SURVEY AND SUMMARY
A case study in cross-talk: the histone lysine
methyltransferases G9a and GLP
Robert Collins1,* and Xiaodong Cheng2,*
1Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT 06520 and
2Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA
Received September 4, 2009; Revised January 21, 2010; Accepted January 27, 2010
The histone code hypothesis predicts that the
bring about distinct chromatin states, and it there-
fore serves a key regulatory role in chromatin
biology. The impact of one mark on another has
been termed cross-talk. Some marks are mutually
exclusive, while others act in concert. As multiple
marks contributing to one outcome are generally
brought about by complexes containing multiple
catalytic and binding domains, it appears regulation
of chromatin involves a web of writers and readers
of histone modifications, chromatin remodeling
activities and DNA methylation. Here, we focus on
the protein lysine methyltransferases G9a and GLP
as examples of this extended cross-talk. G9a and
GLP can catalyze the formation of and bind to the
consider the impact of other histone modifications
activities within G9a/GLP containing complexes.
binding on product specificity and on maintenance
and propagation of the methyl mark. Lastly, we
examine the recruitment of other silencing factors
by G9a/GLP. Regulated assembly of specific com-
plexes around key marks may reinforce or alter the
biological outcome associated with given histone
post-translational modification of histones can directly
impact chromatin structure or can be read by effector
modules. Certain modifications have been correlated
with unique transcriptional outcomes and chromatin
states. Individually, the modifications of the histone
code are bound with weak affinity. Binding modules
with the same specificity are found in complexes with dif-
fering, and sometimes opposing, activities. For example,
G9a and GLP have been described as both co-repressors
(1–11) and co-activators (1,12,13). This has led some to
doubt the validity of the histone code hypothesis, as they
question the ability of a single modification to affect a
biological outcome (14–16). Multivalent interactions
have been proposed as the answer, where no single mod-
ification, but rather patterns of modifications and the
network of interacting proteins that can read and write
them are responsible for the regulation of chromatin
(17,18). We adopt this extended view, and we focus on
G9a/GLP as an example of this, because they feature
the ability to bind their own product and to serve as an
organizing hub for diverse activities.
The impact of one mark of the histone code on another
is frequently observed. With the structural and biochemi-
cal characterization of more of the players in histone mod-
ification, the underlying mechanisms of cross-talk, and the
interplay of histone cross-talk and other pathways, such as
chromatin remodeling and DNA methylation and repair,
are becoming clear (19–25). Mechanistically, cross-talk
occurs when one or more binding modules and catalytic
domains reside in the same complex or polypeptide,
allowing coordination of different activities. Cross-talk
can occur prior to catalysis, in which case the recognition
of one mark (or its absence) can serve to recruit an enzyme
to its substrate in the generation or removal of a second
mark. This is essential for targeting and coordination of
activities. We detail a second type of cross-talk, product
binding, where binding by a reader module follows catal-
ysis. G9a and GLP can bind their products via a domain
distinct from the catalytic domain. In other cases, product
binding has proved essential to maintain marks after their
*To whom correspondence should be addressed. Tel: +1 203 432 9841; Fax: +1 203 432 5767; Email: email@example.com
Correspondence may also be addressed to Xiaodong Cheng. Tel: +1 404 727 8491; Fax: +1 404 727 3746; Email: firstname.lastname@example.org
Published online 16 February 2010Nucleic Acids Research, 2010, Vol. 38, No. 113503–3511
? The Author(s) 2010. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
establishment or to spread the modifications. As sequence-
and structure-specific DNA-binding modules within the
modifier complexes may also play a large role in
contributing specificity, we also consider their role in
mediating gene-specific silencing in G9a/GLP complexes.
Product binding also allows for more complicated signal-
ing on chromatin, where a complex, bound to its product,
can act as a platform to integrate diverse signals.
G9a-like protein (GLP, also known as EuHMT1) and
G9a (also known as EuHMT2) form a heteromeric
complex, and the loss of either substantially reduces
mono- and dimethylation of H3K9, which is a marker
of silent euchromatin (10,26–28). G9a has also been
recognized for its ability to methylate histone H1.4 and
other non-histone proteins, including itself (29–32). G9a
and GLP bind their H3K9me1 and H3K9me2 products
via ankyrin repeat domains (33), which contain a
modules of diverse folds. This cage binds H3K9me1/2
with approximately equal affinity, but it is too narrow to
In G9a or GLP knockout cells, DNA methylation and
HP1 binding to euchromatin are lost (10,34). The
associated loss of silencing causes embryonic lethality,
and differentiation of ES cells in the absence of G9a
leads to apoptosis (26). G9a is required for the restriction
of cell fate in development, for the silencing of the
homeobox gene Oct3/4, which is required for pluripotency
(4), for NRSF/REST-mediated silencing of neuronal
genes in non-neuronal lineages (35), for PRDI-BF1
(Blimp-1) silencing in B-cell differentiation (5) and for
CDP/cut mediated silencing of genes involved in cell dif-
ferentiation and proliferation (8). The role of G9a/GLP as
a gatekeeper of differentiation has been demonstrated by
knockdown and with a novel inhibitor, BIX-01294
(36–39). In the absence of the repressive H3K9me1/2
mark, genes normally expressed only in stem cells are
induced, and cells are more readily reverted to pluripotent
The diverse functions of G9a/GLP appear to require
targeted to DNA is sufficient for transcriptional silencing
(9,40). Conversely, DNA methylation appears to require
the ankryin repeat domain, but not histone methyl-
transferase activity (34,41,42). Lastly, methyltransferase
activity has little or no effect on the transcriptional acti-
vation of nuclear receptor regulated genes, b-globin or
rDNAs by G9a (1,12,13). We consider potential roles
for G9a product binding (Figure 1), including competi-
tion with methyltransferase activity to restrict procession
to trimethylated H3K9, blocking the back modification
of the H3K9me1/2 marks by lysine demethylases,
spreading of the methyl mark akin to the Su(var)3-9/
HP1 system, and recruiting other proteins including
histone H1, HP1, DNA methyltransferases and transcrip-
The influence of other post-translational modifications
on G9a/GLP activity
Unlike certain other methyltransferases, such as Set1 or
Dot1 (43), G9a/GLP methylation is not dependent on
pre-existing marks, but merely the absence of modifica-
tions to the target lysine or adjacent residues is sufficient
for catalysis. This limited catalytic cross-talk in G9a/GLP
relates to their recognition of a fairly short stretch
(minimally H3T6-H3T11) of H3 for methylation (30).
Acetylated H3K9 and phosphorylated H3S10 or H3T11
arginine methylation at H3R8 blocks activity (30,44).
Product binding is blocked by phosphorylation of
Coordination with other activities
A few studies have probed the spatiotemporal relation-
ships of modifications during transcriptional activation
or silencing. One compelling study traced the silencing
promoter during differentiation of thymocytes and it
clearly established a temporal sequence of events from
active to silent chromatin (45). During the maturation
of the thymocytes, the promoter was stripped of
activating modifications by deacetylation of H3K9 and
demethylation of H3K4. The modifications were first
Conversely, silencing H3K9 methylation nucleated at the
promoter and spread over 10kb outward from the
promoter over time. Transcription was strongly reduced
with the loss of the activating modifications and fully
quenched when dimethylated H3K9 (H3K9me2) reached
methylation increased as the cells terminally differentiated.
Coincident with this process, the gene relocalized to a
region of silent heterochromatin. Interestingly, in a cell
line with reversible Dntt silencing, modifications were
nucleated at the promoter, but did not spread. Notably,
the study did not identify the H3K9 methyltransferases
involved and we include it as a fine example of the coor-
dination of silencing activities, but not of G9a/GLP
function. The developmental silencing of the Oct3/4
promoter follows a similar course, with loss of activating
modifications and gain of G9a/GLP-mediated H3K9
methylation, followed by association of HP1 and DNA
Similar coordination has been seen in other silencing
complexes. Small heterodimer partner (SHP), known for
its ability to silence transcription mediated by a number of
deacetylases (HDACs), a H3K9me2 methyltransferase
(G9a) and a silencing SWI–SNF family complex (3).
It appears that HDAC activity precedes histone lysine
methylation. H3K9me2, in turn, was required for
complex (3). Similarly, a RE1-silencing transcription
methyltransferases (G9a) is targeted to neuron-restrictive
ofitsmaximal value.Lastly,DNA CpG
3504 Nucleic Acids Research, 2010,Vol.38, No. 11
silencing elements (a conserved sequence in the promoters
of a subset of neural genes) (46). In this case, H3K4me3
deacetylation, which was followed by H3K9 methylation
(46). The next challenge is making clear the molecular
mechanisms that underlie these patterns.
Interplay of product binding with degree of methylation
Constitutive heterochromatin is distinguished by a high
facultative heterochromatin is primarily marked with
H3K9me1/2 and limited regions of H3K9me3. In vitro,
G9a rapidly generates both mono- and dimethyllysine,
but it more slowly proceeds to trimethylation (47,48).
Although the G9a and GLP knockouts most noticeably
reduce global H3K9me1 and H3K9me2, G9a may mediate
some trimethylation in vivo. Long residence at a target
site may allow the formation of trimethyllysine, as G9a
tethered to a synthetic mini-locus, as a Gal4 DNA-binding
domain fusion generates H3K9me3 in vivo (49) and G9a
trimethylates itself (29,30,32). In vivo, G9a-mediated
H3K9me3 has been detected, typically in small regions
near promoters. For example, in the absence of G9a,
H3K9me3 levels have been found to be reduced at the
Oct3/4 promoter, at the promoters of paternally imprinted
genes and at a host of other developmentally silenced
genes (4,42,50). H3K9me3 reduction at G9a-silenced pro-
moters (but not non-target promoters) is also observed
when G9a is chemically inhibited, though the knockout
of G9a or GLP had a slightly more potent effect in the
same study (38). Since G9a and GLP appear to bring
about multiple levels of methylation, potentially based
on the duration of their residency on a target, they must
strike a balance between proceeding to trimethylation and
restriction to lower methylation states. Of particular
interest is the distinction between the lower states of
H3K9 methylation (me1 and me2), which the G9a and
GLP ankyrin repeats can bind (33), and higher states
(me2 and me3), which are preferred by HP1 (51). All
three degrees of methylation bring about silencing, as
even a monomethylating mutant G9a (F1205Y) transgene
was able to silence target genes in G9a-null ES cells (47).
The ability to reverse a mark and reactivate transcription
also relates to the degree of methylation, as some
demethylases, such as JHDM2A
demethylate H3K9me1/2 but not H3K9me3 (52,53).
Other demethylases, such as those of the JMJD2 family
that can act on trimethylysine, would be required for
removal of the H3K9me3 mark (54–56).
One untested hypothesis is that the G9a and GLP
ankyrin repeats could compete with the non-processive
catalytic reaction, quenching the reaction at the mono-
and dimethyl state and protecting substrates from
trimethylation by competition with the catalytic domain.
Such competition could come into play whether G9a/GLP
themselves are responsible for catalyzing the H3K9me2 to
SETDB1) carries out this process. The ability to limit
methylation will be variable, as some G9a substrates will
make poor binding partners for the ankyrin repeats.
In particular, for ankyrin repeat binding, H3K9
through H3G13 were observed in the crystal structure.
A diglycine motif (H3G12 and H3G13) is required to
allow for a sharp turn and mutation to alanine reduced
binding (33). These features are largely dispensable for the
methylation, where only H3T6-H3T11 are required for
G9a/GLP SET domain activity (30). H3R8—which is
indispensable for methylation—and preceding residues
do not appear in the ankyrin repeat structure. Although
the substrate requirements for methylation have been
binding specificity of the ankyrin repeats comes from a
another enzyme(such as
reads H3K9me1, me2
H3 peptide H3K9me2
H3 peptide H3K9me0
Figure 1. Domain organization of G9a and GLP.
ankyrin repeats and the catalytic SET domain, which also mediates formation of heteromers (PDB codes 3B95 and 2RFI).
G9a and GLP consist of a N-terminal domain with automethylation site(s), the product-binding
Nucleic Acids Research, 2010,Vol.38, No. 113505
single target. It would be interesting to determine whether
the substrates that are not bound as products are more
prone to conversion to trimethylation, either by G9a/GLP
or another methyltransferase. Taken together, the histone
and non-histone methyltransferase activity of G9a and
GLP, potentially moderated by the product binding
demethylases, may allow a distinction between the ‘per-
manent’ silencing associated with imprinting or restriction
of cell fate, where HP1 dependent chromatin compaction
and DNA methylation are warranted, and more dynamic
silencing in cell types with plastic fates, metabolic
feedback (e.g. bile-acid metabolism) and cell signaling
through nuclear receptors (2,3,6,57).
G9a/GLP product binding and ‘gatekeeping’
A second possibility is that G9a and GLP bind their
products to protect the modification, competing with
(Figure 2). This ‘gatekeeper’ function has been suggested
by studies showing GLP or G9a, among several other
methyltransferases, restrict unliganded nuclear receptors
from activating transcription (57). Liganded nuclear
receptors require a partner demethylase to promote tran-
scription. This begs the question of whether it is the
methyl mark or the physical presence of the methyl-
transferase and associated complex that serves as the
gatekeeper. Treatment with estradiol decreased promoter
occupancy of several methyltransferases and the knock-
down of the methytransferase itself is sufficient to yield
transcriptional activity in the absence of ligand. This
suggests active, promoter specific competition between
methylases and demethylases. Sequestering methylation
through product binding could provide a physical means
of blocking removal of the mark. At the same time, the
physical presence of the methyltransferase complex could
blockpromoter access and
Additionally, since G9a/GLP heteromers could bind tails
of neighboring nucleosomes, substantial local cross-
linking could occur, contributing to compaction of local
chromatin (Figure 3).
Recruiting allies to reinforce silencing
H1.4. This role as a chromatin-compacting gatekeeper is
reinforced by the ability of G9a to recruit other proteins,
including histone H1.4, HP1 and C/EBP (CCAAT/
Enhancer-binding protein), to chromatin. Though there
are several H1 variants, they are generally silencing and
contribute to higher order chromatin structure. H1 posi-
tioning serves to block nuclesome repositioning and
sliding, and it occludes access of other proteins to DNA
[reviewed in (58)]. H1 variants appear to have distinct,
non-overlapping roles, and H1.4 participates in dynamic,
gene specific silencing (59). G9a methylates H1.4 at lysine
26 (H1.4K26), suggesting a cross-talk where the presence
of methylated H3K9 could recruit G9a to methylate
H1.4K26, or vice versa. G9a also appears to participate
directly in recruiting H1.4 to chromatin (59). It will be
interesting to determine if product binding is involved,
as the residues flanking K26 appear ideal for ankyrin
binding of H1.4K26me could facilitate its placement
Figure 3. Compaction
methylation. One member of the dimer, and one H3 tail are shown
for simplicity. Alternatively, G9a methylates H1.4, and facilitates its
binding to chromatin (though the requirement of product binding has
not been formally demonstrated).
Figure 2. Product protection.
H3K9me1,2 from Histone Lysine Demethylases (HKDMs), or conver-
sion to H3K9me3 by Histone Lysine Methyltransferases (HKMTs),
including that of G9a/GLP.
Product binding could protect the
3506 Nucleic Acids Research, 2010,Vol.38, No. 11
onto chromatin and reinforce the alteration of chromatin
methylation of H1 serves as another mark to reinforce
silencing, as methylated H1.4 is a binding platform for
HP1 (59,60), for L3MBTL1, which participates in
chromatin compaction (59) [reviewed in (61)] and for
MSX1,a homeobox transcriptional
Completing the cycle, H1.4 lysine demethylases were
also identified (59).
by H1 (Figure3). The
HP1. G9a and GLP are required for the binding of HP1
to euchromatin, as the knockout of either greatly
diminishes euchromatic HP1 staining, and loss of G9a
reduces HP1 occupancy at specific promoters (4,10,63).
Paradoxically, although the G9a catalytic domain is suffi-
cient for silencing when targeted to a reporter gene, it is
insufficient for HP1 recruitment (9). It appears that G9a
targets HP1 to chromatin by automethylating itself
(Figure 4a), potentially at multiple sites (29,32). The
dominant automethylation site (found in GLP also)
mimics the region around H3K9, even containing a
phosphorylation site that blocks HP1 binding. Mutation
of the target lysine eliminates HP1 binding and causes
automethylation sites to be di- and trimethylated (29,32).
Consistent with our proposal that ankyrin repeat binding
to mono- and dimethylated products might regulate
product specificity, the identified automethylation sites
contain substitutions (KTMSK instead of KSTGG in
H3) that should render binding to ankyrin repeats impos-
sible. Again, degree of methylation is all-important, as
HP1 has a preference for H3K9me2/3 over H3K9me1
(51). The best in vivo demonstration of this specificity is
provided by the overexpression of a demethylase (GASC1)
that removes the H3K9me2/3 marks but cannot act on
H3K9me1 (55). The gain in H3K9me1 at the expense of
H3K9me2/3 was insufficient to retain HP1 binding (55).
The requirement for direct interaction of G9a with HP1 is
reminiscent of Suv39H1 mediated targeting of HP1, where
not only H3K9 methylation, but also a direct interaction
between Suv39H1 and HP1 is required for HP1 residence
As HP1 itself has been implicated in the recruitment of
other protein and DNA methyltransferases in the forma-
tion of notonly silent
euchromatin, this could prove an important gateway
into other mutually reinforcing mechanisms of silencing
(65). Some studies have proposed a direct interaction
between G9a and the DNA methyltransferases (see
below). However, HP1 binding to automethylated G9a
or H3K9me2/3 could serve to recruit DNMT3a and
DNMT3b. Indeed, HP1 knockdown has little effect on
G9a occupancy at the TNFa promoter, but DNMT3a,
DNMT3b and CpG methylation were lost in the
absence of HP1 or G9a. This suggests that G9a is the
key organizer of silencing at that promoter and that
recruitment of DNMTs requires HP1 as a bridge (63).
DNA methyltransferases. There is a surprising lack of
cross-talk involved in G9a-mediated DNA methylation.
G9a and GLP are both required for normal levels of
DNA methylation in facultative heterochromatin and
interact with DNA methyltransferases. An attractive
model emerges, where G9a and GLP might establish
H3K9 methylation, bind to it, and then attract DNA
methyltransferases directly, or through HP1. However,
several studies have shown the catalytic activities of G9a
and GLP to be dispensable for DNA methylation, and
that G9a-mediated DNA and histone methylation are
independent pathways, either of which can accomplish
silencing (34,41,42). This implies that G9a and GLP play
a more structural role in the context of DNA methylation,
where their presence, but not activity, recruits DNA
methyltransferases to target loci. Perhaps they bridge the
DNA-binding partners of G9a (see above) and DNA
methyltransferases. Indeed, G9a appears to interact
DNMT3a and DNMT3b through its ankyrin repeat
domain, even in the absence of the G9a catalytic SET
domain (42) (Figure 4b). Whether this is physiological
or perhaps an alternate pathway that suffices to bring
about some methylation in the absence of a pathway
that does more linearly couple histone and DNA
methylation is unclear.
DNA binding and other target proteins. G9a also has been
reported to inhibitthe
methylates C/EBP-b, blocking its ability to activate tran-
scription. Binding is not observed in the absence of a
HP1 recruitment by automethylation
DNMT recruitment by ANK
Figure 4. Recuritment.
(b) DNA methyltransferases interact with G9a in a manner not depen-
dent on catalytic activity. (c) G9a/GLP have been shown to bind or to
methylate a number of targets. G9a methylation of C/EPBb blocks
chromodomain blocks its ability to bind histone (red X’s). Many
other targets have been identified, some of which may also be bound
Nucleic Acids Research, 2010,Vol.38, No. 113507
functional catalytic domain, which suggests product
binding; however, this was not formally tested (31).
Many other targets for G9a methylation have been
identified (29,30); all of which are known to play roles in
processes involving chromatin (Figure 4c). It is unclear
how many of these substrates can also meet the require-
ment for product binding to the ankyrin repeats. In
general, lysine methylation exerts its effect through
modules that bind the methyl marks. As an exception to
this rule, G9a methylation appears to block directly
the CYDL chromodomain from binding H3K9me3,
apparently by altering the chemical properties of a key
Spreading the methyl mark
G9a and GLP are responsible for broad regions of H3K9
methylation up to 4.9 Mb, in what have been termed ‘large
organized chromatin K9 modifications’ (LOCKS), which
can cover up to nearly 50% of the genome in some
differentiated cell types (66). The finding that there are
few LOCKS in embryonic stem cells, and that they grow
in number with differentiation is under dispute [see (67),
rebuttal in (68)]. However, the existence of LOCKS, their
apparent tissue specificity and the presence of G9a/
GLP-mediated methylation within LOCKS is not (68).
These broad patterns of methylation are at odds with
the narrow targeting of G9a and GLP to primarily
promoter regions. For example, G9a is found in
complex with the CCAAT displacement protein, a tran-
scription factor involved in differentiation and develop-
ment (8), Blimp-1 (also known as PRDI-BF1), a
DNA-binding protein involved in the terminal differenti-
ation of B lymphocytes (5) and is linked to the CtBP
co-repressor viaan adaptor
(7,69). Spreading the modification outward from these
sites could bring about the methylation of large regions
of chromatin adjacent to regions where G9a and GLP are
targeted. In spreading, the binding of product [via their
ankyrin repeats (33)] could position G9a and GLP to
methylate the next neighboring nucleosome, which then
can serve as a binding site for enzyme (Figure 5). The
cyclic repetition of this process would allow the mark to
propagate over great distances. Although this model is
sub-compartments to which genes are recruited during
silencing (70–72). Long non-coding RNAs are best
recognized for silencing clusters of imprinted genes, but
given the volume of non-coding RNAs detected, and their
specific expression patterns in development and differenti-
ation [reviewed in (73,74)] it would not be surprising if the
formation of G9a-containing LOCKS is a RNA-mediated
process. These nuclear sub-structures may distinguish the
large swaths of more permanent silencing associated with
cell differentiation and imprinting from more localized
and reversible domains of G9a- and GLP-mediated
The web of interactions detailed above will likely benefit
from a systems approach like those applied to signaling
networks. Catalytic activity, product binding, RNA
binding and partner binding will have to be analysed. Of
particular interest is defining what non-histone targets of
G9a/GLP can be bound as products. It is unknown what
governs the switch to co-activation by G9a—whether the
presence of another mark like arginine methylation or
Similarly, it is unclear if DNA methylation follows
lysine methylation and HP1 recruitment, or if the two
are independent pathways bridged by G9a/GLP acting
as a scaffold. Lastly, we ask whether the propagation of
the methyl mark occurs by product-binding mediated
spreading, a RNA-mediated process, or both.
G9a and GLP provide a tractable system to test
activity and product binding are defined. One unanswered
Figure 5. Spreading. Like Su(var3-9)/HP1 and yeast Clr4 (SET and
chromodomain), product binding could help propagate the methyl
mark by posing the catalytic domain for attack on the next neighboring
nucleosome. Repetition of this process allows spreading of the mark
from the site of nucleation.
3508 Nucleic Acids Research, 2010,Vol.38, No. 11
question that could complicate these experiments is why,
despite their apparent heteromeric nature, perturbation of
one activity typically has a penetrating effect. In the case
of complete knockouts, loss of one member of the
complex causes the other to degrade (10), but in the case
of more discreet deletions or point mutants, the answer
remains enigmatic. Detailed analyses using mutations in
both partners will likely be essential.
R.E.C. is a fellow of the Jane Coffin Childs Memorial
Fund for Medical Research. The work in the Cheng lab-
oratory is currently supported by the US National
Institutes of Health (GM068680-05, GM049245-16 and
DK-082678-02). Funding for open access charge: waived.
Conflict of interest statement. None declared.
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