Histone Lysine Demethylases and Their Impact on Epigenetics

Article (PDF Available)inCell 125(2):213-7 · May 2006with25 Reads
DOI: 10.1016/j.cell.2006.04.003 · Source: PubMed
Chromatin establishes, maintains, and propagates different patterns of gene expression by storing and organizing genetic information. Histone lysine methylation has been regarded as a stable chromatin modification that together with DNA methylation defines epigenetic programs. Epigenetic phenomena are responsible for the non-Mendelian inheritance of phenotypic alterations. The recent discovery of histone lysine demethylases that reversibly remove methyl marks appears to challenge the epigenetic potential of histone lysine methylation. However, we argue that the reversibility of histone lysine methyl marks does not jeopardize their epigenetic status. We also suggest that not all histone lysine methylation residues are equally reversible and argue that two such residues—present exclusively in multicellular organisms—play important roles in establishing cellular identity.


Leading Edge
Cell 125, April 21, 2006 ©2006 Elsevier Inc. 213
Chromatin establishes, maintains,
and propagates different patterns of
gene expression by storing and orga-
nizing genetic information. Histone
lysine methylation has been regarded
as a stable chromatin modification
that together with DNA methylation
defines epigenetic programs. Epi-
genetic phenomena are responsible
for the non-Mendelian inheritance of
phenotypic alterations. The recent
discovery of histone lysine demeth-
ylases that reversibly remove methyl
marks appears to challenge the epi-
genetic potential of histone lysine
methylation. However, we argue
that the reversibility of histone lysine
methyl marks does not jeopardize
their epigenetic status. We also sug-
gest that not all histone lysine meth-
ylation residues are equally reversible
and argue that two such residues—
present exclusively in multicellular
organisms—play important roles in
establishing cellular identity.
The Complexity of the Histone
Methylation Machinery
“Lower eukaryotes (unicellular
organisms) usually have a relatively
short life cycle, a simple genome
organization and a profile of gene
expression that must adapt rapidly
to environmental cues. An extraor-
dinary step during evolution was the
development of multicellular organ-
isms with their increased genome
size and complexity. This complexity
required the transmission of active”
and “repressed” genetic information
to the next generation. Multicellular-
ity also necessitated that different
cells of an organism became spe-
cialized for certain tasks. Therefore,
a system had to evolve that would be
capable of organizing cellular differ-
entiation (and establishing cellular
identity) through early development
and that maintained a cell’s given
identity in adulthood. This system
is found in the complicated machin-
ery that regulates the chromatin
of higher eukaryotes, in which the
majority of chromatin is condensed
The number of methyltransfer-
ases that add methyl groups to
lysine residues increases dramati-
cally from lower to higher eukary-
otes. Reecting the difference in
global chromatin organization, the
number of methylation sites on
the lysines (K) of histones (H) also
increases from yeast to human.
Methylation marks that are linked
to open chromatin and transcrip-
tional activation (H3K4, H3K36, and
H3K79) are present in all eukary-
otes. In contrast, enzymes target-
ing methylation sites characteris-
tic of condensed chromatin and
transcriptional repression (H3K9,
H3K27, and H4K20) are not pres-
ent in the budding yeast, Saccha-
romyces cerevisiae. Moreover, his-
tone lysine residues can be mono-,
di-, or trimethylated, and a picture
has begun to emerge in which dif-
ferent degrees of methylation on
one particular site could be linked
to different functional outcomes.
Generally, in lower eukaryotes all
three degrees of methylation on a
particular histone methylation site
are regulated by the same enzyme,
whereas in higher eukaryotes his-
tone lysine methyltransferases have
been identied that control only one
degree of methylation. This adds an
additional layer of complexity to the
chromatin regulatory functions of
histone lysine methyltransferases
in higher eukaryotes.
Histone lysine methylation seems
to be an anomaly among histone
modifications. Contrary to other
modifications, the global turnover of
lysine methylation is low, suggest-
ing that the modification is stable.
Until recently, there was little doubt
that stably methylated histone lysine
residues contributed to the estab-
lishment and propagation of differ-
ent patterns of gene expression in
the same genome. Thus, methylated
histone lysine residues have been
considered “epigenetic marks” (Jen-
uwein and Allis, 2001).
Histone Lysine Methylation Is
Although researchers sought evi-
dence for enzymatic demethylation
of histone lysine residues, the tech-
nical difculties in setting up an in
vitro assay for this type of demeth-
ylase activity hindered progress. In
the absence of evidence for histone
lysine demethylases, the appar-
ent loss of histone methyl marks
Histone Lysine Demethylases and Their
Impact on Epigenetics
Patrick Trojer
and Danny Reinberg
Howard Hughes Medical Institute, Department of Biochemistry, Division of Nucleic Acids Enzymology, Robert Wood Johnson
Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854, USA
*Contact: reinbedf@umdnj.edu
DOI 10.1016/j.cell.2006.04.003
Methylation marks on the lysine residues of histone proteins are thought to contribute
to epigenetic phenomena in part because of their apparent irreversibility. Will this view
change with the recent discovery of histone lysine demethylases that reversibly remove
methyl marks?
214 Cell 125, April 21, 2006 ©2006 Elsevier Inc.
was explained either by “passive”
demethylation (that is, every round
of replication “dilutes” the total
number of modified histones) or by
histone exchange. Identification of
the first enzyme responsible for his-
tone lysine demethylation changed
this view.
A groundbreaking study reported
the discovery of H3K4 demethylation
by the amine oxidase family member
LSD1 (Shi et al., 2004). This enzyme
removes with remarkable specificity
one or two methyl groups from H3K4
(H3K4me1/2) but cannot attack tri-
methylated H3K4 (H3K4me3). Sur-
prisingly, LSD1 was also reported
to demethylate H3K9 (H3K9me1/2)
upon interaction with the andro-
gen receptor (Metzger et al., 2005).
(Several excellent review articles
provide a detailed discussion of the
chemistry and substrate specificity
of LSD1 [Bannister and Kouzarides,
2005; Kubicek and Jenuwein, 2004]).
Recently, it was proposed that a fam-
ily of proteins containing Jumonji C
(JmjC) domains demethylates his-
tone lysine residues (Trewick et al.,
2005), and an activity called JHDM1A
that specifically catalyzes demeth-
ylation of H3K36 (H3K36me1/2)
has been purified (Tsukada et al.,
2005). The same group has puri-
fied a homologous JmjC protein that
demethylates H3K9me2 resulting
in unmodified H3K9 (Yamane et al.,
2006). Meanwhile, others have dis-
covered demethylation of H3K9me3:
ectopic expression of the Jmjd2b
protein resulted in a global reduction
of H3K9me3 in vivo (T. Jenuwein,
personal communication). More-
over, various JMJD2 proteins were
found to antagonize H3K9me3 and
H3K36me3 both biochemically and
in vivo, resulting in H3K9me1/2 and
H3K36me1/2, respectively (Whets-
tine et al., 2006). The mammalian Jmj
protein family is large, and it is likely
that there are other histone lysine
demethylases that await discovery.
The identification of histone lysine
demethylases raises the possibility
that all three degrees of methylation
at these sites could be reversible.
Although the discovery of his-
tone lysine demethylases would be
predicted to impact the epigenetic
potential of histone lysine methyla-
tion, we believe it does not. These
are the reasons why: (1) demethyl-
ation of histone lysine residues does
not change the fact that some meth-
ylation marks seem to be very stable
and exhibit low turnover rates (at least
in restricted regions of chromatin or
in certain cellular states). Therefore,
expression and/or activity of histone
lysine demethylases must be tightly
regulated in differentiated cells. (2)
There is accumulating evidence that
Figure 1. Methylation of Histone Lysines
(Top) The enzymes that methylate and demethylate lysine (K) residues of histones H3 and H4 are shown. In higher eukaryotes, methylation of H4K20
(H4K20me1) is catalyzed exclusively by the methyltransferase PR-SET7 (the SUV4-20H1/2 enzymes are responsible for H4K20me2/3). H3K27
methylation (H3K27me, mediated by EZH1/2) and H4K20me1 (an autonomous mark independent from H4K20me2/3) are marks not found in unicel-
lular organisms but which rather appeared with the emergence of multicellularity. Histone demethylases of the LSD1/BHC110 family are absent in
the budding yeast Saccharomyces cerevisiae but are present in the fission yeast Schizosaccharomyces pombe. Certain proteins containing Jumonji
(Jmj) domains, which are conserved from yeast to human, have histone demethylase activity. DOT1 is the only enzyme responsible for methylating
H3K79, a methyl mark that is associated with maintaining open chromatin. H3K79me is also present in S. cerevisiae, and so we speculate that the
epigenetic potential of H3K79me is different from that of H4K20me1 and H3K27me. (Bottom) The transmission of the epigenetic histone methyl
marks H4K20me1 and H3K27me from parental to daughter chromosomes. (Bottom, left) The model proposes that H3K27me marks are transmitted
during DNA replication. A Polycomb dimer binds to H3K27me on the parental chromatin and an unmethylated H3 tail from the newly synthesized
chromatin. EZH2, the H3K27-specific histone lysine methyltransferase, is recruited and methylates H3K27 on the daughter strand. (Bottom, right)
DNA is replicated in S phase, but H4K20me1 is not transmitted to newly synthesized chromosomes before mitosis. PR-SET7, the H4K20me1-specific
histone lysine methyltransferase, is only expressed during mitosis. The enzyme directly (or indirectly through interaction with an unknown H4K20me1
binding protein) recognizes H4K20me1 on the parental chromosome and methylates the appropriate position on the daughter chromosome.
Cell 125, April 21, 2006 ©2006 Elsevier Inc. 215
DNA methylation is reversible yet its
epigenetic potential is not in dispute.
DNA methylation is dependent on
histone lysine methylation in various
cases (Tamaru and Selker, 2001).
Therefore, histone methylation marks
may influence epigenetic memory
formation indirectly by regulating
DNA methylation. (3) We still know
very little about the biology of histone
lysine demethylases. Many chroma-
tin regulatory proteins are dynamic
and are constantly recruited, bound,
and ejected from chromatin. Are his-
tone lysine demethylases also highly
dynamic or are they stably bound to
chromatin to preserve a demethyl-
ated state? (4) We need to assess
the plasticity of histone methyl marks
during development (for example, by
genome-wide ChIP-on-chip analysis
for each lysine methylation site and
for all degrees of methylation). More-
over, analyses must be performed at
different developmental stages (e.g.,
zygote, blastula, gastrula) neces-
sitating technical advances to deal
with such limited material. However,
these techniques are not expected
to detect epigenetic information that
is restricted to limited but develop-
mentally important regions of chro-
matin. (5) There is accumulating evi-
dence that one methyl mark by itself
might have only a limited biological
message. For instance, H3K9me3,
originally considered a hallmark of
constitutive heterochromatin, was
recently reported to be present at
actively transcribed genes. There-
fore, although H3K9me3 by itself
might not correlate directly with tran-
scriptional state, in combination with
other histone marks, it might still con-
tribute to the nal outcome. In gen-
eral, the spatial and temporal context
of histone lysine marks seem to be
important. (6) Thus far, only a small
number of residues (H3K4me1/2,
H3K36me1/2, and H3K9me1/2/3) are
antagonized by a restricted number
of demethylases (see Figure 1). It is
likely that demethylases targeting
other histone lysine residues will be
discovered in the near future, but it
is also possible that some histone
methyl marks are not targeted by
demethylases at all.
Transmission of Epigenetic
Histone Lysine Marks
The crux of an epigenetic mechanism
involves the transmission of infor-
mation via the germline to the next
generation of a multicellular organ-
ism. We still do not fully comprehend
how histone methylation marks are
maintained throughout the cell cycle,
or how epigenetic marks are trans-
mitted through the germline to the
next generation. Although potentially
every histone lysine methyl mark pro-
vides epigenetic information, here we
provide a model of how two specific
histone lysine methyl marks could be
transferred to daughter cells.
The two methyl marks, H3K27me
and H4K20me1, are each established
by one enzyme, EZH2 and PR-SET7,
respectively (other methyl marks may
be established by several enzymes)
(see Figure 1). EZH2 and PR-SET7
emerged at the same time as multi-
cellularity. Moreover, H3K27me and
H4K20me1 are not known targets of
demethylases, and both marks are
present on the inactive X chromo-
some. Transcriptional inactivation of
the X chromosome is an excellent
system with which to study epigene-
tic mechanisms, because one X chro-
mosome must remain silent in all cell
types and throughout all cell divisions
of female mammals. We propose that
H3K27me and H4K20me1 may have
limited reversibility and could be used
to transmit epigenetic information.
An important property for an epi-
genetic histone lysine methyl mark
is that it has to be established and
maintained throughout the cell cycle.
Monomethylation of H4K20 fulfills
several criteria for an epigenetic mark.
The expression of PR-SET7, which
monomethylates H4K20, is strictly
regulated during the cell cycle, being
detectable only during late G2 and
early M phase. The H4K20me1 mark
is present throughout the cell cycle,
suggesting that it is not removed in
interphase cells that do not have PR-
SET7 to replace it. This also suggests
that H4K20me1 is not antagonized by
histone demethylation. Persistence
of H4K20me1 was also observed in
Drosophila embryos lacking PR-Set7.
In these embryos, the maternally
deposited modification is present
until late larval stages, at which point
defects in proper cell division appear
resulting in lethality (Karachentsev et
al., 2005). These data suggest that
H4K20me1 is not erased by histone
lysine demethylases during early
embryonic development. Mitosis is the
final stage for propagating epigenetic
information from parental chromatin
to newly generated chromatin prior
to chromosome segregation and cell
division. It has been proposed that
the physical association of PR-SET7
with mitotic chromosomes places it at
the appropriate position for transmit-
ting H4K20me1 marks from mother to
daughter cells (Reinberg et al., 2004).
This epigenetic mechanism would
necessitate that PR-SET7 somehow
recognizes H4K20me1 on the mother
chromosomes and then “writes” the
same mark on corresponding posi-
tions on the daughter chromosomes
(see Figure 1; D.R. and P.T., unpub-
lished data).
Methylation of H3K27 is performed
solely by EZH2, the mammalian
homolog of the Drosophila protein Ez
(enhancer of zeste). Ez is a member
of the Polycomb (PcG) protein fam-
ily and is crucial for the maintenance
of transcriptional repression of the
developmentally important homeotic
(Hox) genes. Polycomb- and Tritho-
rax-group proteins are known to be
key regulators of proper embryonic
development and are important play-
ers in maintaining cellular identity
established early during development
in multicellular organisms. The finding
that PcG proteins stabilize long-term
transcriptional silencing of homeotic
genes provided the first evidence for
a molecular mechanism of “cellular”
or “epigenetic” memory (Rastelli et
al., 1993). EZH2 executes its histone
lysine methyltransferase activity only
as a component of various multi-
protein complexes ((Reinberg et al.,
2004). Collectively, there is compelling
evidence that EZH2 plays an impor-
tant epigenetic role in establishing
cellular identity and that this function
is ultimately linked to its H3K27 his-
tone lysine methyltransferase activ-
ity. Moreover, EZH2 directly interacts
with DNA methyltransferases in vitro.
216 Cell 125, April 21, 2006 ©2006 Elsevier Inc.
RNAi against EZH2 resulted in a loss
of DNA methyltransferases and CpG
methylation of target genes in vivo
(Vire et al., 2005). Therefore, EZH2
also might have an indirect impact on
epigenetic phenomena by regulating
DNA methylation.
H3K27me3 is impor tant in
imprinted and random X chromo-
some inactivation (Heard, 2005).
Imprinted paternal X chromosomes
are loaded with H3K27me2/3 dur-
ing preimplantation stages and are
maintained in extraembryonic cells,
but the methyl mark is then lost in
the inner cell mass of the embryo at
the blastocyst stage. These changes
in the status of H3K27 methyla-
tion reflect a general plasticity of
this modification and indicate that
an H3K27-specific histone lysine
demethylase might be expressed in
embryonic cells. H3K27me3 is also
observed during random X chro-
mosome inactivation in embryonic
stem cells upon their differentiation.
Although the mark is dependent on
the expression of Xist, a noncoding
RNA that coats the inactivated X
chromosome during the early stages
of differentiation, at late stages the
K27 methyl mark persists even when
Xist expression is blocked (Kohlma-
ier et al., 2004). This corroborates
the role of H3K27me3 as an epigen-
etic mark and suggests that either
a putative H3K27-specific histone
lysine demethylase is not recruited
to the inactive X chromosome or
that it simply does not exist in differ-
entiating embryonic stem cells.
The mechanism by which the
H3K27 methyl mark is transferred
from mother to daughter chromo-
somes is still unclear. However, in
contrast to the case of PR-SET7
where the factor recognizing and
binding to H4K20me1 remains to
be discovered, Polycomb has been
identified as the binding protein for
the H3K27me2/3 mark. Polycomb,
a component of the PRC1 complex,
binds as a dimer to two histone
H3-tails simultaneously; structural
studies demonstrate that the bound
tails are in close proximity (Min et
al., 2003). For steric reasons, it is
unlikely that the two H3 tails come
from the same nucleosome, and this
may provide a mechanism whereby
a Polycomb dimer (or PRC1) could
compact chromatin by binding
simultaneously to two nucleosomes.
We speculate that this mechanism
may transmit the epigenetic H3K27
methyl mark from parental chroma-
tin to newly synthesized chromatin
(see Figure 1). The PRC1 complex
(and Polycomb) binds to unmodified
(or tail-less) nucleosomes (Francis et
al., 2004). In vitro binding studies of
Polycomb and histone H3 tails sug-
gest that H3K27 methylation only
facilitates binding. Therefore, it is
possible that the Polycomb protein
can bind to unmethylated histone
H3 tails. Given that transient inter-
actions between the EZH2 complex
and Polycomb have been reported
(Poux et al., 2001), EZH2 may be
recruited as the “epigenetic indexer
of Polycomb bound histone H3 tails
of daughter chromosomes. Alterna-
tively, DNA-specific binding factors
like zeste or GAGA (components of
PRC1) could mediate transient inter-
actions between PRC1 and EZH2
complexes (Mulholland et al., 2003).
Which stage of the cell cycle then
is selected to transmit the H3K27
methyl mark? EZH2 and its asso-
ciated polypeptides are target
genes of the E2F transcription fac-
tor, and many targets of this fac-
tor are expressed at the transition
from G1 to S phase. Moreover, ezh2
knockdown by RNAi causes severe
defects in cell proliferation, suggest-
ing a role in cell cycle progression.
Recently, binding of the EZH2 com-
plex and PRC1 to the inactivated X
chromosome was shown to be highly
dynamic and cell cycle stage depen-
dent, occurring primarily in early- to
mid-S phase (Hernandez-Munoz et
al., 2005). Collectively, these studies
suggest that, within the context of
our proposed epigenetic mechanism
for the inheritance of the K27 methyl
mark, this mark would likely be
placed during S phase immediately
after DNA replication (see Figure 1).
The major issue is our limited knowl-
edge regarding how Polycomb-group
proteins become recruited in the first
place. In Drosophila, Polycomb-
responsive elements (PRE) consti-
tute specific DNA sequences located
several kilobases upstream of the
transcriptional start sites of the Hox
genes. These PREs are targeted by
Polycomb-group proteins that bind
to DNA and then recruit the EZH2
complexes and PRC1. Surprisingly,
repression by PREs can affect genes
over wide distances in the genome,
and these elements somehow seem
to interact with each other. These
remarkable ndings have led to a
model during replication in which the
PRE bound Polycomb-group com-
plexes loop toward and bind to the
PREs on newly synthesized daughter
chromosomes, thus propagating the
epigenetic marks (Pirrotta, 1998).
However, PRE sequences have not
been found in mammals as yet, and
so this model cannot explain the
recruitment of EZH2 complexes to
chromatin in mammalian cells.
We suggest that two histone lysine
methyltransferases, EZH2 and PR-
SET7, are important epigenetic
regulators given that their specific
substrate residues, H3K27me and
H4K20me1, remain stably methyl-
ated over several cell generations, at
least in particular chromatin regions.
EZH2 and PR-SET7 emerged with the
appearance of multicellularity and a
complex system to regulate cellular
identity. We propose that H4K20me1
and H3K27me are not erased by his-
tone lysine demethylases, at least
not at certain developmental stages.
Rather, H4K20me1 and H3K27me
may be pivotal epigenetic marks,
although we do not doubt that other
histone lysine methyl marks also con-
tain epigenetic information. Finally, we
should not forget that Waddington’s
epigenetic landscape (Waddington,
1957) comprises both peaks and val-
leys (that is, some regions with high
concentrations of epigenetic marks
and others that lack them). Thus, his-
tone lysine demethylases could be
epigenetic factors themselves if they
protect regions from being methyl-
ated, and thus from being converted
from “epigenetic” valleys to “epigen-
etic” peaks.
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    • "Most remarkable was the absence of K9me3 in nonnucleosomal H3.1 and H3.3 (Table S1). These data imply that H3K9me3 occurs at the time of or after chromatin assembly and, if evicted, demethylation occurs immediately (Trojer and Reinberg, 2006 ). Given the low proportion of evicted/stored histones in our experimental conditions , H3 PTMs on nonnucleosomal complexes most likely reflect H3 status prior to incorporation into DNA. "
    Full-text · Dataset · Dec 2015 · Toxicology in Vitro
    • "Most remarkable was the absence of K9me3 in nonnucleosomal H3.1 and H3.3 (Table S1). These data imply that H3K9me3 occurs at the time of or after chromatin assembly and, if evicted, demethylation occurs immediately (Trojer and Reinberg, 2006 ). Given the low proportion of evicted/stored histones in our experimental conditions , H3 PTMs on nonnucleosomal complexes most likely reflect H3 status prior to incorporation into DNA. "
    Full-text · Dataset · Sep 2015 · Toxicology in Vitro
    • "(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) whereas H3K4, H3K36 methylations have been associated with transcriptional activation and a more relaxed and open chromatin structure (Bannister and Kouzarides, 2005; Martens et al., 2005; Trojer and Reinberg, 2006; Wang et al., 2007). We have identified a significant increase in global H3K9me2 and H3K9me3 in NRK-52E cells exposed to 25, 50 and 100 lM of FB1 compared with vehicle controls. "
    [Show abstract] [Hide abstract] ABSTRACT: Fumonisin B1 (FB1) is a Fusarium mycotoxin frequently occurring in maize-based food and feed. Although the effects of FB1 on sphingolipid metabolism are clear, little is known about early molecular changes associated with FB1 carcinogenicity. It has been shown that FB1 disrupts DNA methylation and chromatin modifications in HepG2 cells. We investigated dose- and time-dependent effects of FB1 in global histone modifications such as histone H3 lysine 9 di-, trimethylation (H3K9me2/me3), histone H3 lysine 4 trimethylation (H3K4me3), histone H4 lysine 20 trimethylation (H4K20me3), histone H3 lysine 9 acetylation (H3K9ac) and the enzymes involved in these mechanisms in rat kidney epithelial cells (NRK-52E). The increased levels of global H3K9me2/me3 were observed in FB1 treated cells, while the global levels of H4K20me3 and H3K9ac were decreased. FB1 caused some changes on the activities of H3K9 histone methyltransferase (HMT) and histone acetyltransferase (HAT) at high concentrations in NRK-52E cells. Further, the effects of trichostatin A (TSA), a histone deacetylase inhibitor, were investigated in NRK-52E cells. TSA was found to cause an increase on H3K9ac levels as expected. In this study we suggest that FB1 may disrupt epigenetic events by altering global histone modifications, introducing a novel aspect on the potential mechanism of FB1 carcinogenesis. Copyright © 2015. Published by Elsevier Ltd.
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