and ChIP analysis whose expres-
sion increased with LPS-induced loss of JMJD3 from its
promoter (De Santa et al. 2007). These data indicate that
JMJD3 has functional role as a transcriptional activator
during differentiation (De Santa et al. 2007).
Various studies have identified H3K27 demethylases
as essential for normal development. Depletion of the
two zebrafish UTX orthologs, zUTX1 and zUTX2, by
morpholino treatment caused a decreased expression of
Hox genes, truncations of the posterior notocord, and
abnormal development of the posterior structures (Lan et
al. 2007a). The phenotypes observed in these animals
with disturbed development of the posterior trunk, re-
semble thrithorax-like phenotypes and are consistent
with an antagonistic role of UTX to PcG-mediated si-
lencing (Lan et al. 2007a). Human UTX could partially
rescue the phenotype, whereas a catalytically inactive
form could not, demonstrating that the H3K27-demeth-
ylating activity of the enzyme was responsible for the
observed phenotype (Lan et al. 2007a).
Similarly, analysis of mutant strains and the use of
RNAi have shown that F18E9.5, one of four UTX/JMJD3
orthologs encoded by C. elegans, which have high ho-
mology with human JMJD3, is required for normal go-
nadal development (Agger et al. 2007).
As discussed above the mammalian UTX and JMJD3
proteins have also been shown to have a role in various
differentiation processes in mammalian cells, and in agree-
ment with this notion, JMJD3 is activated by the retinoic
acid receptor in neural stem cells (Jepsen et al. 2007).
Finally, because of the implication of these enzymes in
cell fate decisions, counteracting pluripotency and be-
cause of their putative involvement in the transcrip-
tional regulation of the INK4A-ARF locus these enzymes
are putative tumor suppressors (see further below).
The JARID2 protein also known as Jumonji (JMJ) is phy-
logeneticly closely related to the JARID1 family. As the
JARID1 members, JARID2 features an Arid/Bright domain
in addition to a JmjC and JmjN domain (Fig. 2).
Cloos et al.
1126 GENES & DEVELOPMENT
The Jarid2 gene was originally identified in a genetrap
screen for genes involved in mouse embryonic develop-
ment (for review, see Jung et al. 2005b). Depending on
the genetic background of the mouse strain, Jarid2-null
mice die in utero (Takeuchi et al. 1995, 1997, 1999) or
neonatally (Lee et al. 2000). The likely cause of death
appears to be various heart abnormalities similar to hu-
man congenital heart diseases (CHD), including ven-
tricular septal defects, noncompaction of the ventricular
wall, double outlet right ventricle, and dilated atria (Lee
et al. 2000). In addition, Jarid2-null mice have an abnor-
mal cruciform neural tube morphology (Takeuchi et al.
1995). In situ hybridization studies of markers for cardiac
development indicate that cardiomyocytes in Jarid2-null
mice are differentiated, but that the transcriptional regu-
lation of genes involved in the formation of heart cham-
bers are defective in late-stage embryos (Lee et al. 2000;
Toyoda et al. 2003). Interestingly, Volcik et al. (2004)
found a threefold increased risk for CHD in individuals
carrying single-nucleotide polymorphisms in exon 6 of
the JARID2 gene, suggesting it as a candidate gene for
At the molecular level, JARID2 appears to function as
a repressor of cardiomyocyte proliferation through inter-
action with pRB (Jung et al. 2005a), which along with its
relative p130, are key regulators of cell cycle progression
in these cells (MacLellan et al. 2005). JARID2 has been
shown to repress the expression of Atrial natriuretic fac-
tor (ANF) through physical interaction with two cardiac-
restricted transcription factors, the homeodomain pro-
tein Nkx2.5 and the zinc-finger protein GATA4 (Kim et
al. 2004). More recently, a single-nucleotide polymor-
phism located in the Arid/Bright domain of JARID2 was
associated to schizophrenia, identifying it as a candidate
gene for this psychiatric disorder (Pedrosa et al. 2007). So
far, no demethylation activity has been assigned to
JARID2, but based on its biological function as a tran-
scriptional repressor, JARID2 would be anticipated to re-
move an activating mark. However, the protein does not
feature a fully conserved iron-binding site, and is there-
fore possibly devoid of demethylation activity.
The PHD finger (PHF) cluster
This cluster consists of three proteins that in addition to
the JmjC domain contain a PHD finger domain: PHD
finger protein 2 (PHF2), PHD finger protein 8 (PHF8), and
KIAA1718 (Fig. 2). No enzymatic activities have been
assigned to these proteins, and very little is known about
their biology. Truncating mutations in the PHF8 gene
are associated with XLMR and cleft lip/palate in affected
patients (Siderius et al. 1999; Laumonnier et al. 2005;
Koivisto et al. 2007), indicating an important function of
PHF8 in the development of cognitive functions and
midline formation. Alignment studies and phylogenetic
analysis of the PHF cluster indicates that it is closely
related to the FBXL and JMJD1 families. Moreover, the
residues potentially involved in coordination of the iron
and ?KG cofactors in both PHF8 and KIAA1718, are
identical to the ones found in FBXL11, making these
proteins prime candidates for histone demethylases
(Klose et al. 2006a). The last member of the PHF family
is PHF2; this protein does not feature a conserved HXD/
EXnH motif, and it is therefore unlikely that it has de-
methylase activity. Little is known about the biological
function of this protein, but the PHF2 gene is located on
human chromosome 9q22 within the candidate region
for hereditary neuropathy I (HSN1) (Nicholson et al.
1996; Blair et al. 1997) and multiple self-healing squa-
mosus epitheloma (ESS1) (Goudie et al. 1993).
Interestingly, deletion of the C. elegans PHF2/PHF8
ortholog 4F429 causes embryonic lethality with a low
penetrance, suggesting an important developmental role
of this protein (http://www.wormbase.org).
Heat-shock 27 (Hsp27)-associated protein 1 (HSPBAP1,
also denoted PASS1), is a member of the JmjC family,
most closely related to JMJD5 and FIH (Fig. 2). The pro-
tein features an HXDXnH motif and could potentially be
a histone demethylase. HSPBAP1 is expressed in most
tissues except the brain. The protein is most abundant in
thymus, pancreas, and testis (Liu et al. 2000; Jiang et al.
As suggested by its name, HSPBAP1 was initially iden-
tified in a screen for proteins associated with Hsp27 (Liu
et al. 2000). Functionally, HSPBAP1 appears to be in-
volved in the regulation of stress responses in cells by
inhibiting the function of Hsp27 (Liu et al. 2000; Jiang et
Hsp27 has a neuroprotective effect during experimen-
tally induced epileptic neuropathology. Interestingly, in
this context, HSPBAP1 inhibits the ability of Hsp27 to
protect cells against heat shock (Liu et al. 2000) and it is
abnormally expressed in the anterior temporal neocortex
of patients with intractable epilepsy (IE) (Xi et al. 2007).
These results indicate that deregulation of HSPBAP1
may play a role in the development IE.
HSPBAP1 may also have a role in the development of
cancer, since the gene has been found associated with a
translocation, giving rise to a DIRC3-HSPBAP1 fusion
protein in a family with renal cell cancer (Bodmer et al.
In conclusion, HSPBAP1 appears to be involved in
regulating stress responses in cells by inhibiting Hsp27.
At present, it is unclear how HSPBAP1 abrogates Hsp27
function, but it could potentially be involved in hydrox-
ylation of some critical residues of Hsp27, similar to the
inhibitory activity of FIH on HIF1?.
As originally proposed by Bannister et al. (2002), deimi-
nases might catalyze the reversal of arginine methyla-
tion. Members of the peptidyl arginine deiminase (PADI)
family deiminate arginine residues by converting them
into citrulline (Nakashima et al. 2002; Cloos and Christ-
Erasing the methyl mark
GENES & DEVELOPMENT1127
gau 2004). PADI4 is a nuclear member of the PADI fam-
ily, and was therefore suggested and subsequently shown
to deiminate histones (Cuthbert et al. 2004; Y. Wang et
al. 2004). Strictly speaking, PADI4 does not cause de-
methylation as it catalyzes the conversion of methyl-
arginine to citrulline and not an unmodified arginine;
therefore, PADI4 rather works antagonistically to argi-
PADI4-catalyzed citrullination of histones has been
associated with estrogen-regulated transcription of the
pS2 promoter. This regulation occurs in a cyclic manner;
after an initial phase with augmented transcription, ar-
ginine methylation is decreased together with a con-
comitant increase in histone citrullination and recruit-
ment of PADI4 to the promoter (Bauer et al. 2002; Me-
tivier et al. 2003). Hence, PADI4 action and appears to
antagonize arginine methylation in vivo repressing tran-
Recently, JMJD6 (previously believed to be the phos-
phatidyl serine receptor and therefore denoted PTDSR)
was identified as a histone demethylase with specificity
to H3R2me2 and H4R3me2, demonstrating that also ar-
ginine methylations are reversible (Chang et al. 2007).
Although this study provided little insight into the biol-
ogy of this demethylase, previous studies of the Jmjd6-
null mouse have demonstrated that the gene is essential
for the differentiation and maturation of a large variety
of tissues during embryogenesis (Bose et al. 2004; J.E.
Schneider et al. 2004). Hence, loss of Jmjd6 causes peri-
natal lethality, growth retardation, and a delay in termi-
nal differentiation of multiple organs including the kid-
ney, intestine, liver, and lungs (J.E. Schneider et al. 2004).
Moreover, some Jmjd6-null mice display severe malfor-
mations of the forebrain and nasal structures and/or im-
paired eye formation, ranging from defects in the differ-
entiation of the retina to complete absence of the eyes
(J.E. Schneider et al. 2004). Jmjd6 also appears to be es-
sential for development of the heart controlling ven-
tricular septal, outflow tract, pulmonary artery, and thy-
mus development (Bose et al. 2004). Consistent with a
role for H4R3 demethylation in embryogenesis, H4R3
methylation is lost in metaphase during oocyte develop-
ment and in preimplantation mouse development (Sar-
mento et al. 2004).
Several reports have addressed the importance of
H3R2 methylation, providing possible clues to the bio-
logical role of this mark (Guccione et al. 2007; Kirmizis
et al. 2007). Current data suggests that asymmetric di-
methylation of H3R2 (H3R2me2a) is antagonistic to tri-
methylated H3K4; thus, H3R2me2a appears to be a re-
pressive modification preventing gene transcription
(Guccione et al. 2007; Kirmizis et al. 2007).
Given the opposing action of this mark to H3K4me3,
it may be anticipated that JMJD6 will form a part of
activating complexes, collaborating with or being inte-
grated into Trithorax-like complexes catalyzing K4
methylation. Likewise, depletion of JMJD6 might cause
“loss of derepression” during development explaining
the multitude of severe phenotypes observed in the
Other JmjC proteins
In addition to the JmjC proteins reviewed above, addi-
tional JmjC proteins exist; most of these are, however,
undescribed in terms of biological activity, and all except
for one have not been ascribed any enzymatic activities.
The one exception is the factor inhibiting HIF-1? (FIH),
which has been identified as an asparaginyl hydroxylase
targeting Asp 803 of the transcription factor hypoxia-
inducible factor 1? (HIF-1?), the master switch of cellu-
lar hypoxia response (Lando et al. 2002a,b). FIH is active
under normoxic conditions abrogating the interaction
between HIF-1?- and p300-repressing HIF1? activity.
The biological impact of histone lysine demethylation
The versatile histone demethylases
It is widely accepted that epigenetic mechanisms, in-
cluding signaling involving histone modifications, are
essential for the control of gene expression programs and
cell fate decisions. Although we are only just starting to
uncover the biology of these exciting enzymes, histone
demethylases are rapidly surfacing as essential players in
an array of important biological processes.
By catalyzing the removal of a histone methyl mark,
these enzymes constitute an ideal and versatile regula-
tory instrument, ensuring the dynamic changes in global
gene expression required for diverse cellular processes,
such as transdifferentiation of macrophages in response
to inflammatory cytokines (De Santa et al. 2007) or stem
cell differentiation (Loh et al. 2007). In addition, the fact
that both H3K4 and H3K27 KMTs (MLL2, MLL3, and
EZH2) and demethylases (RBP2, UTX, and JMJD3) asso-
ciate with “bivalent” homeotic target genes, suggest
that histone demethylation is required for “fine-tuning”
transcription by maintaining specific epigenetic states
(Fig. 3; Agger et al. 2007; Pasini et al. 2008). Moreover,
mathematical modeling suggest that the concerted ac-
tion of opposing histone-modifying activities will permit
a highly dynamic control of the chromatin state without
compromising stability (Dodd et al. 2007).
Since the degree of methylation of the histone tail ap-
pears to be an important factor for the regulation of a
number of cellular processes such as X inactivation, dif-
ferentiation, cellular senescence, and DNA damage re-
pair, and histone demethylases take an active part in
this, it is anticipated that the expression levels of these
enzymes are carefully controlled and their localization
exquisitely targeted. Indeed, many demethylases and/or
their binding partners feature histone mark-binding
modules as Tudor, PHD, or Chromo domains, opening
the possibility for specific recruitment of demethylases
to certain chromatin areas and for spreading of demeth-
ylation to neighboring nucleosomal areas. As an example
of this, the PHD domain of JMJD2A has been demon-
strated to bind specifically to H3K4me3 H3K9me3 and
H4K20me3 (Huang et al. 2006; Kim et al. 2006; Lee et al.
2008). Although the biological significance of this obser-
vation is still unclear, this could be one example of de-
methylase targeting to specific chromatin areas. Another
Cloos et al.
1128 GENES & DEVELOPMENT
report has suggested that the interplay between the his-
tone demethylase LSD1 and its complex partner BHC80
may be involved in spreading of histone demethylation.
Remarkably, the PHD finger of BHC80 was shown to
bind unmethylated H3K4, a binding that is specifically
abrogated by methylation of H3K4 (Lan et al. 2007b).
Gene location analysis demonstrated that LSD1 and
BHC80 binding were interdependent, leading to the sug-
gestion that the binding of BHC80 to demethylated K4
may constitute a targeting system leading to the propa-
gation of K4 demethylation and required for the binding
of LSD1 (Lan et al. 2007b).
Thus, demethylases appear to represent a fundamental
piece of equipment in the cells chromatin toolbox, per-
mitting a fast and robust regulation of transcriptional
programs. Evidently, aberrant localization or deregula-
tion of these enzymes may contribute to diseases such as
cancer, for instance, by promoting a pluripotent-like
state or by disruption of senescence induction as dis-
Interaction of histone demethylases with other
histone-modifying enzymes and nuclear receptors
Several reports have documented that demethylases are
often found in large histone-modifying complexes asso-
ciated with other chromatin modifiers such as KMTs
and HDACs. Thus, as mentioned above, the K27 de-
methylase UTX has been found in complex with K4
KMTs MLL2 and MML3, and JMJD3 coimmunoprecipi-
tates with core subunits of K4–methyltransferase com-
Similarly, the K4 histone demethylase JARID1A is as-
sociated with the PRC2 complex, instilling the repres-
sive K27 mark (Fig. 3; Pasini et al. 2008). The fact that
depletion of JARID1A by RNAi decreases the ability of
PRC2 target genes to remain repressed demonstrates the
importance of the concerted action of these activities
(Pasini et al. 2008). The apparent intimate association
between several demethylases and KMTs affecting
marks of complementary activity suggest that this is a
general phenomenon, ensuring a fast and efficient shift
in expression of the affected gene.
It remains to be established whether these examples
constitute a general mechanism for gene regulation. The
recent observation that the K4me3/me2 demethylase
JARID1C is in a complex with the H3K9 methyltrans-
ferase G9a indicates that this may indeed be a general
phenomenon (Tahiliani et al. 2007).
Several reports have highlighted the interaction of de-
methylases and HDACs, and have suggested a possible
interplay or coregulation of such activities. As an ex-
ample of this, one study reported that HR interacts with
HDACs and is localized in matrix-associated deacetylase
bodies, consistent with a role of HR as a transcriptional
corepressor (Potter et al. 2001). Similarly, the JARID1 S.
pombe ortholog Msc1 has been shown to interact with
HDACs (Ahmed et al. 2004). Cells lacking Msc1 have a
20-fold increase in global acetylation of histone H3, in-
dicating that it may be required for the recruitment or
activity of certain HDACs. Moreover, JARID1A has been
shown to be part of a complex that contains HDAC ac-
tivity (Klose et al. 2007). The reintroduction of a cata-
lytically dead JARID1A mutant into Jarid1a-null mice
caused repression of the Sdf1 gene (Klose et al. 2007),
demonstrating that JARID1A-mediated repression can-
not entirely be explained by K4-demethylation activity.
Histone demethylases have also been implicated as es-
sential players in nuclear receptor signaling. Nuclear re-
ceptors are transcription factors whose activity is depen-
dent of the binding of lipophilic ligands, triggering their
translocation to the nucleus. Most nuclear receptors
function by activating transcription of specific target
genes. Nuclear receptor-mediated transcriptional activa-
tion occurs through binding of the ligand to the C-ter-
minal domain, triggering conformational changes of the
receptor into an activator and favoring association with
specific coactivator complexes (for review, see Rosenfeld
et al. 2006). In addition to this, a subgroup of these re-
ceptors including the receptors for retinoic acid, vitamin
D, and thyroid hormone have the additional capacity of
functioning as transcriptional repressors when not
bound to their ligands, primarily by recruiting repressor
complexes through the so-called CORNR domain. More-
over, many nuclear receptors including the ER? and the
AR can be converted to transcriptional activators even
methyltransferases in transcriptional regulation of de-
velopmental genes. Histone methyltransferases and de-
methylases are found in the same complex, which
methylates one mark while removing the opposing
mark. The methylation pattern at a specific gene is de-
termined by the equilibrium between activities of the
two opposing complexes, exemplified here by the acti-
vating MLL2/UTX complex and the repressive PRC2/
RBP2 complex (Agger et al. 2007; Pasini et al. 2008).
Analogously, it has been shown that repressive com-
plexes carrying K9 methyltransferase activity (G9a)
may also contain H3K4 demethylase activities (Tahil-
iani et al. 2007). Correspondingly, it may be envisioned
that H3K9 demethylases may form a part of activating
complexes carrying methyltransferases to activating
marks as H3K4.
Model for the involvement of demethylases/
Erasing the methyl mark
GENES & DEVELOPMENT 1129
in the absence of a ligand, through other signaling path-
The transcriptional control exerted by nuclear recep-
tors is critical for the development and homeostasis of
all mammals and is mediated through their interaction
with various corepressor complexes. Not surprisingly,
given their important biological roles, deregulation of
nuclear receptor function has been implicated in a vari-
ety of diseases. Indeed, inhibition or activation of
nuclear receptors forms the central mechanisms of ac-
tion for many current drugs, and more than a tenth of the
world’s 100 best selling drugs are targeting nuclear re-
As mentioned previously, several reports have pro-
vided evidence linking demethylase function to the regu-
lation of transcription by nuclear receptors (Table 1;
Chan and Hong 2001; Metzger et al. 2005; Yamane et al.
2006; Garcia-Bassets et al. 2007; Wissmann et al. 2007;
Wolf et al. 2007). Thus, LSD1, JMJD1A, JMJD1C, and
JMJD2C have been involved in AR-mediated transcrip-
tion (Fig. 4; Metzger et al. 2005; Yamane et al. 2006;
Wissmann et al. 2007; Wolf et al. 2007). Accordingly,
Garcia-Bassets et al. (2007) found that LSD1 occupied a
significant fraction of estrogen-responsive genes, and
that the occupancy of this promoter by LSD1 was in-
versely correlated to H3K9 dimethylation. Conversely,
Garcia-Bassets et al. (2007) found that RNAi-mediated
depletion of several H3K9 KMTs (RIZ1, ESET, and
EuHMTase1) led to the activation of these promoters.
This activation was independent of the presence of li-
gand (estrogen), but relied on the binding of the unligated
receptor to the promoter. These data led Garcia-Bassets
et al. (2007) to suggest a model where repressive meth-
ylations imposed by KMTs act as gatekeepers inhibiting
inappropriate transcriptional activation of genes by un-
ligated nuclear receptors. In contrast, the ligand-depen-
dent recruitment of LSD1 (and possibly other demethyl-
ases) could act as an activator of ER-mediated transcrip-
tion by reversing the repressive H3K9 methylation
(Garcia-Bassets et al. 2007). This study, taken together
with a number of other studies (Table 1), suggests a gen-
eral regulatory mechanism for transcriptional regulation
by nuclear receptors where a carefully coordinated inter-
play between histone methyltransferases and histone de-
methylases balance repressing and activating histone
modifications, leading to either transcriptional activa-
tion or repression.
Roles of demethylases in development
During cellular differentiation, the transcriptional pro-
gram of cells is rapidly modulated to acquire new lin-
eage-specific phenotypic states. Concurrently or prior to
this, modulation of genome-wide chromatin changes oc-
cur with establishment of new histone marks and era-
sure of others. Chromatin areas featuring both H3K4 and
H3K27 methylations are often seen in ES cells or early
precursor cells, located at genomic regions encoding
transcription factors such as HOX genes that are essen-
tial for development and differentiation. Due to the pres-
ence of both trimethylated H3K4 and H3K27, which are
normally associated with transcriptionally active and si-
lent chromatin, respectively, these areas have been
coined bivalent domains, and been proposed to be poised
for transcription (Bernstein et al. 2006) or poised for per-
manent silencing (Christensen et al. 2007). These biva-
lent marks may provide plasticity to the gene; i.e., the
gene can be either activated (K4-methylated/K27-un-
methylated) or maintained repressed (K27-methylated/
K4-unmethylated) in specific cell lineages during differ-
entiation. Indeed, upon differentiation of ES cells these
bivalent domains are induced in a timed and spatially
controlled manner or remain silent depending on the
phenotypic characteristics of the lineage in question.
ChIP studies of cells before, during, and after differentia-
tion has documented that the status of various histone
methyl marks is rapidly modulated on specific genes
during these processes.
As described above, members of the JARID1 and UTX/
JMJD3 groups appear to have specific roles in this regu-
lation. In fact, the JARID1 and UTX/JMJD3 proteins are
recruited to HOX promoters or displaced from them in
response to retinoic acid differentiation, and they are es-
sential for the correct regulation of the HOX genes
(Klose et al. 2006a; Agger et al. 2007; Christensen et al.
2007; De Santa et al. 2007; Lan et al. 2007a; Lee et al.
2007b), revealing a causal relationship between H3K4
and H3K27 demethylation and transcriptional control
during these processes.
Moreover, as also described previously, JMJD1A and
JMJD2C are involved in regulating ES cell differentiation
(Loh et al. 2007). Oct4 positively regulates both genes
and inhibition of their expression induces ES cell differ-
transcription. When bound to its ligands, androgen (A), the AR
translocates to the nucleus to interact with histone demethyl-
ases on androgen-responsive elements (ARE) on specific genes.
Through its interaction with JMJD2C, LSD1 or JMJD1A de-
methylation is triggered, removing the repressive H3K9 meth-
ylation and leading to the transcriptional induction of these
androgen-responsive genes. Repressive complexes (RCO), possi-
bly featuring H3K9-methyltransferase (KMT), HDAC, and
H3K4 demethylase (JARID1) activities, may potentially act to
prevent ligand-independent activation.
The involvement of demethylases in AR-mediated
Cloos et al.
1130 GENES & DEVELOPMENT
entiation and a concomitant increase of lineage-specific
genes and decrease of ES cell-specific genes (Loh et al.
2007). Moreover, JMJD1A was shown to be a positive
regulator of the pluripotency-associated genes Tcl1,
Tcfcp2l1, and Zpf57, whereas JMJD2C acts as a positive
regulator of Nanog, encoding a key transcription factor
for self-renewal, presumably by removing repressive
H3K9 methylations at the promoters of these genes. In
addition, several other members of the JmjC family dis-
play developmental phenotypes, suggesting putative
roles in cellular differentiation. Examples include PHF8,
JARID2, and JMJD6.
In summary, several examples exist suggesting that
demethylases are at the nexus of cellular differentiation
and development. Importantly, since histone demethyl-
ases appear to take center stage in executing nuclear re-
ceptor function, this protein group may constitute excel-
lent drug targets for novel therapies to cancer and other
Putative roles in senescence
Cellular senescence constitutes a barrier against exces-
sive cell proliferation, and is a specific form for irrevers-
ible growth arrest that can be induced by a number of
different stress inducers, including activation of onco-
genes and DNA damage, as well as telomere erosion.
Several studies have revealed the importance of the se-
nescence process in suppression of tumorigenesis in vivo
(Braig et al. 2005; Collado et al. 2005; Lazzerini Denchi et
al. 2005; Michaloglou et al. 2005). The two tumor sup-
pressor proteins pRB and p53 play a central role in the
induction of senescence. Stabilization of p53 leads to
transcriptional induction of the CDK inhibitor p21,
which negatively affects the cell cycle. pRB is found in
its active hypophosphorylated form in complex with E2F
family members. These complexes induce heterochro-
matin on E2F-responsive promoters during senescence,
resulting in formation of SAHFs (Narita et al. 2003). The
exact mechanism leading to SAHF formation is not clear
at present. However, recent studies suggest a key role for
trimethylation of H3K9 in the establishment of SAHFs
and induction of the senescent state. For instance, the
accelerated formation of lymphomas in transgenic mice
expressing the activated version of the oncogene N-RAS
(RASG12D) as a result of Suv39h1 loss correlates with a
decreased number of SAHFs and ability to enter senes-
cence in primary lymphocytes (Braig et al. 2005). As sug-
gested above, demethylases removing the repressive
H3K9me3 marks may, in theory, act to increase genomic
instability, dissolve SAHFs, and prevent or override se-
nescence induction, and may thus potentially provide
cancer cells with the possibility to evade this important
tumor suppressor mechanism (Cloos et al. 2006).
Another demethylase family, which potentially could
be involved in the senescence process, is the UTX/
JMJD3 family of H3K27 demethylases. Even though in-
duction of heterochromatin and increased levels of re-
pressive histone marks have been clearly linked to se-
nescence, the gain of repressive K27 methylation at
specific genetic loci and concomitant gene silencing has
likewise been associated with immortalization and can-
cer. Thus, the polycomb-repressive complexes PRC2 and
PRC1, which catalyze methylation of H3K27 and are in-
volved in chromatin compaction, respectively, are over-
expressed or amplified in cancer (Valk-Lingbeek et al.
2004). Moreover, a recent study has provided a direct
mechanistic link between PcG activity, H3K27 methyl-
ation, and the transcription of the INK4A–ARF locus in
senescence (Bracken et al. 2007). This study showed that
the PRC2 members, including the H3K27-specific meth-
yltransferase EZH2, and subsequently PRC1 members,
are lost from the INK4A–ARF locus as cells undergo se-
nescence, concomitant with the loss of H3K27 and in-
creased transcription of INK4A and ARF. The INK4A–
ARF locus is a sensor of signals inducing senescence and
the deletion and/or inactivation of the locus, which oc-
curs in a large number of cancers, prevents induction of
cellular senescence. The loss of H3K27me3 at the
INK4A–ARF locus as cells undergo senescence could,
however, be a result of the concerted loss of H3K27 tri-
H3K27me3 demethylases (Fig. 5). In this context, it is
noteworthy that UTX is generally down-regulated in tu-
mors. Thus, a large number of studies providing normal-
ized expression levels for UTX in tumors are available on
the Oncomine online database, and the large majority of
these show a significant decrease in UTX expression in
diverse malignancies such as melanoma, prostate, brain,
and breast carcinoma. Only a limited number of studies
provide JMJD3 data, in the online databases, but the gene
is significantly decreased in hepatocellular carcinoma.
Of particular interest, the JMJD3 gene is located on
chromosome 17 in close proximity to the p53 tumor sup-
pressor gene. Allele loss of the short arm of chromosome
17, where both p53 and JMJD3 map, is among the most
frequently observed genetic lesions in cancer and is
found in a variety of human malignancies (Nigro et al.
1989). The allelic losses at 17p13 are significantly corre-
lated with high proliferative activity and associated with
more aggressive tumor behavior. Detailed mapping of
deletion patterns at this locus and the lack of good cor-
relation between allelic loss at this locus and mutation
at p53 has lead to the suggestion that additional, and as
of yet undiscovered tumor suppressor genes, may be
found in this area that could be responsible for at least a
part of the observed malignant phenotype.
It is pertinent to note that a large part of the genetic
lesions leading to p53 loss likewise causes the deletion of
JMJD3. Thus, given the possibility that JMJD3 may be at
the heart of p16 regulation, it could be envisioned that
allelic loss at 17p13.1 may cause inactivation of both the
p53 and p16 tumor suppressor pathways (Fig. 5A) ex-
plaining the aggressive tumor behavior in these cancers.
In addition to the putative involvement of K27 de-
methylases in the induction of senescence through acti-
vation of the INK4A–ARF locus, these proteins may
have further tumor suppressor activities by maintaining
cells in a nonpluripotent state. Thus, PcG proteins in-
volved in setting the repressive methylation at K27 have
Erasing the methyl mark
GENES & DEVELOPMENT1131
been demonstrated to be key players in regulating “stem-
ness” preserving cellular pluripotency by establishing
and maintaining repression of specific genes implicated
in differentiation (Boyer et al. 2006; Bracken et al. 2006;
T.I. Lee et al. 2006). Correspondingly, it could be envi-
sioned that loss of activity, or aberrant localization of
K27 demethylases could potentially drive cells toward a
more pluripotent and tumorigenic state.
Finally, other demethylases may have roles in senes-
cence, either counteracting it or being involved in its
induction. Remarkably, several JmjC genes, including
JMJD3, JMJD6, and KIAA1718, are transcriptionally in-
duced in response to ectopic expression of the oncogenic
RAS in primary human epithelial cell lines (http://www.
oncomine.org). RAS is known to trigger oncogene-in-
duced senescence in primary mammalian cells, indicat-
ing that the three JmjC proteins may have roles in se-
Putative roles in genomic stability and cancer
JmjC proteins are deleted, translocated, mutated, and ab-
errantly expressed in human cancers. The JMJD3 and
JMJD1B genes are located at 17p13.1 and 5q31, respec-
tively. These are genomic areas that are frequently lost
in various malignancies (17p13.1), and in myeloid leuke-
mias (5q31). As described previously, HSBAP1 has been
identified in a family with renal cell cancer having a
t(2;3)(q35;q21) translocation, resulting in a DIRC3-HSP-
BAP1 fusion transcript (Bodmer et al. 2003). van Zutven
et al. (2006) described a patient with NUP98 transloca-
tions with AML-M7 with a cryptic t(11;21;12)(p15;p13;p13)
complex variant resulting in a NUP98-JARID1A fusion.
The resulting NUP98-JARID1A fusion protein, includ-
ing the first 13 exons of NUP98 and exons 28–31 of
JARID1A contains the Phe–Gly (FG) repeats of the N-
terminal part of NUP98. Moreover, the fusion protein
features the sequence encoding the C-terminal PHD do-
main of JARID1A, which may be involved in securing
the correct localization of the wild-type JARID1A pro-
tein. It may be envisioned that the mutant protein could
contribute to the leukemic phenotype of AML-M7 pa-
tients, perhaps by working as a dominant-negative mu-
tant. In this context, it is noteworthy that ectopic ex-
pression of a catalytic inactive mutant of JARID1A re-
sults in an increase in global H3K4me3 levels in human
cell lines (Christensen et al. 2007).
Another recent study links mutations of JmjC demeth-
ylases to hematopoetic cancer. Hence, Suzuki et al.
(2006) identified two JmjC proteins, FBXL10 and JMJD5,
modifications in senescence. (A) JMJD3
and p53 are transcribed from the same ge-
nomic region: chromosome 17p13.1. In
response to oncogenic, replicative, or
other types of stress (DNA damage, drug
treatment, etc.) the tumor suppressors p53
and INK4A–ARF are induced, triggering
senescence. JMJD3 or UTX may be in-
volved in the transcriptional activation of
the INK4A–ARF tumor suppressor locus
by removing repressive K27 methylation
from the gene (see B). In turn, p16 and
p14ARF lead to the induction of pRB and
p53, respectively, triggering senescence
and/or apoptosis. (B) The compacted, tran-
scriptionally silent chromatin structure of
the INK4A–ARF locus serves to ensure
cell cycle progression in normal “un-
stressed” cells and in some malignant
cells. (Left panel) This silent state is main-
tained by H3K27 methylation mediated by
the PcG proteins, and possibly also by the
repressive activities of JARID1 proteins
and other chromatin modifiers. When
cells with intact tumor suppressor path-
ways are subjected to oncogenic stimuli or
other types of stress, PcG proteins are dis-
placed from the INK4A–ARF locus and
K27 demethylases are possibly recruited to
remove the repressive K27 methylation,
leading to the expression of the INK4A–
ARF locus and the induction of senescence. (C, left panel) In normal “unstressed” cells the transcription factor E2F mediates
transcription, while the tumor suppressor pRB is maintained in an inactive phosphorylated state as a result of low p16 levels. During
senescence induction, p16 is induced, causing hypophoshorylation of pRB. (Right panel) pRB subsequently recruits chromatin modi-
fiers as HDAC, SUV39H1 (setting H3K9 methylation), SUV4H20 (setting H4K20 methylation), HP1, and possibly JARID1 proteins to
silence euchromatic E2F target promoters and form SAHFs.
Cloos et al.
1132 GENES & DEVELOPMENT
as candidate tumor suppressors in a mouse model of leu-
kemia. In agreement with this notion an earlier study
identified two JmjC genes T26A5.5 and C06H2.3 or-
thologs of the human FBXL10 and JMJD5 genes, respec-
tively, in a RNAi screen designed to identify genes that
contribute to genomic stability in C. elegans somatic
cells (Pothof et al. 2003). While the exact mechanisms
causing oncogenesis in response to loss-of-function mu-
tations in FBXL10 and JMJD5 remain unclear, the obser-
vation that depletion of JMJD5 decreased the survival of
cells exposed to N-methyl-N0-nitro-N-nitrosoguanidine
(MNNG), suggests that it might fulfill functions in DNA
mismatch repair. Finally, the JMJD2 genes JMJD2A,
JMJD2B, and JMJD2C are all highly expressed in prostate
cancer (Cloos et al. 2006), and the fact that JMJD2C is
required for the proliferation of cancer cells (Cloos et al.
2006; Wissmann et al. 2007) point to a role of JMJD2
proteins to the development of cancer. In summary, the
observation that genetic alterations of the histone de-
methylases are often found in cancer, and that several of
these have been found to be regulation of cancer cell
lines, suggest that activation or inactivation of these pro-
teins contribute to tumor development (Table 1).
Putative roles of demethylases in X inactivation
One of the two X chromosomes becomes inactivated in
the developing female embryo. In this way gene products
expressed from the X chromosome can be dosage-com-
pensated (Heard 2005). Central to the mechanism of X
inactivation is the noncoding RNA Xist, which is ex-
pressed from a locus within a region of the X chromo-
some called the X-inactivation center (Heard 2005). Xist
is induced 1 or 2 d following ES cell differentiation, and
the Xist transcript subsequently covers the future inac-
tive X chromosome. Xist induction and coating repre-
sent an essential and initial event in the X-inactivation
process. One of the earliest chromosomal changes that
occur during X-chromosome inactivation is the loss of
euchromatic histone modifications. Immediately follow-
ing Xist RNA coating, a reduction in H3K4me3/me2 and
H3K9ac is observed at the X-inactivation center as de-
tected by ChIP and immunofluoresence (Heard et al. 2001;
Okamoto et al. 2004). Subsequently, the PRC2 complex
is transiently recruited to the affected X chromosome,
which is accompanied by an increase in the repressive
H3K27me3 mark. The molecular mechanisms mediat-
ing these early changes in H3K4me3/me2 and H3K9ac
are currently unknown, but it has been speculated that
enzymes such as HDACs and histone demethylases could
be involved in the process (Fig. 6; Heard 2005).
Likewise, little is known about how the PRC2 com-
plex is recruited to the X chromosome. As illustrated in
Figure 6, several features point to an involvement of
H3K4me3/me2 demethylases in establishing these epi-
genetic events. The JARID1 proteins are strong candi-
dates as the enzymes mediating the initial K4 demeth-
ylation wave. Moreover, as mentioned previously, vari-
ous JARID1 members have been reported to interact
with HDACs. This could be the mechanism by which
HDACs are recruited, leading to the secondary deacety-
lation events, which remove transcriptionally permis-
sive acetyl groups from K9 and K27 positions on histone
H3. Finally, the JARID1 members might also be instru-
mental for the subsequent recruitment of PRC2 mem-
JARID1A has been shown to bind the PRC2 complex and
to be required for its full repressive activity on gene tran-
scription (Pasini et al. 2008). An intriguing possibility is
that JARID1 family members could be initially recruited
to Xist covering the X chromosome destined for inacti-
vation. Hence, all JARID1 members feature a so-called
AT-rich interacting domain (ARID) believed to be in-
volved in the binding of DNA or RNA.
K27me3 mark. Thus,
Perspectives and future directions
Considering the involvement of histone demethylases in
essential cellular processes and their putative implica-
tion in various human diseases (Table 1), these enzymes
may constitute attractive drug targets. Recently, several
studies have reported the inhibitory effect of various
small molecules on LSD1 (Culhane et al. 2006; M.G. Lee
et al. 2006; Yang et al. 2006; Y. Huang et al. 2007). Sig-
nificantly, some of these molecules were shown to reac-
tivation. Xist RNA associates with the X chromosome through
unknown factor(s), and recruits an initial silencing activity. De-
methylation of H3K4 is an early event in X inactivation, and
most likely is mediated by JARID1 family members. HDAC
activity is recruited, possibly through JARID1 members to
deacetylate H3K9 and H3K27, probably in preparation for the
subsequent repressive methylation of these marks. The PRC2
complex transiently associates with the inactivating X to meth-
ylate H3K27 and perhaps H3K9. The recruitment of PRC2 could
be mediated through JARID1 members. Subsequently, PRC1
proteins transiently associate with the inactivating X chromo-
some, mediating histone H2A ubiquitinylation. Finally, local-
ized Xist is indispensable for the accumulation of the histone
variants macroH2A1 and macroH2A2 on the inactive X chro-
mosome to form the so-called macrochromatin body.
Demethylases and histone modifications in X inac-
Erasing the methyl mark
GENES & DEVELOPMENT1133
tivate transcription of otherwise silenced tumor suppres-
sor genes, raising hopes for the success of such com-
pounds for cancer therapies (Y. Huang et al. 2007).
Correspondingly, inhibitors targeting specific members
of the JmjC group of histone demethylases may be envi-
sioned with potential for treatment.
While the recent years have provided and revealed
many aspects of histone demethylase biology, several in-
teresting questions remain to be answered: One question
is whether LSD1 is unique, or whether additional FAD-
type demethylases exist. The human genome features a
dozen proteins related to LSD1, which potentially could
be involved in demethylation of histones; however, so
far, no activities have been reported.
Another open question is whether additional classes of
histone demethylases exist. The cupin protein superfam-
ily comprises several other enzymes that could poten-
tially function as histone demethylases, including the
recently characterized fat mass and obesity-associated
(FTO) gene (Gerken et al. 2007) and the previously iden-
tified AlkbH enzymes (Falnes et al. 2002; Trewick et al.
2002) that catalyze the removal of methyl groups from
alkylated nucleotides. In addition, other types of en-
zymes may exist with the ability to demethylate his-
tones. Candidate enzymes include NADH/NADPH-de-
pendent oxidoreductases, which could demethylate his-
tones by cycling the NAD+/NADH cofactor in a
mechanism similar to that used by LSD1; radical S-ad-
enosyl-methionine (SAM) proteins; or enzymes with two
iron centers as ribonucleotide reductases or methane
mono-oxygenase-like proteins, which may hydroxylate a
carbon backbone by using various reactive radicals and
which therefore, in principle, could use a trimethylated
lysine as substrate (Chinenov 2002; Shi and Whetstine
Yet another central question is whether 5-methylcy-
tosine demethylases exist. Given the large importance of
CpG methylation in epigenetic signaling and cancer bi-
ology, DNA demethylases have long been sought. Vari-
ous observations indicate the existence of such enzymat-
ic activities. Thus, methylation of CpG islands in im-
printed genes is erased during early embryogenesis
(embryonic day 10.5–12.5) in primordial germ cells to
permit the subsequent setting of gender-specific methyl-
ation, suggesting that such enzymes exist. Theoretically,
such demethylation could occur “indirectly“ through
the exchange of methylated nucleotides by DNA repair
mechanisms (for review, see Reik 2007). Considering the
speed with which 5-methylcytosine is eliminated from
DNA and the problems in a “genome-wide DNA repair
mechanism,” direct demethylation seems more plau-
sible. Direct demethylation of 5-methylcytosine would
involve breaking a carbon–carbon bond that is energeti-
cally less favorable than the scission of a carbon–nitro-
gen bond as used in histone demethylation. However, it
is still a possibility that iron and ?KG-dependent en-
zymes as the JmjC proteins, or as those described above,
could catalyze such reactions.
A final question we believe is important to address is
if the histone demethylases target other nonhistone sub-
strates. A recent report has demonstrated the involve-
ment of LSD1 in demethylation of the transcription fac-
tor and tumor suppressor p53 (J. Huang et al. 2007).
Similarly, JmjC proteins may be involved in demethyl-
ation or other hydroxylation-derived modifications of
nonhistone proteins and/or nucleotides contributing to
an increased regulatory complexity of these systems.
In conclusion, there is little doubt that the near future
will provide detailed insights into the function and regu-
lation of these enzymes contributing to unravel patho-
genesis of various diseases and eventually pave the way
for the discovery of novel therapeutic targets and treat-
The work in the Helin laboratory is supported by grants from
the Danish Cancer Society, the Danish National Research
Foundation, the Danish Medical Research Council, the Danish
Natural Science Research Council, the Novo Nordisk Founda-
tion, the Villum Kann Rasmussen Foundation, the European
Union Framework 6 program (INTACT and DIAMONDS), and
the Association for International Cancer Research. P.A.C.C. is
supported by a grant from the Benzon Foundation.
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