RBP2 is an MRG15 complex component and down-regulates intragenic histone H3 lysine 4 methylation.

Abstract

MRG15 is a conserved chromodomain protein that associates with histone deacetylases (HDACs) and Tip60-containing histone acetyltransferase (HAT) complexes. Here we further characterize MRG15-containing complexes and show a functional link between MRG15 and histone H3K4 demethylase activity in mammalian cells. MRG15 was predominantly localized to discrete nuclear subdomains enriched for Ser(2)-phosphorylated RNA polymerase II, suggesting it is involved specifically with active transcription. Protein analysis of the MRG15-containing complexes led to the identification of RBP2, a JmjC domain-containing protein. Remarkably, over-expression of RBP2 greatly reduced the H3K4 methylation in culture human cells in vivo, and recombinant RBP2 efficiently removed H3K4 methylation of histone tails in vitro. Knockdown of RBP2 resulted in increased H3K4 methylation levels within transcribed regions of active genes. Our findings demonstrate that RBP2 associated with MRG15 complex to maintain reduced H3K4 methylation at transcribed regions, which may ensure the transcriptional elongation state.

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© 2007 The Authors
Journal compilation © 2007 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Genes to Cells (2007)
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, 811–826
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DOI: 10.1111/j.1365-2443.2007.01089.x
Blackwell Publishing IncMalden, USA GTCGenes to Cells1356-9597© 2007 The AuthorsJournal compilation © 2007 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
XXXOriginal ArticlesMRG15 interacts with H3 demethylase RBP2 T Hayakawa et al.
RBP2 is an MRG15 complex component and down-regulates
intragenic histone H3 lysine 4 methylation
Tomohiro Hayakawa
Fuyuki Ishikawa
1
, Yasuko Ohtani
and Jun-ichi Nakayama
1
, Noriyo Hayakawa
1,
*
1
, Kaori Shinmyozu
2
, Motoki Saito
3
,
3
1
Laboratory for Chromatin Dynamics, and
2
Proteomics Support Unit, Center for Developmental Biology, RIKEN, Kobe, Hyogo 650-0047, Japan
3
Laboratory for Cell Cycle Regulation, Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University,
Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan
MRG15 is a conserved chromodomain protein that associates with histone deacetylases (HDACs)
and Tip60-containing histone acetyltransferase (HAT) complexes. Here we further characterize
MRG15-containing complexes and show a functional link between MRG15 and histone H3K4
demethylase activity in mammalian cells. MRG15 was predominantly localized to discrete nuclear
subdomains enriched for Ser
-phosphorylated RNA polymerase II, suggesting it is involved
specifically with active transcription. Protein analysis of the MRG15-containing complexes led to
the identification of RBP2, a JmjC domain-containing protein. Remarkably, over-expression of
RBP2 greatly reduced the H3K4 methylation in culture human cells
RBP2 efficiently removed H3K4 methylation of histone tails
in increased H3K4 methylation levels within transcribed regions of active genes. Our findings
demonstrate that RBP2 associated with MRG15 complex to maintain reduced H3K4 methylation
at transcribed regions, which may ensure the transcriptional elongation state.
2
in vivo
, and recombinant
in vitro
. Knockdown of RBP2 resulted
Introduction
Histone N-terminal tails are subjected to multiple cova-
lent modifications that affect chromatin structure and
transcription (Strahl & Allis 2000). Histone acetylation at
lysine residues is closely linked to active transcription and
is dynamically controlled by histone acetyltransferases
(HATs) and histone deacetylases (HDACs). Most of the
HATs and HDACs are part of large multiprotein com-
plexes, whose subunits mediate regulation, recruitment,
substrate recognition and other undefined functions.
While histone acetylation is dynamically regulated by
balancing the opposing activities of HATs and HDACs,
histone lysine methylation serves as an additional stable
epigenetic mark, participating in a variety of biological
processes, including both transcriptional activation and
repression (Lachner & Jenuwein 2002). Recent studies
suggest that histone lysine methyl groups are removed by
at least two distinct classes of enzymes: the nuclear amine
oxidase homologs and the JmjC-domain proteins. LSD1
belongs to the first of these classes and was the first histone
lysine demethylase identified; it specifically demethylates
methylated histone H3 lysine 4 (H3K4me) (Shi
Unlike LSD1, which can only remove mono- and di-
methyl lysine modifications, the JmjC-domain-containing
histone demethylases (JHDMs) can reverse all three mono-,
di- and tri-methylation states. It has been shown that
JHDM1 and JHDM2A possess demethylase activity
specific to H3K36me and H3K9me, respectively, and that
JHDM3 demethylates both H3K9me and H3K36me
(Klose
et al.
2006).
Human MRG15 and MRGX were originally identified
as factors closely related to MORF4 (mortality factor
on human chromosome 4), whose transient expression
induces senescence in a subset of human tumor cell
lines (Bertram
et al.
1999). Although these proteins show
extensive sequence similarities and share common motifs
such as helix-loop-helix and leucine zipper regions, only
MRG15 contains an N-terminal chromodomain and has
known orthologs in other eukaryotic species, suggesting
MRGX and MORF4 emerged late in evolution (Bertram
& Pereira-Smith 2001). In the budding yeast
the MRG15 homologue Eaf3 is a component of both the
et al.
2004).
S. cerevisiae
,
Communicated by
*Correspondence
: Fumio Hanaoka
: E-mail: jnakayam@cdb.riken.jp

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NuA4 HAT and Rpd3 HDAC complexes and functions
in transcriptional regulation (Eisen
et al.
2002). We previously showed that the fission yeast
homologue, Alp13, is a component of the Clr6 HDAC
complex and is required for the maintenance of genome
integrity (Nakayama
et al.
2003). Human MRG15 has
also been identified as a stable component of both the
Tip60 HAT (NuA4) and HDAC complexes (Yochum &
Ayer 2002; Cai
et al.
2003; Doyon
these results implicate MRG15 family proteins in modulat-
ing histone acetyl modification for both transcriptional
regulation and DNA double-strand break (DSB) repair
(Doyon & Cote 2004; Kusch
Several lines of evidence suggest that human MRG15
and MRGX are involved in both the activation and
repression of transcription. MRG15 and MRGX inter-
act with RB in nucleoprotein complexes and activate or
repress
β
-Myb promoter activity in a cell-type-dependent
manner (Leung
et al.
2001; Tominaga
also interacts with the mSin3 co-repressor complex, and
the tethering of MRG15 to the promoter region leads to
the repression of a reporter gene (Yochum & Ayer 2002).
Recent studies on
Mrg15
-deficient mice revealed that
the mice die as embryos and have a reduced number
of proliferating cells (Tominaga
MrgX
-null mice have no detectable phenotype, analysis
of
Mrg15
,
MrgX
double-null embryos suggested
that Mrg15 and MrgX have overlapping functions in
early embryogenesis (Tominaga
Promoter and transcribed regions at active gene loci
are marked by characteristic patterns of histone acetylation
and methylation (Millar & Grunstein 2006). The recruit-
ment of HATs to specific promoter regions facilitates
gene expression, and a global pattern of histone H3 and
H4 acetylation is created in which the levels are generally
high in promoter and low in transcribed regions. Recent
studies on
S. cerevisiae
revealed that transcriptional initi-
ation and transcribed regions are differentially marked by
H3K4 and H3K36 methylation, which are mediated,
respectively, by Set1 and Set2 methyltransferases (Hampsey
& Reinberg 2003). Moreover, these modifications are
mechanistically connected to the phosphorylation state
of the carboxy-terminal domain of RNA polymerase II.
In
S. cerevisiae
, the loss of Eaf3, the homologue of MRG15,
causes an increase in histone H3 and H4 acetylation
within transcribed regions (Reid
deacetylation at transcribed regions is considered to
suppress intragenic transcription initiation (Kaplan
2003). Further studies revealed that Eaf3 interacts
with H3K36me through its chromodomain and recruits
Rpd3S, one of the Rpd3-containing complexes, to
H3K36-methylated nucleosomes, that leading to the
et al.
2001; Gavin
et al.
2004). Together,
et al.
2004).
et al.
2003). MRG15
et al.
2005a). Although
–/––/–
et al.
2005b).
et al.
2004). Preferential
et al.
deacetylation of transcribed regions (Carrozza
2005; Joshi & Struhl 2005; Keogh
these studies implicate the MRG15 family proteins in
transcriptional elongation, the exact mechanisms by
which human MRG15 and its related proteins function
in this process have remained elusive.
Here we report the biochemical characterization of
MRG15-associated complexes and show that the JmjC-
domain protein, RBP2 (also known as JARID1A),
specifically interacts with the MRG15 complex. We
further show that the over-expression of RBP2, but not
a catalytically inactive mutant, reduces the H3K4 tri-,
di- and mono-methylation levels in human cultured
cells. Finally, we demonstrate that the RNAi depletion
of RBP2 results in increased H3K4 methylation at the
transcribed regions of active genes. These results suggest
that MRG15-containing complexes target H3K36me-
enriched transcribed regions and further recruit the H3K4
demethylase, RBP2 and HDAC complexes.
et al.
et al.
2005). Although
Results
MRG proteins localize to nuclear subdomains
associated with active transcription
Although recent studies on
specific role for Eaf3 in maintaining the hypoacetylation
at transcribed regions, the precise role of human MRG15
and its related proteins (MRGX, a highly homologous
protein lacking the N-terminal chromodomain; and
MRGBP, a tightly associated partner protein) in tran-
scriptional regulation is incompletely understood. To
probe the function of MRG proteins in transcriptional
regulation, we first analyzed their subcellular localization
in human cell lines using polyclonal antibodies raised
against each recombinant protein. These antibodies
specifically recognized endogenous proteins and no cross-
reactive bands were observed in Western analysis (data
not shown). MRG15 was distributed throughout the
nucleoplasm in formaldehyde-fixed HeLa cells (Fig. 1A,
formaldehyde); a similar nucleoplasmic distribution was
observed for transiently-expressed GFP-MRG15 protein
in living cultured cells (data not shown). If the cells were
fixed with methanol, the MRG15 signals were detected
as dozens of nuclear foci resembling “splicing speckles”
with nucleoplasmic distribution (Fig. 1A, methanol).
These MRG15 signals were largely excluded from
chromatin regions intensely stained with DAPI (Fig. 1A,
merge). Similar fixation-dependent distribution was
observed for MRGX and MRGBP, and their signals in
methanol-fixed cells overlapped with those of MRG15,
partly for MRGX and more so for MRGBP (Fig. 1B,C).
S. cerevisiae
uncovered a

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MRG15 interacts with H3 demethylase RBP2
© 2007 The Authors
Journal compilation © 2007 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
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These localization patterns were confirmed in different
cell types, including human HEK293T, A431 and
mouse NIH3T3 cells (Fig. 1A and data not shown). One
interpretation of these data is that the nucleoplasmic
distribution exhibited following different fixation con-
ditions represents distinct subfractions of MRG15.
Hyperphosphorylated RNA polymerase II exhibits
fixation-dependent distribution analogous to that of
MRG proteins (Bregman
et al.
To gain further insight into the relationship between
MRG15 and transcription, we investigated whether
MRG15 foci co-localized with signals for RNA polymerase
II and histone methyl modifications associated with active
transcription. RNA polymerase II engaged in transcrip-
tional elongation step is hyperphosphorylated on Ser
of its C-terminal domain and is specifically recognized
in this state by monoclonal antibody, H5 (Bregman
1995; Guillot
et al.
2004). Immunostaining of RNA poly-
1995; Guillot
et al.
2004).
2
et al.
merase II using this antibody exhibited a punctate
nuclear distribution in methanol-fixed HeLa cells that
resembled that of MRG15 (Fig. 1D; Guillot
Indeed, these RNA polymerase II signals preferentially
co-localized with those of MRG15 (Fig. 1D). Moreover,
MRG15 signals also overlapped with those of H3K4me3
and H3K36me3 (Supplementary Fig. S1A, S1B). Taken
together, these results suggest that the MRG15-enriched
nuclear subdomains are associated with the sites of active
transcription, and further support the idea that MRG15—
and presumably MRGX and MRGBP—play a critical
role in the transcriptional elongation.
et al.
2004).
Characterization of MRG15-, MRGX- and
MRGBP-containing complexes
Human MRG15 is known to be a component of both
the Tip60 HAT and HDAC complexes (Yochum & Ayer
Figure 1 MRG15 localizes to the transcriptionally active nuclear domains. (A) Immunofluorescence images of HeLa (upper) and 293T
(lower) cells. Cells were fixed with cold methanol (upper row) or formaldehyde (lower row) followed by indirect immunostaining using
an anti-MRG15 antibody (left column). Chromatin was visualized by DAPI (middle column), and merged images of the green (MRG15)
and magenta (chromatin) staining are shown in the right column. Overlapped signal of the green and magenta becomes white. (B, C)
Immunofluorescence images of HeLa cells expressing FLAG-tagged MRG15 (F-MRG15). Cells were fixed with cold methanol,
followed by immunostaining with an anti-MRGX or MRGBP antibody (left) or anti-FLAG M2 antibody for F-MRG15 (middle).
Merged images of green (MRGX or MRGBP) and magenta (MRG15) staining are shown on the right. (D) Immunofluorescence images
of HeLa (upper) and A431 (lower) cells. Cells were fixed with cold methanol and then stained using anti-MRG15 (left column) and anti-
RNA Pol II (H5) antibodies (middle column). Merged images of the green (MRG15) and magenta (RNA Pol II) staining are shown in
the right column. Scale bar, 10 µm.

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2002; Cai
its specific role in these complexes nor the relationships
with MRGX or MRGBP are clear. To investigate the
distinctive functions of the MRG family proteins, we
purified and analyzed MRG15-, MRGX- and MRGBP-
containing complexes. We produced HeLa cell lines that
expressed N-terminal FLAG-tagged fusions (F-MRG15,
F-MRGX or F-MRGBP) in a tetracycline-regulated
manner. The lines were subjected to immune affinity
et al.
2003; Doyon
et al.
2004), but neither
chromatography using anti-FLAG antibodies and purified
proteins were analyzed by SDS-PAGE (Fig. 2A) and
Western blotting using cognate antibodies (Fig. 2B).
The profiles of the F-MRG15- and F-MRGX-associated
complexes were largely superimposable (Fig. 2A), although
F-MRG15-associated complexes did not include native
MRGX or vice versa (Fig. 2B, IP). These results suggest
that MRG15 and MRGX are mutually exclusive to
complexes with an otherwise similar composition. Since
Figure 2 Immunoaffinity purification of
MRG protein complexes. (A) Affinity-
purified MRG15-, MRGX- and MRGBP-
complexes from HeLa cells were subjected
to 4%–20% SDS-PAGE followed by
silver staining (left). Each protein band
was excised from the gel and subjected to
LC-MS/MS analysis. Interacting proteins
identified by LC-MS/MS analysis are
indicated on the right. Newly identified
proteins are highlighted in pink. Mock
indicates the purification results using cells
transformed with an empty expression
vector. (B) MRG15 and MRGX form
distinct complexes that are mutually exclu-
sive. Nuclear lysates (input) of HeLa cells
expressing each of the FLAG-tagged
MRG proteins and immunoprecipitated
fractions (IP) using anti-FLAG M2 agarose,
were subjected to Western blot analysis
using specific antibodies to the MRG
proteins. Bands in the mock lane of input
indicate endogenous MRG proteins.

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MRG15 interacts with H3 demethylase RBP2
© 2007 The Authors
Journal compilation © 2007 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Genes to Cells (2007)
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MRGX lacks a chromodomain, this further indicates
that the chromodomain is not required for complex
formation. The profile of the F-MRGBP-associated
proteins was similar to that of F-MRG15 and F-MRGX
(Fig. 2A), and native MRG15 and MRGX were present
in the F-MRGBP-precipitated fraction (Fig. 2B, IP).
This result is consistent with previous observations (Cai
et al.
2003) and suggests that MRGBP is a shared
component of MRG15- and MRGX-containing
complexes. Interestingly, we noticed that the expression
of F-MRGBP caused an increase in the protein levels of
MRG15 and MRGX (Fig. 2B, input, F-MRGBP). This
result implies that MRGBP plays a role in regulating
synthesis and/or stability of MRG15 and MRGX.
RBP2 is associated with MRG15- and
MRGX-containing complexes
To investigate further the composition of MRG15-
and MRGX-containing complexes, each constituent band
following SDS-PAGE was excised and analyzed by
nano-liquid chromatography tandem mass (LC-MS/
MS) spectrometry. This analysis detected most of the
proteins previously identified as being associated with
MRG15 or MRGBP (Cai
et al.
and confirms that MRG15- and MRGX-containing
complexes are associated with both Tip60 HAT- and
HDAC-associated factors (Fig. 2A). However, our
analysis also revealed several novel proteins, including the
retinoblastoma (RB) binding protein RBP2, ZNF131,
Mbt domain containing 1 (MBTD1), hnRNP K, hnRNP
M and hnRNP U, stably associated with MRG15- or
MRGX-containing complexes (Fig. 2A, highlighted
in pink). Although any functional link between these
newly identified factors and MRG proteins remains to
be shown, this observation forms a physical basis for the
functional correlation between MRG15 and RBP2-
mediated transcriptional regulation (Kim
& Hong 2001; Benevolenskaya
RBP2 possesses a conserved JmjC domain, recently
shown to correspond to the catalytic domain of histone
demethylase (Klose
et al.
2006), implying that it could be
involved in the dynamics of histone methyl modifica-
tions. Therefore, we focused on RBP2 and its relation-
ship with MRG proteins and transcriptional regulation.
We first analyzed their association by gel filtration
chromatography. MRG15, MRGX and MRGBP in
HeLa nuclear extract were eluted with distinct profiles
and part of them was detected in fractions corresponding
to more than 600 kDa (Fig. 3A). RBP2 and HDAC2 were
also eluted in fractions for higher molecular weight. These
results suggest that MRG15, MRGX and MRGBP are
2003; Doyon
et al.
2004)
et al.
1994; Chan
et al.
2005). In addition,
a component of large protein complexes and their
elution profiles were overlapped with that of RBP2.
To assess in greater detail the specificity of interaction
between RBP2 and the MRG proteins, we performed
immunoprecipitation (IP) of F-MRG proteins and
analyzed precipitated RBP2 by Western blotting using an
anti-RBP2 antibody. We found that RBP2 interacted
with F-MRG15 and F-MRGX, but not with F-MRGBP
(Fig. 3B). Similar specificity was observed for Sin3B
and HDAC2. This was consistent with our LC-MS/MS
analysis, in which no RBP2, Sin3B or HDAC2 peptide
was detected in the F-MRGBP-precipitated fraction
(Fig. 2A). These results suggest that MRG15 and MRGX
are components of distinct HAT and HDAC complexes
and that RBP2 specifically associated HDAC complexes
containing either MRG15 or MRGX. MRGBP appeared
to be specific to Tip60-containing HAT complexes (Figs 2A
and 3B), in good agreement with the identification of
Eaf7, an MRGBP homolog in
but not in Rpd3-containing HDAC complexes (Krogan
et al.
2004). Under the conditions of these IP Westerns,
the level of interacting RBP2 was relatively higher
in the F-MRG15-precipitated fraction than in that of
F-MRGX (Fig. 2B, RBP2). To corroborate this pattern of
specificity, we expressed N-terminal FLAG-tagged RBP2
(F-RBP2) in HeLa cells. F-RBP2-associated proteins
were isolated from nuclear extracts by immunoaffinity
purification and identified by Western blotting (Fig. 3C,D).
This revealed that F-RBP2 belonged to stable complexes
containing MRG15, Sin3B and HDAC2 (Fig. 3C), but
not Tip60, MRGX or MRGBP (Fig. 3D). Together,
these reciprocal IP-Western experiments further
demonstrate that, although RBP2 was detected in
co-immunoprecipitates containing either F-MRG15 or
F-MRGX (Figs 2A and 3A), it preferentially associates
with the MRG15-containing HDAC complex.
S. cerevisiae
, in NuA4 HAT,
RBP2 over-expression induces histone H3K4
demethylation
in vivo
We next examined the subcellular localization of RBP2.
Since the anti-RBP2 antibody was not applicable to
immunostaining, due to its high background, we
examined RBP2 localization using HeLa cells that stably
expressed tetracyclin-regulated F-RBP2. F-RBP2 in
HeLa cells predominantly localizes to discrete nuclear
foci and partially co-localized with MRG15 or Ser
phosphorylated RNA polymerase II (Fig. 3E,F), sug-
gesting its involvement with transcriptional regulation.
MRG15 binds H3K36-methylated histone tail (Zhang
et al.
2006) and it is therefore suggested that MRG15
plays a role in transcribed regions to recruit the HDAC
2
-