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| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online February 20, 2009
doi: 10.1242/10.1242/dev.020966
Review |
1 Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
2 Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.
* Author for correspondence (e-mail: yang_shi{at}hms.harvard.edu)
SUMMARY
Since the discovery of the first histone lysine demethylase in 2004, two protein families with numerous members have been identified that demethylate various histone lysine residues. Initial studies of the histone lysine demethylases focused on their in vitro enzymatic activity but, more recently, model organisms have been used to examine the roles of these enzymes in vivo. Here, we review recent insights into the roles of the histone lysine demethylases in multiple aspects of development across various species, including in germline maintenance and meiosis, in early embryonic development and differentiation, and in hormone receptor-mediated transcriptional regulation.
Introduction
Due to physical and functional constraints, the genetic material must be packaged within the cell in a way that both efficiently minimizes the space it occupies and allows it to be accessed in a regulated manner. The basic unit of this packaging is the nucleosome, which consists of 146 bp of DNA wrapped around a histone octamer. Each histone octamer contains two subunits of four types of histones: H3, H4, H2A and H2B. The DNA between histones is associated with the linker histone H1.
The four histone subunits can be post-translationally modified in a variety
of ways, including by phosphorylation, ubiquitylation, sumoylation,
acetylation and methylation. Many of these modifications are correlated with
particular chromatin environments and biological outcomes (for reviews, see
Kouzarides, 2007
;
Li et al., 2007;
Margueron et al., 2005).
Certain modifications, such as acetylation, are generally associated with
euchromatin. Among the histone modifications, histone lysine methylation is of
particular interest given the diverse set of methylation-associated biological
processes that exist, including transcriptional activation and repression,
heterochromatin-mediated transcriptional silencing, the DNA damage response
and X chromosome inactivation (Margueron
et al., 2005; Martin and
Zhang, 2005).
Each of the methylated lysines exists in four distinct states: un-, mono-,
di- or tri-methylated, and each of these states can have independent functions
(see Table 1). The most-studied
sites of histone lysine methylation are histone H3 lysines 4, 9, 27, 36 and
79, and histone H4 lysine 20 (Martin and
Zhang, 2005). Only those four lysines closest to the N-terminus of
histone H3 have known demethylases. H3K79- and H4K20-specific demethylases
have not yet been identified and there is some controversy over their
existence (Frederiks et al.,
2008).
|
), histone
methyltransferases (KMTs) and histone lysine demethylases (KDMs) have only
recently been identified. The existence of enzymes that methylate or
demethylate histone lysines is perhaps unsurprising. However, prior to the
discovery of the KDMs, the process of histone methylation was widely
considered to be irreversible (reviewed by
Bannister et al., 2002). In
2004, the amine oxidase-domain-containing mammalian protein LSD1/KDM1 [for
lysine-specific demethylase 1, later renamed lysine (K) demethylase 1 to
standardize nomenclature; also known as AOF2] (see
Table 2) was identified as the
first histone lysine demethylase. LSD1/KDM1 demethylates both di- and
mono-methylated K4 (H3K4me2/1) (Shi et
al., 2004). In 2006, the first jumonji-domain-containing
demethylase, JHDM1A/KDM2A (FBXL11), was identified as an H3K36me2/1
demethylase (Tsukada et al.,
2006). The jumonji (or JmjC)-domain-containing proteins belong to
the dioxygenase superfamily and use a demethylation mechanism distinct from
that of LSD1/KDM1 (Anand and Marmorstein,
2007) (see Box 1).
These enzymes were predicted to be capable of tri-methyl demethylation
(Schneider and Shilatifard,
2006; Trewick et al.,
2005), and a tri-methyl demethylase family, the JMJD2/KDM4 family
(Fodor et al., 2006;
Klose et al., 2006;
Whetstine et al., 2006), was
indeed reported soon after the first jumonji-domain-containing demethylase was
discovered. Over the next few years, a series of studies identified additional
jumonji-domain-containing families that have K4me, K9me, K27me and K36me as
their substrates (Table 2).
|
; Cloos et al.,
2008; Secombe and Eisenman,
2007; Shi, 2007)
and so is not reviewed in detail here. Histone methylation and demethylation in the germline
In the germline – the immortal lineage of cells that gives rise to the haploid gametes in sexually reproducing organisms – numerous DNA-directed events must be coordinated and tightly controlled, both spatially and temporally, as nuclei progress through meiosis. Of the histone modifications examined during meiosis in the mouse and in Caenorhabditis elegans, the methylation of two residues in particular, H3K4me and H3K9me, shows especially interesting dynamics during the early stages of meiotic prophase that correspond to chromosomal pairing, synapsis and the initiation/resolution of DNA double-strand breaks (DSBs). Recent studies are now implicating both KMTs and KDMs in this dynamic regulation.
During early stages of both oogenesis and spermatogenesis, the replicated
sister chromatids must find, pair and synapse with their homologs, as well as
undergo programmed DSBs that lead to crossover recombination. The
identification of paired versus unpaired chromosomes is therefore an essential
meiotic process, with meiotic silencing of unpaired chromosomes (MSUC) being
an important consequence (reviewed by
Turner, 2007). The single X
chromosome that is present in mammalian and C. elegans males appears
to trigger a version of MSUC that is specific to the sex chromosomes, known as
meiotic sex chromosome inactivation (MSCI)
(Turner, 2007). H3K9me2 coats
unpaired chromosomes in C. elegans pachytene nuclei, including the
lone X chromosome in males (Bean et al.,
2004; Kelly et al.,
2002; Reuben and Lin,
2002; Schaner and Kelly,
2006), suggesting that this methylation state is associated with
chromosome mis-alignment and asynapsis. Interestingly, a mutation in the
C. elegans him-17 gene (which encodes a chromatin-associated protein
that contains six repeats of a putative DNA-binding motif that is also found
in proteins implicated in chromatin regulation via their genetic interactions
with LIN-35/Rb) leads to mis-patterning of H3K9me2 and to gross defects in DSB
initiation and DSB repair (DSBR) progression, as well as to impaired sex
chromosome segregation (Reddy and
Villeneuve, 2004). These mutant phenotypes highlight the links
between the detection of proper chromosome pairing, DSBR and histone
methylation in meiosis.
The levels of H3K4me also show distinct and sometimes dramatic changes
during early meiosis in both C. elegans and mammals. H3K4me is
present on the autosomes and is excluded from the silenced X chromosomes in
both sexes in C. elegans (Schaner
and Kelly, 2006). Moreover, H3K4me shows a striking global
upregulation at the start of male meiosis in mice, followed by a
downregulation at the zygotene/pachytene stage
(Godmann et al., 2007). This
pattern of upregulation and downregulation corresponds to the stages at which
chromosomes synapse and DSBR starts to progress.
Given these dynamic methylation patterns, it is not surprising that several
enzymes that act on H3K4me and H3K9me have been identified to have meiotic
roles. Two classes of KDMs have been identified that recognize H3K4me as a
substrate: the amine oxidase-domain-containing LSD1/KDM1
(Shi et al., 2004) and the
jumonji-domain-containing JARID1/KDM5 family
(Christensen et al., 2007;
Iwase et al., 2007;
Lee et al., 2007a;
Lee et al., 2007c;
Secombe et al., 2007;
Seward et al., 2007;
Tahiliani et al., 2007;
Xiang et al., 2007b;
Yamane et al., 2007). In
metazoans, LSD1/KDM1 acts on H3K4me2/1 (Di
Stefano et al., 2007; Rudolph
et al., 2007; Shi et al.,
2004) and plays important roles in the germline (see below). The
KDM5 family acts on H3K4me3/2 (Christensen
et al., 2007; Iwase et al.,
2007; Lee et al.,
2007a; Lee et al.,
2007c; Secombe et al.,
2007; Seward et al.,
2007; Tahiliani et al.,
2007; Xiang et al.,
2007b; Yamane et al.,
2007) and has a less well-defined role in the germline. However,
KDM5 mutants in budding yeast (Seward et
al., 2007) and in corn fungus
(Quadbeck-Seeger et al., 2000)
have sporulation defects, hinting at meiotic roles for this demethylase family
in these organisms as well. Intriguingly, a recent study in mice has reported
that a spermatogenesis-specific association exists between SMCY/KDM5D
(JARID1D) and MSH5, a meiosis-specific protein required for the progression of
synapsis and for crossover completion
(Akimoto et al., 2008). This
potential connection between a regulator of H3K4me2/3 and these essential
meiotic processes warrants further study.
Homologs of LSD1/KDM1 in C. elegans
(Shin-i and Kohara, 2005) and
in Drosophila melanogaster (Di
Stefano et al., 2007; Rudolph
et al., 2007) are also expressed in the germline, and the
Drosophila KDM1 mutants have a germline-specific phenotype
(Di Stefano et al., 2007;
Rudolph et al., 2007). Rudolph
et al. identified Drosophila LSD1/KDM1 as SU(VAR)3-3, a suppressor of
heterochromatic silencing (Rudolph et al.,
2007). Purified SU(VAR)3-3 protein is a H3K4me2/1 demethylase in
vitro, and Su(var)3-3 null larvae have increased levels of H3K4me2.
Surprisingly, H3K9me is reduced in these animals in heterochromatic regions
where SU(VAR)3-3 is not detected, indicating that a finely tuned balance
exists between these modifications, in which the absence of a H3K4me2 KDM
indirectly influences H3K9me levels
(Rudolph et al., 2007). They
also reported a severe sterility defect in both male and female
Su(var)3-3 mutant flies, with a complete absence of oocytes in the
females and severe defects in spermatogenesis in the males
(Rudolph et al., 2007). Di
Stefano et al. also examined SU(VAR)3-3 (referred to in that study as dLSD1)
mutants and reported a similar phenotype of female fertility defects together
with H3K4me misregulation. However, unlike Rudolph et al.
(Rudolph et al., 2007), they
also observed a male-specific embryonic lethality
(Di Stefano et al., 2007),
perhaps owing to the use of different mutant alleles. Both groups support a
model in which the mutation of LSD1/KDM1 leads to the disruption of
heterochromatic/euchromatic boundary regions and to a disruption of germline
development and meiotic processes. A mechanistic connection between these two
phenotypes is still lacking at present, but one possibility is that these
phenotypes reflect the transcriptional misregulation of LSD1/KDM1 target genes
that are located in these boundary regions.
A set of recent studies in Arabidopsis thaliana supports a
transcriptional role for LSD1 in regulating target genes important for
reproductive function. These studies show that, FLOWERING LOCUS D (FLD), one
of the Arabidopsis LSD1 homologs, promotes the transition from the
vegetative to the reproductive phase (flowering) by repressing transcription
of the floral repressors FLOWERING LOCUS C (FLC) and FWA
(Jiang et al., 2007;
Liu et al., 2007). Liu et al.
also showed a functional interaction between FLD and the RNA recognition motif
(RRM)-domain-containing protein FCA in repressing FLC
(Liu et al., 2007).
Interestingly, purification of the Schizosaccharomyces pombe LSD1
also identified an RRM-domain-containing protein (SPBPJ758.01)
(Nicolas et al., 2006).
Perhaps RNA interactions might affect LSD1 function in these protein
complexes, but future studies are needed to understand this potential
connection between LSD1 and RNA-mediated regulation, chromatin boundary and
meiotic function in Arabidopsis and other organisms.
| Box 1. Classes of histone demethylase enzymes Two classes of enzymes have been reported with histone lysine demethylase activity. The first class of enzyme is solely represented by LSD1/KDM1, whereas the remaining known demethylases fall into the jumonji (JmjC)-domain-containing class. The proposed enzymatic mechanisms of these two distinct classes offer insights into their substrate specificity and necessary co-factors. Demethylases have been identified for histone H3 lysine 4 (H3K4), H3K9, H3K27 and H3K36; in these examples, we use H3K4 as a substrate. Amine oxidase-domain-containing mechanism: LSD1/KDM1 contains a flavin adenine dinucleotide (FAD)-dependent amine oxidase domain that is responsible for its demethylase activity. The enzyme uses FAD as a co-factor to catalyze an amine oxidation of the protonated nitrogen (blue), creating an iminium (N+) ion. This iminium ion spontaneously hydrolyzes to release a formaldehyde molecule, resulting in a mono-methylated lysine (H3K4me1). This mono-methylated lysine can also undergo the same reaction to become unmethylated. An important difference between this reaction mechanism and that of the JmjC-domain-containing proteins is that LSD1/KDM1 requires a protonated nitrogen as a hydrogen donor. The nitrogen of a tri-methylated lysine is not protonated and therefore cannot be demethylated by LSD1/KDM1. R, amino acid backbone. JmjC-domain-containing mechanism:
The JmjC-domain-containing enzymes were predicted to be capable of histone
demethylation based on their homology to the AlkB dioxygenases, which use
oxidation to remove damage-induced alkylation from DNA. The multiple
co-factors required for JmjC-mediated demethylation [Fe(II), O2 and
|
|
|
As with the enzymes that regulate H3K4me, several H3K9me KMTs and KDMs have
been identified that have meiotic functions. For example, in S.
pombe, LSD1/KDM1 functions as an H3K9me2/1 demethylase
(Lan et al., 2007b;
Opel et al., 2007), and loss
of one of the two S. pombe LSD1/KDM1 homologs results in a
sporulation defect (Lan et al.,
2007b). Both homologs are also important for maintaining
heterochromatic boundaries (Gordon et al.,
2007; Lan et al.,
2007b), which indicates that LSD1 proteins have a conserved role
in meiosis and chromatin boundary maintenance across widely divergent
organisms. In mice, the H3K9me2/1 methyltransferase G9a/KMT1C (EHMT2) is
temporally expressed during the early stages of spermatogenesis, being present
only in the pre-meiotic spermatogonia and the early leptotene stage of meiosis
(Tachibana et al., 2007). A
germline-specific G9a/Kmt1c genetic inactivation produces completely
sterile males and partially sterile females, with male germ cell nuclei
showing defects in synapsis and DSBR progression, leading to pachytene arrest
and apoptosis (Tachibana et al.,
2007). The authors also reported a decrease in H3K9me levels as
nuclei enter the pachytene stage, coincident with the expression of the
testis-specific H3K9me2/1 demethylase JHDM2A/KDM3A (JMJD1A)
(Okada et al., 2007;
Tachibana et al., 2007),
indicating that these enzymes antagonistically regulate this modification
during spermatogenesis (Fig.
1).
JHDM2A/KDM3A mediates the demethylation of H3K9me2, and its expression
appears to begin in late pachytene and peaks in intensity in the round
spermatids (Okada et al.,
2007), correlating well with a reduction of the H3K9me2
modification at this stage of meiosis. Male mice that lack JHDM2A/KDM3A are
viable but sterile, with smaller testes and severe defects in spermatogenesis,
but only in post-meiotic cells (Okada et
al., 2007). A defect in chromatin condensation exists in the few
detectable mature sperm present in these mutants, and given the normal
appearance of pre-meiotic and meiotic cells, the authors concluded that
JHDM2A/KDM3A functions primarily after the completion of meiosis
(Okada et al., 2007)
(Fig. 1). Multiple genes have
been shown to affect chromatin condensation in maturing sperm, so the authors
examined the expression of several of these genes in Jhdm2a/Kdm3a
mutant testes. They observed a downregulation of two essential sperm
chromatin-packaging genes: transition nuclear protein 1 (Tnp1) and
protamine 1 (Prm1) (Okada et al.,
2007). JHDM2A/KDM3A binds to the promoters of both Tnp1
and Prm1 in round spermatids, and Jhdm2a/Kdm3a mutant
spermatids show increased H3K9me at these promoters. These data suggest that
the normal function of JHDM2A/KDM3A is to activate chromatin condensation
genes upon sperm maturation by removing the repressive H3K92/1me modification
from their promoters.
|
;
Klose et al., 2006;
Whetstine et al., 2006). The
C. elegans KDM4 homolog, JMJD-2, appears to share this substrate
specificity, as its depletion by RNAi leads to a general increase in germline
H3K9me3 staining and to an increase in H3K36me3 on one end of the X chromosome
during meiotic prophase I (Whetstine et
al., 2006) – an intriguing pattern of unknown significance.
Although pairing and synapsis appear to be normal, the JMJD-2-depleted animals
have a germline phenotype of increased apoptosis and perturbed DSBR,
indicating that a potential connection exists between this enzyme, its target
modifications and meiotic DSBR (Whetstine
et al., 2006). It remains unclear whether the misregulation of one
or both tri-methyl modifications is responsible for the meiotic phenotype
observed in JMJD-2-depleted C. elegans, opening a novel avenue of
research for potential roles of H3K9me3 and/or H3K36me3 in DSBR. In summary, studies in multiple model systems have shown that H3K4me and H3K9me, in particular, are present in chromatin in defined patterns during meiosis, and that the perturbation of the enzymes that regulate these modifications leads to defects in many essential meiotic steps. Collectively, these studies suggest that the dynamic regulation of histone methylation by both KMTs and KDMs might be one way to monitor meiotic progression. The mechanisms that guide the interplay between histone demethylation and meiotic processes are unknown, and future studies are necessary to explore these interactions. Does the regulation of other histone methylations, such as H3K27me, play important roles in meiosis as well? Although future work might identify unsuspected roles for this modification and the enzymes that regulate it in germline function, as we discuss below, H3K27me KDMs appear to primarily function later in differentiation and development.
Demethylase roles in differentiation
Pluripotency: a role for demethylases?
Studies of several demethylases suggest that they help to modulate the
progression of pluripotent progenitor cell types into differentiated cell
lineages during development. LSD1/KDM1 has a particularly interesting
expression pattern in early mammalian development. Its maternally stored
transcript is no longer detectable by the maternal-to-embryo transition (MET),
when the embryo becomes transcriptionally competent. LSD1/KDM1 expression then
recovers to oocyte levels by the blastula stage
(McGraw et al., 2007). In
flies, loss of LSD1/KDM1 causes embryonic lethality
(Di Stefano et al., 2007;
Rudolph et al., 2007), as it
does in mice at an early embryonic stage
(Wang et al., 2007). The
application of the LSD1/KDM1 chemical inhibitor bisguanidine 1c, which is a
polyamine analog, to in vitro fertilized mouse embryos not only increases
global H3K4me2 (as visualized by immunofluorescence), but induces irreversible
arrest at the two-cell stage (Shao et al.,
2008), which occurs after the MET in mouse
(McGraw et al., 2007),
providing further evidence that LSD1/KDM1 has an embryonic role. The authors
show that inappropriate upregulation of the pluripotency-maintaining factor
OCT4 (POU5F1) occurs in embryos after this chemical inhibition, which
indicates a potential mechanism for their early arrest
(Shao et al., 2008). However,
whether the observed effect brought about by bisguanidine 1c treatment is
entirely due to the inhibition of LSD1/KDM1 function requires further
investigation.
Several other demethylases are preferentially expressed in undifferentiated
cell types, hinting at their having roles in maintaining pluripotency. Of the
JMJD2/KDM4 family members, for example, JMJD2C/KDM4C is preferentially
expressed in undifferentiated human and mouse ES cells, as compared with
blastocysts and both hepatocyte and neural progenitor cells
(Katoh and Katoh, 2007). Both
JMJD2C/KDM4C and another KDM3 family member, JHDM2A/KDM3A, have been recently
implicated in the maintenance of pluripotency in mouse ES cells
(Loh et al., 2007). The
authors identified predicted OCT4 binding sites within the introns of both the
JMJD2C/KDM4C and JHDM2A/KDM3A genes and showed that OCT4-activated
transcription occurred via these sites, as assessed by a reporter assay
(Loh et al., 2007), findings
which together indicate that OCT4 positively regulates the expression of both
these demethylases. The RNAi depletion of JMJD2C/KDM4C and JHDM2A/KDM3A in
mouse ES cells affected global levels of H3K9me3 and H3K9me2, respectively,
and led to a dramatic change in ES cell morphology that is reminiscent of
fibroblast differentiation (Loh et al.,
2007). Colony formation assays showed that both demethylases were
crucial for the maintenance of ES cell pluripotency
(Loh et al., 2007). Moreover,
the depletion of both JMJD2C/KDM4C and JHDM2A/KDM3A correlated with decreased
levels of the pluripotency factors OCT4, SOX2 and NANOG. The depletion of
JMJD2C/KDM4C alone led to the increased expression of certain genes, including
the endodermal lineage markers Gata4 and Gata6, whereas the
depletion of JHDM2A/KDM3A alone led to the induction of genes, including the
mesodermal lineage marker brachyury (Loh
et al., 2007). JHDM2A/KDM3A was also specifically implicated as a
positive regulator of the self-renewal regulatory gene T-cell lymphoma
breakpoint 1 (Tcl1), and was shown to be required for the binding of
endogenous OCT4 to the Tcl1 promoter
(Loh et al., 2007). Although
the recruitment mechanism remains to be determined, JMJD2C/KDM4C has been
found in association with the Nanog promoter, indicating that
Nanog might be directly regulated by this demethylase
(Loh et al., 2007). These data
suggest a model in which JMJD2C/KDM4C and JHDM2A/KDM3A are downstream targets
of the pluripotency factor OCT4 that act in a positive-feedback manner to
promote pluripotency by activating target genes, including Nanog and
Tcl1, respectively.
In support of this model, the overexpression of JHDM2A/KDM3A in adult
neural stem cells (NSCs) has been shown to induce Oct4 expression
(Ma et al., 2008). This is
consistent with the idea that these H3K9 demethylases might promote
pluripotency through a positive-feedback loop that involves both NANOG and
OCT4 (Fig. 2A).
|
), but the timing of irreversible arrest in mouse embryos that
have been exposed to a LSD1/KDM1 inhibitor
(Shao et al., 2008) suggests
that it is the embryonic expression of this demethylase that is essential for
early differentiation events. The observed upregulation of the pluripotency
factor OCT4 after mouse embryos are treated with a LSD1/KDM1 inhibitor
suggests a possible mechanism (yet to be experimentally addressed) in which
LSD1/KDM1 acts to remove the activating H3K4me modification from the
Oct4 promoter under normal developmental conditions, repressing the
gene and allowing cells to differentiate. In contrast to LSD1/KDM1, the H3K9me
demethylases JMJD2C/KDM4C and JHDM2A/KDM3A are expressed in undifferentiated
ES cells and appear to act in the maintenance of pluripotency, potentially
through the transcriptional activation of pluripotency-maintaining factors,
such as OCT4, NANOG and TCL1 (Katoh and
Katoh, 2007; Loh et al.,
2007). Taken together, these studies hint at a potentially general
mechanism for the regulation of developmental events by KDMs, by which
antagonistically acting demethylases transcriptionally regulate target genes
that are important for pluripotency or differentiation. Further examples of
this mode of action are discussed below.
Hox gene regulation: a model for opposing demethylase roles in differentiation
The Hox genes are a developmentally regulated set of evolutionarily
conserved genes that are responsible for body plan patterning (reviewed by
Akam, 1995). They are
transcriptionally silenced in ES cells, but become activated in various
lineage-specific patterns upon differentiation (reviewed by
Soshnikova and Duboule, 2008).
Studies in Drosophila first showed that the regulation of many
developmentally important genes, including the Hox genes, is accomplished by
the opposing Trithorax (Trx) and Polycomb group (PcG) proteins, which form
complexes that are responsible for the transcriptional activation and
repression of their targets, respectively (reviewed by
Schuettengruber et al., 2007).
Both Trx and PcG complexes contain many chromatin-directed activities, such as
the H3K4me3-specific histone methyltransferase Mixed-lineage leukemia
(MLL/KMT2) family, which can be found in Trx complexes, and the
methyltransferase Enhancer of zeste [E(Z)/KMT6], which is found in PcG
complexes and is responsible for H3K27 methylation. Surprisingly, given that
Trx proteins are gene activators with associated H3K4me3 methylation activity,
one Trx group protein, LID, is a Drosophila KDM5 family homolog and
an H3K4me3-specific demethylase, which would be expected to repress
transcription (Eissenberg et al.,
2007; Lee et al.,
2007c; Secombe et al.,
2007). LID appears to interact with the cell growth regulator dMYC
(also known as DM), which binds the jumonji domain of LID to inhibit its
demethylase activity (Secombe et al.,
2007), but whether and how this connection pertains to Hox gene
regulation remains to be determined.
Does the gain or loss of various lysine methylations via these protein
complexes play a role in differentiation? Genome-wide studies of the chromatin
status of cells as they lose pluripotency and begin to differentiate has
proven to be a fruitful means of uncovering key regulatory histone
modifications important for differentiation decisions. For example, the
unexpected identification of the bivalent domains in the pluripotent genomes
of mouse and human ES cells, which contain both `active' H3K4me3 and
`repressive' H3K27me3 modifications
(Bernstein et al., 2006), is
indicative of a novel means of chromatin-based gene regulation that is
believed to be especially relevant for development and differentiation. The
resolution of these bivalent domains into either H3K4me3 or H3K27me3 upon
differentiation is believed to stably mark these regions for activation or
repression, respectively (Bernstein et
al., 2006; Mikkelsen et al.,
2007) (Fig. 2B). In
mouse ES cells, these bivalent domains are enriched on the developmentally
essential Hox gene loci (Bernstein et al.,
2006), rendering these genes an especially useful model for
understanding the roles of these unique chromatin domains.
Although H3K4me3 and H3K27me3 co-exist at Hox gene loci in undifferentiated
ES cells, upon differentiation only H3K4me3 is found at activated Hox genes,
whereas only H3K27me3 is present at silenced Hox genes
(Mikkelsen et al., 2007). Is
there any connection between the methyltransferases responsible for
maintaining these modifications and the demethylases that might act to
selectively remove H3K4me3 or H3K27me3? Evidence for coordinated KDMs and KMTs
that act to maintain H3K4me3 or H3K27me3 has been found in mammalian systems,
as discussed below.
For example, the H3K27me3 demethylase ubiquitously transcribed
tetratricopeptide repeat, X chromosome (UTX/KDM6A)
(Agger et al., 2007;
Lan et al., 2007a;
Xiang et al., 2007a) has been
identified in the H3K4me3 methyltransferase (MLL/KMT2)-containing complex
(Cho et al., 2007;
Lee et al., 2007b). Both
UTX/KDM6A and the closely related demethylase, JMJD3/KDM6B, bind Hox gene
promoters upon differentiating (activating) conditions
(Agger et al., 2007;
De Santa et al., 2007;
Lan et al., 2007a;
Lee et al., 2007b)
(Fig. 2B). Zebrafish embryos
depleted of the UTX/KDM6A homolog (Lan et
al., 2007a) and Xenopus embryos that lack the H3K4
methylation patterning protein WDR5
(Wysocka et al., 2005), show
surprisingly similar phenotypes of defective somitic patterning (defective
posterior development in the zebrafish and axial defects leading to shortened
tadpoles in Xenopus) that are indicative of Hox gene misregulation
(Lan et al., 2007a;
Wysocka et al., 2005). This
suggests that H3K27me3 removal is functionally coordinated with H3K4me3
addition, a model that is supported by the physical association of an H3K27me3
demethylase with an H3K4me3 methyltransferase, as well as by the similarities
in phenotypes between animals defective in either H3K27me3 demethylation or
H3K4me3 patterning.
There is evidence supporting H3K4me3 KDM and H3K27me3 KMT coordination. The
H3K4me3 demethylase retinoblastoma binding protein 2 (RBP2/KDM5A; also known
as JARID1A) physically interacts with the H3K27me3
methyltransferase-containing Polycomb group repressive complex 2 (PRC2)
(Pasini et al., 2008).
RBP2/KDM5A and PcG proteins also show an extensive overlap of target genes,
and although depletion of RBP2/KDM5A does not prevent PRC2 from binding to its
target genes, it appears to inhibit their repression
(Pasini et al., 2008).
However, the depletion of PRC2 proteins reduces RBP2/KDM5A promoter binding
(Pasini et al., 2008),
suggesting that the PRC2 complex recruits the demethylase. During ES cell
differentiation, both PRC2 and RBP2/KDM5A are lost from the promoters of genes
that are induced upon differentiation
(Pasini et al., 2008)
(Fig. 2B), suggesting that the
loss of both the H3K27me3 KMT and the H3K4me3 KDM is required for
transcriptional activation. Consistent with this, an earlier study showed that
a specific loss of RBP2/KDM5A occurs from Hox gene loci upon ES cell
differentiation (Christensen et al.,
2007).
In summary, it appears that in mammals, two large biochemical complexes,
analogous to the Trx and PcG complexes that exist in flies, might act to
coordinately regulate H3K4me3 and H3K27me3 so that only one modification is
dominant on the target gene in differentiated cells. These complexes therefore
contain opposing enzymatic activities that control and coordinate the balance
of histone methylation at H3K4 and K3K27
(Fig. 2B). One complex,
analogous to the Trx complex, contains both the H3K4me3 methyltransferase
MLL/KMT2 and the H3K27 demethylase UTX/KDM6A. The other, analogous to the PcG
complex PRC2, contains the opposite enzymatic capabilities, with the H3K27
methyltransferase subcomplex PRC2/KMT6 and the H3K4me3 demethylase RBP2/KDM5A.
Both demethylases are expressed in undifferentiated mouse ES cells
(Christensen et al., 2007;
Lan et al., 2007a) and
RBP2/KDM5A has been reported to bind Hox gene loci before ES cells undergo
differentiation (Christensen et al.,
2007). Interestingly, the H3K27 demethylase UTX, although
expressed in ES cells, appears to be excluded from the Hox gene loci
(Lan et al., 2007a),
suggesting a possible mechanism for the protection of the bivalent domain in
ES cells. How the bivalent domain is protected in stem cells and how it is
resolved during differentiation are questions of significant interest and
importance, and are likely to involve the relevant histone methylases and
demethylases. An answer to these questions might lie in the control of the
recruitment of these enzymes and/or their activation upon differentiation,
which are poorly understood at the present time.
Demethylases in neural differentiation and disease
In addition to the transition of ES cells from pluripotency to
differentiation, both H3K4me3 and H3K27me3 demethylases have been reported to
play key roles in neuronal development. A role for H3K27me3 demethylation in
mammalian neuronal development has been identified through studies of the
co-repressor, silencing mediator for retinoid and thyroid hormone receptor
(SMRT; also known as NCOR2), which is crucial for mouse forebrain development
and for the maintenance of pluripotency
(Jepsen et al., 2007). These
authors identified JMJD3/KDM6B as a transcriptional target of SMRT when mouse
embryonic NSCs were induced to differentiate with retinoic acid (RA). They
also showed that the overexpression of JMJD3/KDM6B in a neuronal stem cell
culture induced the expression of neuronal subtype genes, such as distal-less
homeobox 5 (Dlx5) (Jepsen et al.,
2007), supporting a role for JMJD3/KDM6B in promoting neuronal
differentiation. JMJD3/KDM6B is also induced upon macrophage terminal
differentiation (De Santa et al.,
2007) and is both necessary and sufficient for epidermal
differentiation in human keratinocytes
(Sen et al., 2008), hinting at
a general mechanism in which an H3K27me3 demethylase is activated to remove a
pluripotency-maintaining modification upon differentiation
(Fig. 2B).
Zebrafish embryos depleted of the H3K4me3 demethylase homolog, selected
mouse cDNA on the X (SMCX/KDM5C; also known as JARID1C), show decreased
neuronal survival during embryonic development
(Iwase et al., 2007), and rat
neuronal cultures depleted of SMCX/KDM5C develop defects in dendritic growth
(Iwase et al., 2007),
indicating a potentially specific role for this demethylase in neuronal
development. Consistent with this, mouse studies have also identified the
enriched expression of SMCX/KDM5C in the hippocampus, hypothalamus, cerebellum
and in differentiated neurons (Xu et al.,
2008). SMCX/KDM5C also appears to play a role in the genetic
disorder X-linked mental retardation (XLMR), as mutations that compromise its
demethylase activity are associated with this disorder
(Iwase et al., 2007;
Tahiliani et al., 2007)
(reviewed by Agger et al.,
2008; Cloos et al.,
2008; Shi,
2007).
Given the shared involvement of H3K4me3 and H3K27me3 in Hox gene regulation
and neural differentiation, it is possible that neuronal lineage-specific
genes, such as Dlx5, show other regulatory traits in common with Hox
genes, including the bivalent domain chromatin architecture. Interestingly, a
recent study also identified a non-coding RNA, HOTAIR, in the regulation of
Hox gene transcription (Rinn et al.,
2007). Whether and how the action of histone-modifying enzymes is
coordinated with that of non-coding RNAs in the regulation of the epigenetic
states of Hox loci is an exciting area of future investigation.
Demethylases in organogenesis
Epigenetic regulation clearly plays a role in the earliest stages of
development, as discussed above, but there is also a need for the stable
patterning of gene regulation as tissues and organs are specified. The
regulation of histone methylation at subsets of lineage-specific genes is one
way that transcription networks can be guided during development. Methylation
appears to be a relatively stable histone modification, and the active
addition or removal of this mark might very well play important roles in
stably maintaining cellular identity during development and maintenance of
organ identity. Not surprisingly, in addition to more general roles early in
development, some demethylases also appear to have tissue-specific roles in
later development.
Studies of pituitary organogenesis support a role for LSD1/KDM1 as a
regulator of differentiation later in development
(Wang et al., 2007). Given the
early embryonic lethality of mouse LSD1/KDM1 gene knockouts, the authors
created a tissue-specific pituitary knockout (because of the high LSD1/KDM1
levels of expression in the adult pituitary) to explore the role of LSD1/KDM1
in organogenesis (Wang et al.,
2007). Pituitary gland development was grossly normal in these
mutants, but they lacked certain terminal differentiation- and
cell-type-specific markers, suggesting a role for LSD1/KDM1 in late
differentiation events (Wang et al.,
2007). During anterior pituitary development, the growth hormone
(GH)-secreting somatotrope cells and the prolactin-secreting lactotrope cells
arise from a common lineage (reviewed by
Rhodes et al., 1994). The
Gh gene is a somatotrope lineage marker that is repressed in
lactotropes. In the activated somatotrope condition, LSD1/KDM1 and the H3K4me3
KMT MLL3/KMT2C were found to be co-recruited to the Gh promoter. Upon
lactotrope specification (and Gh repression), LSD1/KDM1 co-localizes
with the ZEB1-CtBP-CoREST (RCOR2) repressive complex
(Wang et al., 2007). These
data suggest that LSD1/KDM1 is constitutively present on certain promoters,
where it acts as an integral component of both differentially recruited
co-activator and co-repressor complexes. Further studies are required to
address whether and/or how LSD1/KDM1 demethylase activity is modulated in the
context of the various protein complexes it associates with.
LSD1/KDM1 also plays a role in hematopoietic differentiation through
interactions with the growth factor-independent (Gfi) proteins
(Saleque et al., 2007), which
are known to promote the expression of lineage-specific genes. LSD1/KDM1
interacts with GFI1B and is present on 80% of GFI1B binding sites
(Saleque et al., 2007). The
RNAi depletion of LSD1/KDM1 impaired the differentiation of several different
mouse hematopoietic cell types, including erythroid and megakaryocyte cells
(Saleque et al., 2007).
Surprisingly, LSD1/KDM1 depletion actually induced spontaneous granulocyte
differentiation (Saleque et al.,
2007), suggesting that it has lineage-specific activities.
However, LSD1/KDM1 appears to act predominantly in a pro-differentiation
manner in these assays.
The KDM6 family (which includes UTX, UTY and JMJD3 in mammals), as
discussed above, has a role in Hox gene regulation. At least one member of
this enzyme family has a role in C. elegans gonadal development as
well (Agger et al., 2007).
During C. elegans larval development, the distal tip cells of the two
developing gonad arms lead the migration/elongation of the gonad arms, first
distally, away from a mid-ventral location, then proximally back to a mid-body
location. Each arm thus forms a U-shape, at which point the gonad is fully
formed (Schedl, 1997). Defects
in this process can signal pathfinding and/or proliferation defects. Two
independent mutations of one of the C. elegans JMJD3/KDM6B homologs,
F18E9.5, lead to defects in gonad migration and oocyte accumulation
(Agger et al., 2007). It is
presently unclear which pathways underlie these phenotypes, but F18E9.5 is an
active H3K27me3 demethylase (Agger et al.,
2007), indicating a potential connection between H3K27 methylation
regulation and this phenotype.
The C. elegans vulva is a valuable model in which to study
organogenesis (reviewed by Sternberg,
2005; Sternberg and Horvitz,
1991). As such, studies of the severe vulval defect in KDM5 C.
elegans mutants (Christensen et al.,
2007) might uncover general mechanisms for these H3K4me3
demethylases in development. C. elegans has only a single KDM5
homolog, rbr-2, which is responsible for global levels of H3K4me3 in
later larval stages and adulthood, and the purified protein possesses in vitro
H3K4me3 demethylase activity (Christensen
et al., 2007). rbr-2 mutants show a highly penetrant
(80%) phenotype of either undeveloped or multiple vulvas, a phenotype
reminiscent of the synthetic multivulva (synMuv) genes
(Fay and Han, 2000;
Sternberg, 2005). Given that
many of the synMuv genes are homologous to those involved in the mammalian
tumor suppressor and cell cycle regulator pRB/E2F complex (which RBP2/KDM5A
was originally identified to associate with), the authors genetically
addressed a role for rbr-2 in this pathway using various synMuv
mutants (Christensen et al.,
2007). rbr-2(RNAi) or rbr-2 double mutants
showed no enhancement of phenotype with either class of synMuv mutant
(Christensen et al., 2007).
However, in the genetic background of the sensitized synMuv mutant
lin-15(n765ts), rbr-2 depletion dramatically enhanced the multivulva
phenotype at the permissive temperature
(Christensen et al., 2007),
showing a potential role for rbr-2 in this pathway, although its
precise position needs to be determined.
Histone demethylases: key regulators of nuclear hormone signaling?
Hormone signaling plays an important role in animal development and
differentiation (reviewed by van der Burg
et al., 1999). Importantly, several histone demethylases have been
shown to participate in hormone receptor (HR)-mediated transcription,
particularly androgen receptor (AR) and estrogen receptor (ER) signaling,
hinting that they have important roles in the various tissues that express
these proteins. For example, before its demethylase activity was reported, the
H3K4me3 demethylase RBP2/KDM5A was shown to associate with several nuclear
HRs, including ER and retinoic acid receptor (RAR), and to enhance their
transcriptional activities (Chan and Hong,
2001). Exactly how an H3K4me3 demethylase helps transcriptional
activation remains to be determined. Many lines of evidence also support a
role for LSD1/KDM1 in HR-mediated transcription (as discussed below).
Although LSD1/KDM1 was initially identified as an H3K4me2/1-specific
demethylase (Shi et al.,
2004), later studies also identified a role for LSD1/KDM1 in
demethylating H3K9me2/1 when it is associated with the AR
(Metzger et al., 2005) and
other nuclear hormone receptors
(Garcia-Bassets et al., 2007),
although in vitro biochemical evidence of mammalian LSD1/KDM1 having an
H3K9me2/1 activity is still lacking. Additional demethylases were soon
identified as being associated with LSD1/KDM1 and/or the AR complex and as
participating in AR-mediated transcriptional regulation. For example, both
LSD1/KDM1 and the H3K9me2-specific demethylase JHDM2A/KDM3A were found to
co-regulate the AR-responsive genes encoding prostate-specific antigen (PSA;
also known as KLK3) and NK3 transcription factor, locus 1 (NKX3.1)
(Yamane et al., 2006). The
closely related putative demethylase JMJD1C was originally identified as a
thyroid hormone receptor-interacting protein
(Lee et al., 1995), and later
shown to act as an AR co-activator (Wolf
et al., 2007). The H3K9/K36me3/2 demethylase JMJD2C/KDM4C was also
found to cooperatively participate in LSD1/KDM1 activation of AR-responsive
genes (Wissmann et al., 2007),
highlighting the importance of collaboration between me3- and me2-specific
KDMs in regulating histone methylation and the expression of genes that are
important for tissue-specific function, such as PSA in prostate tissue.
LSD1/KDM1 also participates in ER-mediated gene activation, as shown by its
presence at ER target genes and by its requirement for ER ligand stimulation,
where it participates in H3K9me2 demethylation
(Garcia-Bassets et al., 2007).
A recent study of LSD1/KDM1 in ER-mediated activation has uncovered
fascinating and unexpected evidence that links histone demethylation by
LSD1/KDM1 to a localized form of DNA damage that triggers chromatin
conformation changes that promote transcription
(Perillo et al., 2008)
(Fig. 3), a mechanism that
might prove to be relevant for the activities of other demethylases at nuclear
hormone receptors.
|
). The authors found that LSD1/KDM1 is continuously present at
these loci and is responsible for H3K4me2 demethylation, but only demethylates
H3K9me2 upon ER stimulation (Perillo et
al., 2008). Unexpectedly, they discovered that LSD1/KDM1 activity
was required for the DNA looping process
(Perillo et al., 2008). The
authors investigated whether the chemical by-product of the demethylase
reaction, hydrogen peroxide, might act as a signal by creating an 8-oxoguanine
modification of the DNA (Perillo et al.,
2008). Further experiments determined that estrogen treatment
induces 8-oxoguanine foci in cells in an ER- and LSD1/KDM1-dependent manner,
and that an 8-oxoguanine-specific glycosylase (OGG1) and topoisomerase
IIβ are both recruited under these conditions
(Perillo et al., 2008).
LSD1/KDM1 therefore appears to play a role in ER-induced intragenic movement,
bringing elements within the same gene into close proximity. A recent study
has reported a role for LSD1/KDM1 in modulating ER-induced chromosomal
movement into the interchromatin granules, where active transcription is
facilitated by the existing splicing machinery, transcriptional elongation
factors and chromatin remodeling activities
(Hu et al., 2008), hinting at
a potential role for demethylases in modulating both small-scale and
large-scale movements of chromatin in response to HR signaling.
A possible difference between differentiation-induced gene regulation and
HR signaling is that the demethylases are constitutively present on HR target
genes, but demethylase-influenced transcriptional activity seems to be subject
to rapid and transient regulation, possibly by protein-protein interactions
and/or post-translational modifications
(Fig. 3). For example,
LSD1/KDM1 is constitutively present at the ER target genes examined but only
demethylates H3K9me2 in the presence of ER signaling
(Perillo et al., 2008). By
contrast, during differentiation, KDM functions appear to be regulated
predominantly by recruitment mechanisms, such as through the recruitment of
UTX/JMJD6A to the Hox genes during ES cell differentiation
(Lan et al., 2007a). It is
tempting to speculate that this difference contributes to the relative
permanence of methylation patterns after differentiation, as opposed to the
transient nature of HR-induced gene regulation.
Conclusion
From the earliest developmental stages, demethylases have been implicated
in the decision to maintain pluripotency or to commit to differentiation (see
examples in Fig. 2), and these
studies have hinted at wider questions that surround histone demethylation
regulation. A comparison of the roles of KDMs in regulating pluripotency genes
versus lineage-specific genes shows some intriguing differences. Before
differentiation, the H3K9 KDMs act to maintain pluripotency genes in an active
state, and upon differentiation their activity is reduced and the pluripotency
genes become silenced, presumably through contributions of H3K9 KMTs and
H3K4me KDMs (Fig. 2A). This
model suggests that a KDM-influenced switch occurs between H3K4me and H3K9me.
The H3K4me3/H3K27me3 bivalent domain that is present in ES cells on the
lineage-specific Hox genes appears to undergo a different mode of KDM
regulation. Specifically, both H3K4me3 and H3K27me3 KDMs are expressed in ES
cells and are either absent or present at the target gene promoters at a low
frequency in order to protect the `poised' genes from inappropriate activation
(Agger et al., 2007;
Lan et al., 2007a;
Lee et al., 2007b;
Pasini et al., 2008) (see
model in Fig. 2B). Upon
differentiation, an unknown mechanism acts to resolve the bivalent domain to
either H3K4me3 or H3K27me3 through coordinate regulation of the appropriate
KMTs and KDMs. These studies have primarily been performed in cell culture,
but the C. elegans organogenesis studies suggest that KDM gene
regulation is important for multiple aspects of development, although
experiments connecting these proposed molecular mechanisms to the phenotypic
observations remain to be done.
Histone demethylases have only been identified and studied for four years,
but much has already been uncovered about their important roles in development
and disease. However, many questions remain. Specifically, understanding how
these demethylases are recruited to target genomic locations and how their
activities are regulated is of great importance. Some regulation occurs at the
level of demethylase gene expression, but in those cell types in which the
demethylase is expressed, there are presumably multiple layers of regulation
required to control where the enzymatic activity is directed. Recruitment
might involve sequence-specific DNA factors and non-coding RNAs. The local
chromatin environment might also contribute to effective recruitment by
regulating the stability of protein occupancy (reviewed by
Lan et al., 2008). Regulation
of demethylase activity may involve post-translational modifications,
non-coding RNAs and/or associated partners. These types of regulation might
play a role in modulating the H3K4me2/1 to H3K9me2/1 switch in demethylase
activity for LSD1/KDM1, which occurs upon its association with the AR.
Biochemical purification of the known demethylases from various cell types
will help to initiate studies that will ultimately provide mechanistic
insights into their recruitment and regulation.
Another question concerns whether histone demethylases exist that target
H4K20me and H3K79me. A recent study in yeast suggesting the non-processivity
of the only known H3K79me KMT, Dot1, along with the apparent functional
redundancy of the three H3K79me states, argues against the existence of
H3K79me KDMs (Frederiks et al.,
2008). However, like the other methyl modifications with known
KDMs, H4K20me does show differing distributions of mono-, di- and tri-methyl
states (Karachentsev et al.,
2007) and is methylated by SET-domain-containing KMTs. Therefore,
at present there is no compelling argument against the existence of H4K20me
KDMs. Several jumonji-domain-containing proteins have no known substrates but
are believed to be enzymatically active
(Shi and Whetstine, 2007;
Takeuchi et al., 2006);
perhaps one of these or a new chemical class of demethylases will prove to be
H4K20me-specific.
Finally, much has been achieved in vitro and in cell culture towards understanding the enzymatic activities of these demethylases, but the truly illuminating studies will be those that seek to understand their roles and mechanism of action in the context of the whole organism. Work has already been done in many model systems that has substantially increased our understanding of the biological roles of these enzymes, and depletion or mutant studies for the as yet undiscovered histone demethylases will no doubt prove equally fruitful.
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-ketoglutarate] are believed to act coordinately to hydroxylate the
methyl group. The reaction then proceeds analogously to a LSD1/KDM1
demethylation, with the unstable carbinolamine group being spontaneously
released as formaldehyde. The JmjC domain is capable of demethylating mono-,
di- and tri-methylated substrates.
