DEVELOPMENTAL DYNAMICS 235:2449 2459, 2006

SPECIAL ISSUE REVIEWSA PEER REVIEWED FORUM

Roles of jumonji and jumonji Family Genes in

Chromatin Regulation and Development
Takashi Takeuchi,1,2* Yutaka Watanabe,3 Toshiyuki Takano-Shimizu,3,4 and Shunzo Kondo1

The jumonji (jmj) gene was identied by a mouse gene trap approach and has essential roles in the
development of multiple tissues. The Jmj protein has a DNA binding domain, ARID, and two conserved jmj
domains (jmjN and jmjC). In many diverse species including bacteria, fungi, plants, and animals, there are
many jumonji family proteins that have only the jmjC domain or both jmj domains. Recently, Jmj protein
was found to be a transcriptional repressor. Several proteins in the jumonji family are involved in
transcriptional repression and/or chromatin regulation. Most recently, one of the human members has been
shown to be a histone demethylase, and the jmjC domain is essential for the demethylase activity.
Meanwhile, more and more evidence indicating that the jumonji family proteins play important roles
during development is accumulating. Many proteins in the jumonji family may regulate chromatin and gene
expression, and control development through various signaling pathways. Here, we highlight the roles of
jmj and jumonji family proteins in chromatin regulation and development. Developmental Dynamics 235:
2449 2459, 2006. 2006 Wiley-Liss, Inc.
Key words: jumonji; jumonji family; jmjC; chromatin remodeling; histone demethylase; cell proliferation; cell
differentiation
Accepted 19 April 2006

INTRODUCTION
In the past, we have attempted to
identify novel genes that have important roles in development using a
mouse gene trap method and have
successfully identied jumonji (jmj;
Takeuchi et al., 1995; Takeuchi,
1997). The gene was named for the
morphology produced by the normal
neural groove and abnormal grooves
on the neural plates of jmj mutant
mice. The morphology resembles a
cross, and jumonji means cruciform
in Japanese (Takeuchi et al., 1995).
We rst reported that Jmj protein
has two conserved domains (Takeuchi

et al., 1995). Later, one was further

divided into two domains, a jmjN domain and an AT-rich interaction domain (ARID), because there are many
proteins that have only one of these
two domains (Balciunas and Ronne,
2000; Kortschak et al., 2000). ARID is
a DNA binding domain, and proteins
that have ARID belong to the ARID
family. Another conserved domain in
Jmj is designated as jmjC (Balciunas
and Ronne, 2000).
More than 100 proteins from many
species, including bacteria, fungi,
plants, and animals have been shown to
have the jmjC domain or both the jmjN

and jmjC domains (Balciunas and

Ronne, 2000; Clissold and Ponting,
2001; http://www.ebi.ac.uk/interpro/
DisplayIproEntry?acIPR003347).
In the present study, we dene a jumonji family as a family whose members have the jmjC domain.
We have demonstrated previously
that Jmj is a transcriptional repressor
and represses cyclin D1 transcription
in the embryonic heart and that this
repression is required for normal cardiogenesis (Toyoda et al., 2003). Several reports also suggested that jumonji family members are involved in
transcriptional or chromatin regula-

Mitsubishi Kagaku Institute of Life Sciences (MITILS), Machida, Tokyo, Japan

Graduate School of Environment and Information Sciences, Yokohama National University, Hodogaya, Yokohama, Kanagawa, Japan
3
Department of Population Genetics, National Institute of Genetics, Mishima, Shizuoka, Japan
4
Department of Biosystems Science, Graduate University for Advanced Studies (SOKENDAI), Hayama, Kanagawa, Japan

Dr. Kondos present address is JEOL Ltd. 3-1-2 Musashino, Akishima, Tokyo 196-8558 Japan.
*Correspondence to: Takashi Takeuchi, Mitsubishi Kagaku Institute of Life Sciences (MITILS), 11 Minamiooya, Machida,
Tokyo, 194-8511 Japan. E-mail: take@libra.ls.m-kagaku.co.jp
2

DOI 10.1002/dvdy.20851
Published online 16 May 2006 in Wiley InterScience (www.interscience.wiley.com).

2006 Wiley-Liss, Inc.

2450 TAKEUCHI ET AL.

tion. In fact, most recently, Tsukada

et al. showed that a human family
protein (JHDM1A, previous name;
FBXL11) is a histone demethylase
and that the jmjC is required for the
enzymatic activity (Tsukada et al.,
2006). This nding revealed one function of the jmjC domain, and provided
the rst evidence that jumonji family
members can act as enzymes for histone modication.
Our studies have shown that jmj
has essential roles in the development
of multiple tissues. In addition, many
reports suggest that proteins in the
jumonji family play important roles
during development. Many proteins in
the jumonji family may regulate chromatin and gene expression and control development through various signaling pathways. In this paper, we
review the roles of Jmj and other jumonji family proteins in chromatin
regulation and development.

STRUCTURE AND
EVOLUTION OF JUMONJI
FAMILY PROTEINS
The jumonji family proteins comprise a large family. We constructed
a dendrogram of human, Drosophila,
and yeast jumonji family proteins
(Fig. 1) based on their jmjC domain
sequences. Almost all proteins containing jmjC domain in Pfam (http://
pfam.wustl.edu/cgi-bin/getdesc?name
JmjC), SMART (http://smart.emblheidelberg.de/smart/do_annotation.
pl?DOMAIN
JmjC&BLAST
DUMMY), and InterPro (http://www.
ebi.ac.uk/interpro/DisplayIproEntry?
acIPR003347) data bases were
analyzed after redundant, variant,
hypothetical, or low score (e1.0E04, SMART program) proteins were
removed. Recent structure analysis
of jmjC of HIF1AN/FIH, which has a
2-oxoglutarate (2-OG)-Fe(II) dependent dioxygenase activity, revealed
residues binding to the cofactors
2-OG and Fe(II), and showed that
the domain contains a doublestranded -helix motif (Elkins et al.,
2003). A sequence alignment of the
jmjC domains of various jumonji
family proteins suggested that these
features are conserved (Elkins et al.,
2003; Trewick et al., 2005). Recently,
one jumonji family protein, FBXL11/
JHDM1A was shown to be a 2-OG-

Fe(II) dependent histone demethylase (Tsukada et al., 2006). The

alignment of sequences for the dendrogram was made by taking previously predicted conservative residues such as cofactor binding sites
(Clissold et al, 2001; Trewick et al,
2005; SMART and Pfam databases;
Fig. 2).
The family contains distantly related members, as shown in Figure 1.
Note, where we could not place a root.
We can see several distinct clusters in
the dendrogram based on the high
bootstrap values ( 99%). Human
JMJD2A-D and JARID1 and 2 constitute a single major cluster, which is
referred to as cluster 1 hereafter. We
also pay attention to three clusters: a
cluster containing UTX, UTY, and
JMJD3 (cluster 2); one containing
JMJD1A-C and Hairless (cluster 3);
and one containing PHF2 and -8, and
FBXL10, -11 (JHDM1A, B; cluster 4).
Figure 1 also shows the domains
that each member contains. Of interest, most proteins in cluster 1, 2, 3,
and 4 have jmjN, tetratrico peptide
repeat (TPR), C2HC4 zinc-nger and
plant homeodomain (PHD) nger domain domains, respectively (Fig. 1).
Three proteins in the cluster 4 also
have F-box, leucine-rich repeat
(LRR1) and CXXC zinc-nger domains. Function of the jmjN domain
remains unknown. The TPR domain
mediates proteinprotein interactions
and the assembly of multiprotein complexes (DAndrea and Regan, 2003).
PHD nger is a C4HC3 zinc-nger
like motif found in nuclear proteins
thought to be involved in chromatinmediated transcriptional regulation
(Bienz, 2006). Remaining proteins
such as PTDSR or JMJD4 do not have
any apparent distinct domains other
than jmjC (Fig. 1).
Proteins in cluster 1 are further divided into two groups, depending on
the presence of the DNA binding domain ARID (Fig. 1). Only cluster 1
members contain ARID as well as
jmjN domains in the jumonji family
(Fig. 1). ARID proteins are involved in
transcriptional regulation and a variety of biological processes, including
development (Kortschak et al., 2000;
Wilsker et al., 2005). Recently, the human and mouse proteins that have
jmjN, jmjC, and ARID sequences were
named JARID proteins, which are

composed of ve proteins: JARID1A/

RBP2, JARID1B/PLU-1, JARID1C/
SMCX, JARID1D/SMCY, and JARID2/JUMONJI (Wilsker et al.,
2005). In this review, the original
names are used to avoid confusion.
Human JARID1 proteins (RBP2,
PLU-1, SMCX, and SMCY) are closely
related to each other and also have a
PHD nger domain and a C5HC2 type
Zn-nger domain. Q9VMJ7, little
imaginal discs (lid), may be a Drosophila common homologue of JARID1 proteins. On the other hand, JUMONJI protein has a C5HC2 type Znnger domain but not PHD nger. The
amino acid sequence homologies of
jmjN, jmjC, and ARID are not high
between the jumonji and JARID1 proteins. In addition, the positions of
jmjN and ARID in JARID1 proteins
are very close to the N-terminal; however, those of JUMONJI are at the
center in the protein sequence. This
feature is shared by the Q9VT00 Drosophila protein. The relatively high
homologies of jmjN, jmjC, and ARID of
Q9VT00 with those of human and
mouse jumonji protein suggest that
the Q9VT00 protein is a Drosophila
homologue of the JUMONJI protein.
Although all JARID1 proteins and
JUMONJI protein have ARID, the
dendrogram suggests that the jmjC
sequences of JARID1 proteins are
more similar to those of JMJD2 proteins, which do not have ARID, than
to that of JUMONJI (Fig. 1). We hypothesize that the ancient protein had
only the jmjC domain, and then some
of the descendant proteins acquired
jmjN and ARID. Although we could
not come to a denite conclusion with
the low bootstrap probabilities, the
dendrogram suggests that the acquisition occurred before diversication
of the cluster 1 genes and then the
JMJD2 group lost ARID during the
course of evolution. Balciunas and
Ronne have proposed that domain
swapping may have occurred during
evolution of the jumonji family proteins (Balciunas and Ronne, 2000). In
the case of the JMJD2 group, JMD2A,
B, and C have tudor domains instead
of ARID (Fig. 1). Of interest, the tudor
domain is found in many proteins that
colocalize with ribonucleoprotein or
single-strand DNA-associated complexes in the nucleus.
JMJD proteins scatter in the family

JUMONJI IN CHROMATIN REGULATION AND DEVELOPMENT 2451

and do not show a monophyly (Fig. 1).

Because there is no apparent relationship between classication (nomenclature of A-D) of the proteins and
positions in the dendrogram, the nomenclature should be reconsidered
based on the phylogenetic relationships or functions.
Because the family contains many
distantly related proteins, investigation of functional or structural conservation is required to ascertain
whether these proteins form one functional family. The 2-OG-Fe(II) dependent dioxygenase activity of jumonji
family proteins is especially worth analyzing. HIF1AN/FIH and FBXL11/
JHDM1A catalyze protein hydroxylation and histone demethylation,
respectively, through the dioxygenase
activity (Hewitson et al., 2002;
Tsukada et al., 2006). In addition, the
structure of jmjC involved in the enzymatic activity appears to be conserved in many members (Trewick et
al., 2005; Fig. 2).

Fig. 1. Unrooted neighbor-joining tree for 44 jumonji family proteins based on jmjC domain sequences. The tree was constructed by using Poisson correction distance for amino acid sequences and
the MEGA package. Percentage bootstrap values ( 90%) are shown on internal branches. Protein
names in humans, Drosophila, and budding and ssion yeasts are represented as black, magenta, blue
and orange, respectively. Accession numbers are presented in Drosophila and yeast proteins; those of
human proteins are given in Figure 2. Boxes beside protein names show domains that each protein
contains. Colored boxes show characteristic domains in each cluster. Asterisks mark proteins that have
been reported to be involved in gene repression or histone modication.

FUNCTIONS OF JUMONJI
FAMILY PROTEINS IN
TRANSCRIPTION AND
CHROMATIN REGULATION
Many jumonji family proteins have
domains involved in DNA binding,
chromatin binding, or transcription,
such as ARID (Kortschak et al., 2000;
Wilsker et al., 2005), PHD nger
(Bienz, 2006), or Zn-ngers, and jmjC
domains showing metalloenzyme-like
structures, suggesting that jumonji
family proteins regulate transcription
or chromatin function or both (Balciunas and Ronne, 2000; Clissold and
Ponting, 2001; Trewick et al., 2005).
In fact, the potential activities of

Fig. 2. Multiple sequence alignment of jmjC

domains. The regions of jmjC domains in
SMART database were analyzed. The alignment was performed by using the MEGA program with a few modications by eye. In this
gure, only human proteins are shown as examples. Asterisks and # mark the Fe (II) and
2-OG binding residues, respectively (predicted;
Trewick et al., 2005), of which identical residues
with HIF1AN/FIH are highlighted with a red
background. Residues that the frequency of the
most common residue is 0.5 or the frequency
of the most common group of similar residues
(Dayhoff et al., 1979) is 0.7 are highlighted
with a blue background.
Fig. 2.

2452 TAKEUCHI ET AL.

Plu-1 or JMJD2A for transcriptional

repression have been reported (Tan et
al., 2003; Zhang et al., 2005). Hairless
functions as a nuclear receptor corepressor (Potter et al., 2001). In addition, mouse Jmj protein was shown to
be a transcriptional repressor that directly represses cyclin D1 expression
(Toyoda et al., 2003). Lees group also
reported that Jmj is a transcriptional
repressor that can repress the promoter activities of atrial natriuretic
factor and alpha-cardiac myosin
heavy chain genes (Kim et al., 2003,
2004, 2005). The region that is necessary for the repression is located in
the N-terminal of Jmj (Kim et al.,
2003; Toyoda et al., 2003). Further
roles of the region are unknown; however, it is possible that the region
binds to cofactors involved in repression, such as histone modication.
Covalent modications of histone
tails, such as methylation, acetylation, and phosphorylation have important roles in regulating chromatin dynamics (Strahl and Allis, 2000).
Indeed, many residues in histone tails
are modied. For instance, several lysine and arginine residues are methylated. With regard to lysine, methylation occurs on at least ve residues of
H3 (H3-K4, -K9, -K27, -K36, and
-K79) and a single lysine residue of H4
(H4-K20). The effects of methylation
are dependent on the particular lysine
residue (Martin and Zhang, 2005). For
example, methylation of H3-K4 and
H3-K9 is linked to transcriptional activation and repression, respectively.
In addition, the biological effect of
methylation can differ, depending on
the state of methylation (mono-, di-, or
tri-methylation), even within the
same lysine residue (Martin and
Zhang, 2005).
Although the extent of histone acetylation is determined by both acetyltransferases and deacetylases, it is
still unclear whether histone methylation is also regulated by enzymes
with opposing activities. Shi et al.
showed that LSD1, a nuclear amine
oxidase, found in several histone
deacetylase complexes, specically demethylates mono- or di-methyl-H3-K4
(H3-K4me1 or H3-K4me2, respectively) in a avin adenine dinucleotide-dependent oxidase reaction (Shi et
al., 2004). However, this type of en-

zyme cannot demethylate tri-methylated residues.

Trewich et al. proposed that certain
jumonji family proteins may be other
types
of
histone
demethylases
(Trewick et al., 2005). Their concept
appears to be based on two points.
First, a jumonji family protein Epe1 in
the ssion yeast is required for heterochromatin integrity. Inactivation of
Epe1 promoted continuous spreading
of heterochromatin-associated histone
modications such as methylation of
H3-K9 (Ayoub et al., 2003), suggesting
that Epe1 can act as a histone modier. Second, jmjC domains in Epe1
and other jumonji family proteins can
be modeled onto the structure of the
jmjC domain of HIF1AN/FIH, a
2-OG-Fe (II) dependent dioxygenase,
that is a member of the jumonji family. HIF1AN/FIH mediates the hydroxylation of an asparagine residue
in hypoxia-inducible factor (HIF;
Hewitson et al., 2002) and inhibits the
transactivation by HIF. The structure of jmjC of HIF1AN/FIH (Elkins et
al., 2003) and alignment of the aminoacid sequence of Epe1 and jumonji
family proteins suggested that many
jumonji family proteins have structures and enzymatic activities similar
to those of HIF1AN/FIH. Escherichia
coli AlkB family proteins, which remove methyl groups in DNA through
oxidative demethylation (Falnes et al.,
2002; Trewick et al., 2002), are also
2-OG-Fe (II) dependent dioxygenases. The above hypothesis was proposed based on analogy of the mechanisms of demethylation by AlkB
proteins.
Most recently, Tsukada et al. also
speculated that the mechanism of a
new type of histone demethylation is
similar to that of DNA demethylation
by the AlkB family proteins, and puried a jumonji family protein,
FBXL11 as a histone demethylase
from a human cell line using a biochemical assay (Tsukada et al., 2006).
They renamed the protein JHDM1A
(jmjC domain-containing histone demethylase 1A). In the presence of
2-oxoglutarate and Fe (II), JHDM1 demethylates H3-K36me2. The jmjC domain is critical for demethylase activity. The authors also showed that the
same activity is detected in a highly
related protein of JHDM1A, FBXL10
(which they renamed to JHDM1B),

and a homologue in the budding yeast,

scJHDM1.
Studies in yeast have shown that
methylation of H3-K36 by SET2 recruits histone deacetylase to transcribed regions and links to phosphorylation of the C-terminal domain of
RNA polymerase II and the process of
transcriptional elongation (Carrozza
et al., 2005; Joshi and Struhl, 2005;
Keogh et al., 2005). Of interest, Carrozza et al. reported that the histone
deacetylation occurs at coding regions
and suppresses intragenic transcription. These results suggest that
JHDM1 modulates this regulation
and controls transcription quantitatively or qualitatively.
The LSD1 protein family contains
approximately 10 related proteins,
and these proteins cannot demethylate tri-methylated residues. In contrast, the jumonji family is a large
family as described above, and the
2-OG-Fe (II) dependent dioxygenase
activity of jmjC has the potential to
demethylate tri-methylated residues,
suggesting that the jumonji family
proteins can demethylate various residues in three methylation states of
histones (Tsukada et al., 2006).
The jumonji family proteins or the
protein complexes may also be able to
modulate histones in other ways. For
example, a ssion yeast jumonji family protein, Msc1, forms a complex exhibiting histone deacetylase activity
and the lack of Msc1 enhanced acetylation of histone H3 tails (Ahmed et
al., 2004). JMJD2A, a member of the
JMJD2 subfamily, binds to pRb and
HDAC (histone deacetylase) -1 and -3,
and has the potential to perform pRbmediated repression of E2F-regulated
promoters (Gray et al., 2005). Hairless, a nuclear receptor corepressor,
also binds to HDAC-1, -3, and -5 (Potter et al., 2001). Taken together, it is
most likely that jumonji family proteins and/or their protein complexes
can modulate the diverse range of histone modications and regulate the
expression of genes epigenetically and
can control various biological events
including development.

FUNCTIONS OF JUMONJI
IN DEVELOPMENT
As described above, the jumonji (jmj)
gene was identied by a mouse gene

JUMONJI IN CHROMATIN REGULATION AND DEVELOPMENT 2453

Fig. 3. Expression pattern of jmj gene in the

lens. jmj expression was monitored using lacZ,
which was introduced into the jmj gene. jmj
expression is not detected substantially at embryonic day 11.5 (E11.5). The expression can
be detected rst in the posterior elongated cells
forming the primary lens bers at E12.5. jmj
expression is also detected at the equatorial
regions and the intensity increases in the lens
bers, but the expression cannot be detected in
the lens epithelial cells at E13.5 and E14.5.
Arrowheads and LE show the equatorial region
of the lens vesicle and the lens epithelial cells,
respectively. Scale bar 50 m.

Fig. 4. Enhanced expression of cyclin D1 and

abnormal structure in the neural epithelial cells
of jmj mutant embryos. AD: Frozen sagittal
sections of wild-type (Wt, A, C) and jmj mutant
embryos (Hm, B, D) at embryonic day 8.5 (E8.5)
were analyzed by immunohistochemistry using
an antibody against cyclin D1. C,D: High-magnication images of A and B, respectively.
E: Ultrastructural analysis of neural epithelial
cells of jmj mutant embryos at E8.5. p and a
represent posterior and anterior positions, respectively. B: The arrow shows the position of
the abnormal groove. D,E: Bars indicate the
posterior region of the abnormal groove where
jmj expression is lost and cyclin D1 expression
is enhanced. The neural epithelial cells showed
abnormal cell accumulation at the posterior region of the groove. Scale bar 50 m.

2454 TAKEUCHI ET AL.

trap strategy (Takeuchi et al.,

1995).The roles during development
have been investigated most intensively among jumonji family genes.

jmj Expression Pattern

During Development
jmj is expressed in a wide range of
adult tissues in mice (unpublished
data) and humans (Berge-Lefranc et
al., 1996), and jmj is strongly expressed in embryonic stem cells. During development, very weak expression is observed in the whole
embryonic body at embryonic day 8
(E8), after which, a band-like expression pattern is detected at the future
midbrain hindbrain boundary at
E8.5. The expression becomes stronger and shows a ring-like pattern after
neural tube closure at E9 (Takeuchi et
al., 1995). In addition, weak expression is seen in the forebrain and stronger expression is also detected in the
bulbous cordis (future outow tract
and right ventricle) of the heart, the
primitive pharynx, and around the
posterior neuropore in the tail. Subsequent to this, the regions where jmj is
expressed expand gradually and the
expression is detected in almost all
adult tissues, although the intensities
are different among cell types. It is
difcult to identify the rules explaining all spatial and temporal expression patterns of jmj in various tissues
and cell types; however, the expression pattern tends to be involved in
cell proliferation or differentiation at
least in several tissues. For example,
strong expression can be detected in
many neurons after nal mitosis in
the cerebrum and cerebellum (Takeuchi et al., 1995; Takeuchi, 1997). jmj
expression starts when the levels of
cell proliferation start to decrease in
cardiac myocytes in the ventricles
(Toyoda et al., 2003). A more typical
expression pattern can be seen in the
lens (Fig. 3). jmj expression is not detected substantially at E11.5 when
cells in whole lens vesicles proliferate
(Fig. 3). However, the expression can
be detected rst in the posterior cells
that exit the cell cycle and form the
primary lens bers, whereas expression cannot be detected in anterior
lens epithelial cells that are still proliferating at E12.5 (Fig. 3). The lens
epithelial cells then move toward the
equatorial region of the vesicle and

exit the cell cycle. These cells become

gradually incorporated into the lens
proper, and subsequently develop into
secondary lens bers. jmj expression
starts at the equatorial regions and
the intensity increases in lens bers;
however, the expression cannot be detected in the lens epithelial cells at
E13.5 and E14.5 (Fig. 3). These patterns suggest a correlation of jmj expression with cell proliferation or differentiation of lens cells.

jmj Mutant Strains and

Genetic Backgrounds
The function of jmj gene has been investigated using its mutant mice that
were also obtained by the gene trap
strategy. Two jmj mutant strains have
been used for functional analyses
(Takeuchi et al., 1995; Baker et al.,
1997). In the case of our mutant mice,
we showed by expression analyses, including in situ hybridization (unpublished data) and Western blotting
(Toyoda et al., 2000), that the trap
vector disrupted the jmj gene in a
whole body. On the other hand, jmj
was disrupted in a limited number of
tissues, such as the heart, in another
jmj mutant strain (Lee et al., 2000).
Although the mutant mice used by
Lees group showed abnormalities in
only the heart (Lee et al., 2000) probably because of this limitation, our jmj
mutant mice show developmental abnormalities in various tissues and die
in utero (Takeuchi et al., 1995; Motoyama et al., 1997; Kitajima et al.,
1999, 2001; Takeuchi et al., 1999; Anzai et al., 2003).
jmj phenotypes are inuenced by
mouse genetic backgrounds. Therefore, we established jmj mutant
strains on several genetic backgrounds. On a C3H/He background
and probably a 129/Ola background,
the mutant embryos show abnormal
groove formation on the neural plate,
neural tube defects, abnormal morphology in the right ventricle and enhanced proliferation of trabecular cardiac myocytes in the left and right
ventricles, and die around E11.5
(Takeuchi et al., 1995, 1999). All of
these phenotypes were rescued by exogenous expression of jmj by transgenesis (Takahashi et al., 2004; Takahashi et al., unpublished data),
indicating that the phenotypes result

from mutation of jmj gene. In addition, a rescue experiment in only the

heart showed that abnormalities in
the heart cause lethality around
E11.5 (Takahashi et al., 2004).
On BALB/c, C57BL/6, and DBA/2
backgrounds, the mutant embryos can
survive until E15.5. Although most
phenotypes observed in the mice on a
C3H/He background cannot be observed, this survival enables us to analyze jmj phenotypes in midgestation.
The mutant embryos show edema,
hemorrhage, and hypoplasia of the
liver, spleen, and thymus (Motoyama
et al., 1997), and we found that denitive hematopoiesis is impaired (Kitajima et al., 1999).
Thus, jmj phenotypes are clearly divided into two types: C3H and BALB.
However, mutant embryos on a C3H
background would also have BALBtype phenotypes. Mutant embryos on
a C3H background, with exogenous
expression of jmj in the heart, survive
but nally die around E15.5 and these
embryos show similar phenotypes to
mutant embryos on a BALB background (Takahashi et al., 2004). Most
likely, BALB type phenotypes are
masked by lethality around E11.5 in
mutant embryos on a C3H background. Differences in the activities of
unknown gene(s) (modier) would
cause in differences of phenotypes
(Ohno et al., 2004).

jmj Functions in Cardiac

Development
Abnormalities in the heart of jmj mutant mice have been investigated intensively. The cardiac ventricles are
composed of two layers of cardiac myocytes: trabecular and compact layers.
Cardiac myocytes in trabecular layer
showed hyperproliferation in our jmj
mutant mice (Takeuchi et al., 1999;
Toyoda et al., 2003). From analysis of
the hyperproliferation, we found that
jmj expression starts at the stages
when cell proliferation of cardiac myocytes starts to decrease in normal embryos and that cell proliferation and
expression of cyclin D1, the gene that
encodes one of the G1 cyclins, are not
repressed in jmj embryos (Toyoda et
al., 2003). jmj overexpression by
transgenesis represses cyclin D1 expression in the heart. cyclin D1 overexpression by transgenesis causes

JUMONJI IN CHROMATIN REGULATION AND DEVELOPMENT 2455

hyperproliferation in the cardiac myocytes, but analysis of cyclin D1 and

jmj double mutant mice showed that
the absence of cyclin D1 in jmj mutant
embryos rescues the hyperproliferation. Therefore, cyclin D1 is a critical
downstream gene of jmj for repression
of cardiac myocyte proliferation. From
these results, we hypothesized that
jmj protein directly represses transcription of cyclin D1. In fact, Jmj protein binds to cyclin D1 promoter in
vivo and represses cyclin D1 promoter
activity (Toyoda et al., 2003). Lees
group also reported that jmj has repressor activity with respect to several promoters (Kim et al., 2003, 2004,
2005) and that their jmj mutant mice
on a mixed background showed hyperproliferation of trabecular myocytes
and enhanced expression of cyclin D1
(Jung et al., 2005).
As well as hyperproliferation, cardiac myocyte differentiation is affected in jmj mutant embryos (Takeuchi et al., 1999). The expression levels
of cardiac myocytes markers such as
myosin heavy chain, myosin light
chain, and -cardiac actin decrease
drastically. It is important to determine whether and how hyperproliferation or enhanced expression of cyclin D1 results in abnormalities in
differentiation. Proliferation and differentiation are closely related to each
other in many developing cell types.
These analyses of jmj mutant embryos
would be helpful for understanding
the molecular mechanisms regulating
proliferation and differentiation.
Most likely, a jmj-cyclin D1 pathway represses proliferation and maintains differentiation in both trabecular and compact layers, because jmj
expression starts when the levels of
cell proliferation start to decrease in
the compact layer as well as in the
trabecular layer (Toyoda et al., 2003)
and cyclin D1 overexpression affected
proliferation and differentiation in
both layers (unpublished data).
jmj mutant embryos exhibit other
abnormalities in the cardiovascular
system. Lee et al. reported that their
jmj mutant embryos on a mixed background (C57BL/6 X 129/sv) exhibited
double-outlet right ventricle (DORV)
and ventricular septal defect (VSD;
Lee et al., 2000). We also observed
these abnormalities in our mutant
mice on a BALB/c background. These

abnormalities are common human

congenital heart defects and have
been observed in numerous knockout
(KO) mice. Defects in various cell
types and various signaling pathways
could cause the abnormalities. In addition, secondary defects should be
noted because one defect in the heart,
which works as an essential pump,
could easily cause other defects. To
clarify the roles of jmj in the cardiovascular system at the late stages, it
is important to examine what cell
types are primarily affected and what
signaling pathways are involved.

jmj Functions in Brain

Development
As described in the Introduction section, jmj mutant embryos have abnormal grooves at the future midbrain
hindbrain boundary on the neural
plate at E8 E8.5 (Takeuchi et al.,
1995). Expression of the lacZ knocked
into the jmj gene is detected at the
posterior region of the groove, indicating that jmj is expressed at the corresponding region in normal embryos
(Takeuchi et al., 1995). We observed
that cyclin D1 expression was enhanced at the region (Fig. 4B,D).
Moreover, neural epithelial cells
showed a round shape and abnormal
cell accumulation and formed a multilayer at the posterior region of the
groove (Fig. 4E). The neural epithelial
cells of normal embryos and cells at
other regions of jmj mutant embryos
showed an elongated shape and form a
monolayer. These results suggest that
jmj represses cell proliferation by repression of cyclin D1 expression at the
future midbrain hindbrain boundary
as in cardiac myocytes and that repression of cell proliferation is required specically at the boundary.
In fact, recent studies have shown
that the cells in the boundary region
proliferate less rapidly than the surrounding cells at E10.5 (Trokovic et
al., 2005). Of interest, a cyclin-dependent kinase inhibitor, p21Cip1 is also
expressed at the same region as jmj.
Expression of p21Cip1 and jmj decreased largely or disappeared while
expression of cyclin D1 as well as cyclin D2 is enhanced in Fgfr1 KO embryos at E9.5E10.5 (Trokovic et al.,
2005). These results suggest that
Fgfr1 signals enhance or maintain the

expression of jmj and p21Cip1 and repress expression of cyclin D1 and D2

at a distinct region in the midbrain
hindbrain boundary, resulting in slow
proliferation at the region. Because
the Fgfr1 mutant embryos lack isthmic constriction, slow proliferation
would be necessary for development of
the isthmic constriction. It is conceivable that jmj contributes to the mechanisms by repression of cyclin D1.

jmj Functions in Cell

Proliferation
As described above, jmj negatively
regulates proliferation of cardiac myocytes (Toyoda et al., 2003) and would
repress the proliferation of neuroepithelial cells at the midbrain hindbrain boundary (see above and Trokovic et al., 2005). Further evidence for
a role for jmj in cell proliferation has
been obtained in other cells as follows.
First of all, transfection of jmj cDNA
was found to down-regulate cell proliferation in NIH3T3 and COS cells
(Toyoda et al., 2000). The number of
megakaryocyte lineage cells increased
in the liver of jmj mutant embryos,
and a delay of growth arrest in these
cells was observed in colony formation
assays (Motoyama et al., 1997; Kitajima et al., 2001).
Together with the expression patterns negatively correlated with cell
proliferation, these results suggest a
negative role for jmj in cell proliferation during development. However, all
phenotypes of jmj mutant embryos
cannot be explained by this role. For
example, despite the impaired differentiation, the proliferation of hepatocytes was not affected (Anzai et al.,
2003). In addition, a lack of cyclin D1
did not rescue all phenotypes of jmj
mutant embryos. For example, neural
tube defects were not rescued (Toyoda
et al., 2003). Although it is possible
that other cell cycle regulators are affected, these results suggest that jmj
has other roles in addition to the negative control of cell proliferation. Jmj
likely regulates the expression of several or many genes; however, cyclin
D1 is the only downstream target gene
veried in vivo so far (Toyoda et al.,
2003). Therefore, we know a portion of
the functions of jmj. Finding other target genes and related pathways as

2456 TAKEUCHI ET AL.

well as further analyses of mutant

phenotypes are necessary.
The early lethality of jmj mutant
embryos prevents us from analyzing
jmj functions in many tissues after
midgestation. For example, we do not
know the signicance of jmj expression in neurons or cardiac myocytes
after birth. Therefore, analysis of conditional KO mice is required.
Finally, the study of molecular
mechanisms in transcriptional repression by Jmj is important. Because Jmj
conserves almost no residues binding
to the cofactors 2-OG and Fe(II) (Fig.
2), Jmj itself might not have any
2-OG-Fe(II) dependent dioxygenase
activity, especially histone demethylase. However, our recent studies
showed that Jmj binds to several proteins highly involved in chromatin
regulation (unpublished data), suggesting that Jmj protein complex(es)
regulate transcription through epigenetic modication even if Jmj protein
does not have histone demethylase activity.

FUNCTIONS OF OTHER
JUMONJI FAMILY
PROTEINS IN
DEVELOPMENT
RBP2
RBP2 is a member of the JARID1 subfamily (Fig. 1). It is ubiquitously expressed in adult tissues and has jmjN,
jmjC, PHD, and ARID sequences.
Among the over 100 pRB binding proteins, RBP1 and RBP2 were identied
as the rst cellular pRB binding proteins (Defeo-Jones et al., 1991). Although the function has remained unknown for a long time, roles in cell
differentiation were recently proposed
(Benevolenskaya et al., 2005). It is
well known that pRB regulates cell
differentiation in addition to cell proliferation. Benevolenskaya et al. suggested that RBP2 represses expression of the genes required for
differentiation of various types of cells
such as myeloid, bone, and muscle,
and binding to pRB neutralizes the
functions of free RBP2 and then promotes differentiation. They also suggested that the binding is involved in
euchromatin maintenance. It is interesting to consider that pRB may act as
a negative regulator of RBP2 in cell

differentiation. Activity in transcriptional repression and involvement in

euchromatin maintenance suggest
that RBP2 or RBP2 protein complex
probably regulates chromatin remodeling. It would be interesting to examine whether RBP2 itself has histone
demethylation activity.

PTDSR
PTDSR has only a jmjC domain as
an apparent domain (Fig. 1). The
protein was initially identied as the
receptor of phosphatidylserine, a
specic marker present at the surface of apoptotic cells, and is involved in apoptotic cell phagocytosis
(Fadok et al., 2000). However,
PTDSR is localized in the nucleus
(Cikala et al., 2004; Cui et al., 2004).
Moreover, an antibody against phosphatidylserine receptor, used for
original screening, still recognizes
the receptor in mice lacking PTDSR
(Bose et al., 2004). These results
strongly suggest that PTDSR is not
the phosphatidylserine receptor and,
therefore, that the name should be
changed.
Several studies using KO or
knockdown animals (worm, zebrash, and mouse) have shown that
PTDSR is required for clearance of
apoptotic cells (Li et al., 2003; Wang
et al., 2003; Hong et al., 2004; Kunisaki et al., 2004). However, PTDSR
was not essential for clearance of apoptotic cells in an analysis of another
PTDSR KO mouse line (Bose et al.,
2004). Apart from the function related to the clearance of apoptotic
cells, PTDSR would be required for
normal embryogenesis, at least in
zebra shes and mice (Li et al., 2003;
Bose et al., 2004; Hong et al., 2004;
Kunisaki et al., 2004; Schneider et
al., 2004). PTDSR KO mice died at
the perinatal stage and showed various abnormalities in many tissues
such as the heart, brain, lung, kidney, eye, and hematopoietic system.
However, these phenotypes are also
different among KO mouse strains.
For example, neuroepithelial cells in
the retina showed enhanced proliferation in Flavells KO mice (Li et al.,
2003). On the other hand, 14.1% of
Lengelings KO mice lacked eyes
unilaterally or bilaterally (anophthalmia), or the retinal development

was temporally delayed and morphology of the inner granular layer

was abnormal, despite normal external eye structure. The abnormal
morphology observed in Flavells KO
mice does not appear to be seen in
these mice (Bose et al., 2004).
Together with the phenotypes regarding clearance of apoptotic cells,
it is unknown why the phenotypes
are quite different among KO mouse
strains. Although the possibility
that it resulted from differences in
the genetic backgrounds could not be
excluded, whether these phenotypes
of the KO mice resulted from the
lack of the PTDSR should be substantiated by rescue experiments using transgenic mice or other methods. In addition, it is important to
elucidate the molecular functions of
PTDSR, not as a transmembrane receptor but as a nuclear protein.

Hairless
Hairless is in cluster 3 and has a jmjC
domain as the only apparent domain
(Fig. 1). The gene was identied as the
responsible gene for the mouse mutant hairless (Cachon-Gonzalez et al.,
1994). The hairless mouse was rst
recognized in 1926 for its characteristic hair loss phenotype, in which initial hair growth is normal, but after
shedding, the hair does not grow back
(Brooke, 1926), suggesting abnormalities in hair follicle regeneration. Mutations of the human hairless gene revealed congenital hair loss disorders.
Some mutant alleles in both mice and
humans show skin wrinkling and papular rash (Panteleyev et al., 1998).
Hairless null KO mice show both hair
and skin phenotypes (Zarach et al.,
2004). Analysis of the KO mice revealed increased proliferation and
changes of cell types in the epidermis,
suggesting that Hairless is required
for normal balance of cell proliferation
and differentiation in the epidermis
cells (Zarach et al., 2004). The rescue
experiments using transgenic mice
showed that expression of Hairless in
progenitor keratinocytes is required
for hair follicle regeneration (Beaudoin et al., 2005). Hairless represses
expression of Wise, a modulator of
Wnt signaling, coincident with the
timing of follicle regeneration. From
these studies, a model in which Hair-

JUMONJI IN CHROMATIN REGULATION AND DEVELOPMENT 2457

less regulates the precise timing of

Wnt signaling required for hair follicle
regeneration was proposed (Beaudoin
et al., 2005).
Hairless protein functions as a nuclear receptor corepressor and interacts with multiple nuclear receptors
such as thyroid hormone receptor,
retinoic acid receptor-related orphan
receptor , and vitamin D receptor
(Potter et al., 2001; Moraitis et al.,
2002; Hsieh et al., 2003). Hairless represses transcriptional activities in
the context of these nuclear receptors
(Potter et al., 2001; Moraitis et al.,
2002; Hsieh et al., 2003). Of interest,
Hairless as well as Jmj have transcriptional repression activity, although these two proteins might not
have 2-OG-Fe(II) dependent histone
demethylase activity, because cofactor
binding sites are not conserved (Fig.
2). Because Hairless is a transcriptional repressor, altered expression of
genes would cause the phenotypes of
Hairless mutants. The expression of
several genes such as keratinocyte differentiation markers and wise, was
up-regulated in Hairless KO mice
(Zarach et al., 2004; Beaudoin et al.,
2005). Hairless represses the promoter activity of wise (Beaudoin et al.,
2005). These studies link the molecular functions of Hairless to the functions in skin and hair development.

FUTURE PERSPECTIVES
The molecular functions of jmjC and
jumonji family proteins will be analyzed extensively and intensively.
However, it will also be important to
link the molecular functions to biological events such as development. Several points or questions that require
attention when conducting these studies are discussed below.

What Molecular Functions

Do Each Jumonji Family
Protein or the Protein
Complex Have?
There is little doubt that the histone
demethylase activities of many jumonji family proteins will be examined. However, it is possible that jumonji family proteins and the protein
complexes have functions of not only
histone demethylases but also other
chromatin modiers, or other en-

zymes that are not directly related to

chromatin regulation.
One of the jumonji family proteins,
HIF1AN/FIH, hydroxylates an asparagine residue in HIF, and it is unknown whether HIF1AN/FIH is active
in histone demethylation or other
chromatin modication. Several jumonji family proteins do not have conserved residues at the predicted binding sites for the cofactors, 2-OG or
Fe(II) (Trewick et al., 2005; Tsukada
et al., 2006; Fig. 2). Therefore, these
proteins would not have 2-OG-Fe(II)
dependent dioxygenase activity that
would be required for histone demethylation by JHDM1 and hydroxylation
by HIF1AN/FIH. Of interest, jumonji
family proteins exist in bacteria that
do not have histones. These facts suggest that jumonji family proteins can
have various functions other than as
histone demethylases. For example, it
is exciting if some members have DNA
modication activity, such as a DNA
demethylase. As described above, bacterial AlkB protein, a 2-OG-Fe(II) dependent dioxygenase, is involved in
DNA repair by DNA demethylation
(Falnes et al., 2002; Trewick et al.,
2002), suggesting the possibility. Because DNA modication as well as histone modication are very important
events for making epigenetic signatures, the possibility is very interesting.
In addition, forming complexes with
other proteins would add other functions in histone or chromatin modication to jumonji family proteins (see
the section entitled Functions of Jumonji Family Proteins in Transcription and Chromatin Regulation). Several large protein complexes would
have multiple or sequential functions
(Ogawa et al., 2002), similar to that of
a factory. It is possible that jumonji
family proteins are one of the members in the factory. In this case, it is
important to examine what distinct
functions the jumonji family proteins
have in the complexes.

What Molecules or Proteins,

and Moreover, Which Sites
in the Molecules Are Targets
of Jumonji Family Proteins?
If a certain jumonji family protein or
the protein complex has histone modication activities, including histone

demethylation, which residues and

states of premodication (for example,
mono-, or di-, or tri-methylation) in
the histones are modied by the proteins should be examined, because we
can infer specic functions from modied residues and states.

Expression of What Genes

Jumonji Family Members
Regulate and How Is
Expression Regulated?
If a certain jumonji family protein is
shown to have a function in chromatin
regulation, the next step is to answer
the questions. It is also important to
ascertain whether the jumonji family
proteins regulate limited genes in a
part of the genome, or many genes in
more extensive regions. It would be
interesting to study whether jumonji
family proteins are involved in the
regulation, such as formation or maintenance of euchromatin or heterochromatin, or imprinting or X-chromosome inactivation. In fact, Epe1 is
required for heterochromatin integrity (Ayoub et al., 2003).

What Biological Events In

Vivo Including Development
Are Linked to Molecular
Functions of Jumonji Family
Proteins?
Basic biological events can be analyzed in cell cultures, however, studies
in organisms are necessary to analyze
more higher or complicated functions
in vivo. Especially, using mutant organisms such as KO mice is one of the
most powerful methods, because we
can analyze which cell behaviors such
as cell differentiation, cell proliferation, cell migration or cell adhesion
are involved, and in which tissues the
proteins are required. That mutant
mice of two jumonji family proteins,
Jmj and PTDSR, show abnormalities
in multiple tissues (see above) suggests that at least some of the jumonji
family proteins are required for the
regulation of many genes or that these
proteins have common essential functions in the development of diverse
tissues.
Of course, it is difcult to elucidate
the links between molecular functions
and biological events completely by

2458 TAKEUCHI ET AL.

analysis of mutant organisms only.

Therefore, a good combination of studies at the molecular and organism levels would be necessary. We believe
that such studies on the jumonji family will contribute signicantly to our
understanding of the mechanisms of
chromatin regulation and development.

NOTE
Most recently, ve jumonji family
members, JMJD1A/TSGA (renamed
to JHDM2A in the following paper)
and JMJD2A-D have been shown to be
H3K9 demethylases (Whetstine et al.,
2006; Yamane et al., 2006). JMJD2A
and JMJD2C are also H3K36 demethylases (Whetstine et al., 2006). Importantly, JMJD1A/TSGA can demethylate mono- and di-methyl H3K9,
and
JMJD2
family
can
demethylate di- and tri-methyl H3K9/K36. These results showed that jumonji family members can demethylate residues in three methylation
states of histones.

ACKNOWLEDGMENT
We thank Ms. Mizuyo Kojima and Ms.
Kuniko Nakajima for their excellent
technical assistance.

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