UHRF1 binds G9a and participates in p21 transcriptional regulation in mammalian cells.

Abstract

UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is a multi-domain protein associated with cellular proliferation and epigenetic regulation. The UHRF1 binds to methylated CpG dinucleotides and recruits transcriptional repressors DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1) through its distinct domains. However, the molecular basis of UHRF1-mediated transcriptional regulation via chromatin modifications is yet to be fully understood. Here we show that UHRF1 binds histone lysine methyltransferase G9a, and both are co-localized in the nucleus in a cell-cycle-dependent manner. Concurrent with the cell-cycle progression, gradual deposition of UHRF1 and G9a was observed, which mirrored H3K9me2 accumulation on chromatin. Murine Uhrf1-null embryonic stem (ES) cells displayed a reduced amount of G9a and H3K9me2 on chromatin. UHRF1 recruited and cooperated with G9a to inhibit the p21 promoter activity, which correlated with the elevated p21 protein level in both human UHRF1 siRNA-transfected HeLa cells and murine Uhrf1-null ES cells. Furthermore, endogenous p21 promoter remained bound to UHRF1, G9a, DNMT1 and HDAC1, and knockdown of UHRF1 impaired the association of all three chromatin modifiers with the promoter. Thus, our results suggest that UHRF1 may serve as a focal point of transcriptional regulation mediated by G9a and other chromatin modification enzymes.

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Published online 4 December 2008Nucleic Acids Research, 2009, Vol. 37, No. 2 493–505
doi:10.1093/nar/gkn961
UHRF1 binds G9a and participates in p21
transcriptional regulation in mammalian cells
Jong Kyong Kim1, Pierre-Olivier Este `ve1, Steven E. Jacobsen2and Sriharsa Pradhan1,*
1New England Biolabs, Ipswich, MA 01938-2723 and2Department of Molecular Cell and Developmental Biology,
Howard Hughes Medical Institute, University of California, Los Angeles, CA 90095, USA
Received September 29, 2008; Revised October 27, 2008; Accepted November 12, 2008
ABSTRACT
UHRF1 (ubiquitin-like, containing PHD and RING
finger domains 1) is a multi-domain protein asso-
ciated with cellular proliferation and epigenetic
regulation. The UHRF1 binds to methylated CpG
dinucleotides and recruits transcriptional repres-
sors DNA methyltransferase 1 (DNMT1) and histone
deacetylase 1 (HDAC1) through its distinct domains.
However, the molecular basis of UHRF1-mediated
transcriptional regulation via chromatin modifica-
tions is yet to be fully understood. Here we show
that UHRF1 binds histone lysine methyltransferase
G9a, and both are co-localized in the nucleus in
a cell-cycle-dependent manner. Concurrent with
the cell-cycle progression, gradual deposition of
UHRF1 and G9a was observed, which mirrored
H3K9me2accumulation
Uhrf1-null embryonic stem (ES) cells displayed a
reduced amount of G9a and H3K9me2 on chromatin.
UHRF1 recruited and cooperated with G9a to inhibit
the p21 promoter activity, which correlated with the
elevated p21 protein level in both human UHRF1
siRNA-transfected HeLa cells and murine Uhrf1-
null ES cells. Furthermore, endogenous p21 promo-
ter remained bound to UHRF1, G9a, DNMT1 and
HDAC1, and knockdown of UHRF1 impaired the
association of all three chromatin modifiers with
the promoter. Thus,our results
UHRF1 may serve as a focal point of transcriptional
regulation mediated by G9a and other chromatin
modification enzymes.
on chromatin.Murine
suggest that
INTRODUCTION
The Uhrf1 gene encodes a member of RING-finger E3
ubiquitin ligase that is overexpressed in cancers (1).
Mammalian UHRF1, previously known as ICBP90
(inverted CCAAT box binding protein of 90 kDa) in
human (2) and Np95 (nuclear protein of 95 kDa) in
mouse (3), possesses a UBI (ubiquitin-like), PHD (plant
homeodomain), SRA (SET and RING associated) and
RING (really interesting new gene) domains. These four
distinct domains of the protein serve different functions.
The UBI domain exhibits a typical a/b ubiquitin fold
along with surface lysine residues similar to those of ubi-
quitin molecule. The PHD domain is placed between the
UBI and SRA domains. Both PHD and SRA domains
participate in di- and trimethyl histone H3K9 binding
(4). Although the PHD domain determines the binding
specificity, SRA domain promotes binding activity.
Furthermore, both domains are essential for heterochro-
maticlocalization ofhuman
regulation of UHRF1 in both human and mouse cells
resulted in disrupted distribution of H3K9me3 and
Hp1a, two known heterochromatic marks on the mamma-
lian genome (4). The SRA domain of mouse UHRF1 was
also shown to bind histones (5), and depletion of UHRF1
in murine cells resulted in hyperacetylated histone H4 and
increased transcription of major satellites, demonstrating
a role of UHRF1 in pericentromeric heterochromatin
formation (6). In relevance to this observation, a recent
study demonstrated that the PHD domain of mouse
UHRF1 plays a role in large-scale reorganization of peri-
centromeric heterochromatin (7). Apart from binding to
histones, the SRA domain of UHRF1 can bind to methyl-
CpG dinucleotides with a preference for hemimethylated
CpG sites (8,9). Similarly, in Arabidopsis, a UHRF1
homolog VIM1 has methyl cytosine binding properties
via its SRA domain and plays a crucial role in mainte-
nance of heterochromatin (10). The RING domain of
UHRF1 was found to be essential for its E3 ubiquitin
ligase activity for histones (4,5). Deletion of the RING
domain was shown to sensitize cells to the effects of che-
motherapeutics such as etoposide and cis-platinum (11).
Chromatin modifications exert a significant impact on
gene expression. Several chromatin-modifying enzymes
have been identified and known to catalyze specific
UHRF1,anddown-
*To whom correspondence should be addressed. Tel: +1 978 380 7227; Fax: +1 978 921 1350; Email: pradhan@neb.com
? 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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modifications including methylation, acetylation, phos-
phorylationand ubiquitination
remove the modifications have been also identified, reflect-
ing the dynamic situations in chromatin structure and
function. Most of these enzymes have an intrinsic affinity
to specific target substrates; however, such preference
is not considered to be sufficient to carry out efficient
and specific chromatin modifications to accommodate
dynamic changesin chromatin
during normal cell growth. In fact, additional nuclear pro-
teins have been identified and shown to direct these var-
ious chromatin-modifying enzymes to specific targets to
facilitate timely and efficient modifications on chromatin.
Among these additional factors, UHRF1 was found to
form a complex with HDAC1 and bind to methylated
promoter regions of tumor suppressor genes such as p16
and p14 in cancer cells (8). Recently, UHRF1 was also
shown to bind mammalian DNA methyltransferase
DNMT1 (9,13,14). The interaction between DNMT1
and UHRF1 facilitates and maintains DNA methylation
in both human and mouse genomes (9,14). This interac-
tion was shown to maintain global and local DNA
methylation and demonstrated to repress the transcription
of retrotransposons and imprinted genes. The loss of
UHRF1 resulted in 75% reduction in genomic methyla-
tion in mouse embryonic stem (ES) cells (15).
The UHRF1, a known cell-cycle regulator and tran-
scriptional activator of topoisomerase IIa expression,
was also shown to be a regulator for retinoblastoma
(Rb)expression (2,16). Overexpression
resulted in down-regulation of pRb, and the UHRF1
can form complexes with pRb in human cells, indicating
that the protein complexes may affect pRb-regulated pro-
moters. Indeed, the presence of a macromolecular com-
plex of pRb2/p130 on estrogen receptor-a (ER-a)
promoter correlated with DNA methylation status of the
gene, and the same study suggested that pRb2/p130 could
cooperate with UHRF1 and DNA methyltransferases
in maintaining a specific methylation pattern of ER-a
gene (17). Further experimental evidence of UHRF1-
associated nuclear proteins involved in DNA repair and
chromatin modification was recently documented by
mass-spectometric analyses of UHRF1 pull-down com-
plexes (14). The UHRF1-associated protein complexes
suggest that UHRF1 is involved in regulation of local
and global epigenome. Therefore, UHRF1 appears to be
a transcriptional regulator via regulating DNA methyl-
ation and/or recruitment of transcriptional repressors.
Despite significant progress toward understanding the
role of UHRF1 in gene expression, the precise role of
UHRF1 in transcriptional gene regulation is not fully
understood. There are both speculations and evidences
of UHRF1 being a focal point of chromatin modification
due to its close association with DNMT1 and HDAC1.
Here, we demonstrate that UHRF1 recruits G9a, an
essential component of histone H3K9 modification. This
cooperative binding of UHRF1 and G9a was observed on
the endogenous p21 promoter to repress the gene in cancer
cells. Therefore, a new mechanism of UHRF1-based gene
silencing via histone methyltransferase recruitment is
discussed.
(12). Enzymesthat
structureoccurring
ofUHRF1
MATERIALS AND METHODS
Cell culture, transfections andRNA interference
HeLa, COS-7 and HEK293 cells were obtained from
American Type Culture Collection (ATCC) and main-
tained in Dulbecco’s modified Eagle’s medium containing
10% fetal bovine serum (FBS) (Hyclone). Mouse ES cells
(a generous gift from Haruhiko Koseki and Masahiro
Muto), E14 and mUhrf1?/?(19-4), were cultured on
0.1% gelatin (StemCell Technologies Inc) in Glasgow’s
Minimal Essential Medium supplemented with 50 U/ml
mouse Leukemia Inhibitory Factor (LIF) (Chemicon),
10% FBS, 1X non-essential amino acids (Invitrogen),
1 mM sodium pyruvate and 55mM b-mercaptoethanol.
All media (Invitrogen) were supplemented with 2mM
L-glutamine and 1% antibiotic solution (ATCC). DNA
transfections for luciferase assays were performed using
FuGENE 6 (Roche Applied Science) according to the
manufacturer’s instructions. For siRNA transfections,
hUHRF1 (Invitrogen), G9a (New England Biolabs),
DNMT1 (New England Biolabs) and control Litmus
(New England Biolabs) siRNAs were transfected into
HeLa cells twice to a final concentration of 20–100nM
for 4 days using RNAiFECT reagent (Qiagen). Cell syn-
chronization at G1/S was performed essentially following
the double thymidine block protocol (18) except that aphi-
dicolin (3mg/ml) instead of thymidine (2mM) was used for
the first block.
Plasmid construction and antibodies
GFP fusions of full-length human UHRF1 (GFP-
hUHRF1) and mouse UHRF1 (GFP-mUHRF1) were
described previously (9). All GST-hUHRF1 constructs
were generated by PCR-based cloning procedures, using
the GFP-hUHRF1 as a template and cloning the resulting
products into EcoRI/XhoI sites of pGEX5X-1 (GE
Healthcare Life Sciences). DsRed-G9a and all GST-G9a
constructs were previously described (19). To obtain pur-
ified hUHRF1 for GST pull-down assays, the full-length
hUHRF1 fragment was cloned into NdeI/XhoI sites of
pET-28a (Novagen). A 5X Gal4-cdc2 luciferase reporter
(pG5-cdc2-luc) was constructed by inserting the cdc2 pro-
moter fragment (?912 to +33) from pcdc2-luc (20) into
NheI/BglII sites of pG5luc (Promega). To generate a Gal4
DNA-binding domain (Gal4DBD) fusion of hUHRF1
(pG4-hUHRF1), the full-length hUHRF1 fragment was
cloned into SalI/XbaI sites of pBIND (Promega). The
EGFP-fused N-terminal deletion mutant of G9a (EGFP-
N?G9a) was previously described (21). The DsRed-
N?G9a was generated by PCR amplification of coding
sequence for G9a lacking the N-terminal 394 amino
acids and subcloning the PCR product into EcoRI/
BamHI sites of pDsRed2-C1 (Clontech). Antibodies
(Ab) used for immunoprecipitation and western analyses
were as follows: anti-GFP Ab (Roche Applied Science),
anti-hUHRF1 Ab (BDBiosciences),
(Sigma),anti-humanp21
Technology), anti-mouse p21 Ab (Abcam), anti-dimethyl
histone H3 (Lys9) Ab (Millipore), anti-histone H3 Ab
(Cell Signaling Technology), anti-phospho-histone H3
anti-G9a
(Cell
Ab
AbSignaling
494
Nucleic Acids Research, 2009, Vol. 37, No. 2

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(Ser10) Ab (Cell Signaling Technology), anti-actin Ab
(Sigma) and anti-DNMT1 Ab (New England Biolabs).
Coimmunoprecipitation and western blot analysis
After 48h of transfection, COS-7 cells were washed with
PBS once and lysed in ice-cold RIPA buffer with protei-
nase inhibitor cocktail (Sigma). Cleared cell lysates
(1.2mg) were pre-incubated with BSA-blocked protein
G-magnetic beads (New England Biolabs) for 1h at 48C
to reduce non-specific binding of proteins to the beads.
After brief spin, the precleared cell lysates were incubated
with 2mg of indicated antibodies for 2h at 48C before
precipitation of the immune complexes with protein
G-magnetic beads for 1h. Immunoprecipitates were ana-
lyzed using western blot as described previously (19). For
coimmunoprecipitation of endogenous proteins, HEK293
cells were synchronized by serum starvation for 20h and
the subsequent release into 10% FBS-containing medium
for 15h before cell harvest. Immunoprecipitation was per-
formed following the same procedure described earlier.
Purification of GST-fusion proteins and GST
pull-down assays
Purification of GST-fusion proteins and pull-down assays
were described previously (22). Purified hUHRF1 protein
was obtained by bacterial expression of 6xHis-tagged
hUHRF1 and Ni-sepharose chromatography. G9a was
expressed and purified from baculovirus-infected Sf9
cells (New England Biolabs).
Cytochemistry
COS-7 cells were cotransfected with DsRed-G9a and
GFP-hUHRF1 orGFP-mUHRF1
TransPass D2 reagent (New England Biolabs) for 48h.
Fluorescence microscopy was performed as described pre-
viously (19). Cells were visualized with a Zeiss 200M
microscope with a 63? oil objective lens at 488nm for
GFP-hUHRF1 and GFP-mUHRF1 proteins, 568nm for
DsRed-G9a detection and 460nm for DNA staining with
Hoechst 33342.
plasmidswith
Chromatin isolation and chromatin
immunoprecipitation (ChIP)
Chromatin isolation procedure was described previously
(9). The chromatin in buffer C (1% SDS, 10 mM EDTA,
50 mM Tris–HCl, pH 8.0) was used for either western-blot
analyses or ChIP. For ChIP, the chromatin fractions were
diluted 10-fold in ChIP dilution buffer (16.7 mM Tris–
HCl, pH 8.0, 167 mM NaCl, 1.2 mM EDTA, 1.1%
Triton X-100, 0.01% SDS) and subjected to brief sonica-
tion for 20s followed by centrifugation at 4000?g for
5min. The cleared chromatin fractions (30–40 mg based
on DNA per IP) were used for immunoprecipitation
with 2 mg of indicated antibodies overnight at 48C. The
immune complexes were precipitated by pre-blocked pro-
tein G-magnetic beads (New England Biolabs) for 2h and
subjected to sequential washes with low salt buffer (50 mM
Tris–HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1%
Triton X-100, 0.1% deoxycholate, 0.1% SDS) three
times and high salt buffer (50mM Tris–HCl, pH 8.0,
500mM NaCl, 5mM EDTA, 1% Triton X-100, 0.1%
deoxycholate, 0.1% SDS) once. DNA was eluted in 1%
SDS elution buffer (1% SDS, 10mM EDTA, 50mM
Tris–HCl, pH 8.0) and de-crosslinked at 658C overnight.
DNA was purified by using QIAquick PCR purification
kit (Qiagen) and eluted in 50ml of TE buffer. The recov-
ered DNA was analyzed by conventional PCR or quanti-
tative PCR (Q-PCR) by using specific primers to the
proximal and distal regions of p21 promoter (23). Before
each IP, 5% input chromatin was taken and used to nor-
malize the Q-PCR data as % input. The relative promoter
occupancy after knockdown (KD) experiments was calcu-
lated by dividing the % input of each experimental group
by that of control KD (CTL KD).
Luciferase assay
COS-7 cells were cotransfected with pG5-cdc2-luc repor-
ter plus various combinations of pG4-hUHRF1 and
GFP-G9a plasmids in six-well plates as indicated in
the figure legend (Figures 4 and 6). Similarly, the p21
promoter–luciferase construct [pGL2–p21 (24), a kind
gift from Jane B. Trepel] was cotransfected with different
combinations of EGFP-hUHRF1 and EGFP-G9a plas-
mids. Empty vector DNA was included in the transfec-
tions to ensure that the same amounts of the DNA are
introduced to the cells, and the pCMV-Gluc control plas-
mid (New England Biolabs) was used to normalize the
transfection efficiency. After 48h transfection, firefly luci-
ferase activity was determined by Luciferase Assay System
(Promega) and normalized by Gaussia luciferase activity
measured by Gaussia Luciferase Assay Kit (New England
Biolabs). Each transfection was performed in duplicate
and repeated at least three times.
Quantitative RT–PCR
Total RNA was isolated from siRNA-transfected HeLa
cells with RNeasy Micro Kit (Qiagen) and used for
cDNA synthesis by DyNAmo SYBR Green 2-step
qRT-PCR Kit (Finnzymes). The Q-PCR was performed
by a Bio-Rad iCycler using iQ SYBR Green Supermix
(Bio-Rad). The amount of p21 mRNA from each
sample was normalized by the amount of GAPDH (gly-
ceraldehyde-3-phosphate dehydrogenase) as an internal
control. The primer sequences for p21 are 50-ATGGA
ACTTCGACTTTGTCACC-30and 50-AGGCACAAGG
GTACAAGACAGT-30(220 bp). The primer sequences
for GAPDH were described previously (20). Each quanti-
tative RT-PCR was performed in triplicate using four
independent sets of cDNA.
Cell-proliferation assay
Wild-type (+/+) and mUhrf1-null (?/?) ES cells were
seeded on six-well plates at 1?105cells per well, and
cell growth was monitored for 3 days by counting cells
from six replicates for each group. For BrdU (bromo-
deoxyuridine)-incorporation assay, cells were plated in a
96-well format at 1000 cells/well. After 24h incubation,
BrdU labeling was performed for 2h and determined by
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using cell proliferation ELISA kit (Roche) according to
the manufacturer’s instruction.
Bisulfite sequencing
Bisulfite conversion of genomic DNA (2mg) was per-
formed by using EpiTect Bisulfite kit (Qiagen) follow-
ing the manufacturer’s instruction. The primers for
p21 promoter region (?398 to +11) are as follows:
50-TTTTTGTTTGTTAGAGTGGGTTAG-30and 50- AC
AACTACTCACACCTCAACTAA-30. The PCR products
were ligated into pCR2.1-TOPO by using the TOPO TA
cloning system (Invitrogen), and at least 20 separate clones
per group were sequenced.
RESULTS
hUHRF1 interacts withG9a invivo andin vitro
Human UHRF1 (hUHRF1) was shown to form a com-
plex with HDAC1 (8). Both hUHRF1 and mUHRF1
(mouse UHRF1) were found to interact with DNMT1
and contribute to maintenance DNA methylation (9,14).
These observations led to a possibility that UHRF1 may
have an extended role for recruiting a broad range of
chromatin modification enzymes during epigenetic regula-
tion. To establish possible interactions between hUHRF1
and G9a, COS-7 cells were transfected with an expression
vector encoding GFP-fused full-length G9a (GFP-G9a) to
overcome a low abundance of G9a relative to hUHRF1.
The cell extracts were used for immunoprecipitation with
either anti-GFP antibody or anti-hUHRF1 antibody, and
the immunoprecipitates were analyzed by western blot
with specific antibodies to detect the in vivo association
of the GFP-G9a and endogenous hUHRF1 proteins.
Both proteins were detected by reciprocal immunoprecipi-
tation when cells were transfected with GFP-G9a as
opposed to transfection with GFP alone, indicating the
presenceofspecific association
hUHRF1 (Figure 1A). In a similar experiment, we were
able to detect the coimmunoprecipitation of the endogen-
ous G9a and hUHRF1 in synchronized HEK293 cells
(Figure 1B). We also performed GST pull-down assays
to determine whether there are direct physical interactions
between the two proteins in vitro and to map the regions
on the proteins involved in the interactions. Various GST-
fusion fragments of hUHRF1 and G9a were generated
and incubated with purified G9a or hUHRF1. The
hUHRF1 was found to directly interact with G9a predo-
minantly through its C-terminus covering the SRA and
RING domains with a stronger binding affinity of the
RING domain-containing fragment to G9a (Figure 1C).
A similar approach was taken to identify the region of the
G9a involved in a direct interaction with hUHRF1, and
revealed that G9a interacts with hUHRF1 through its N-
terminus (Figure 1D). Taken together, we demonstrated
that hUHRF1 interacts with G9a in vivo and in vitro.
betweenG9aand
UHRF1colocalizes withG9a invivo
The observation that hUHRF1 interacts with G9a
prompted us to examine the subnuclear localization of
the two proteins by cotransfecting COS-7 cells with
GFP-hUHRF1 and DsRed-G9a. The transfected cells
were synchronized by treatment of aphidicolin, released
from G1/S border and followed for the indicated time
points to monitor the localization of the two proteins.
Although both proteins were present in a diffused pattern
throughout the nucleus at 0h time point (G1/S border),
they formed more distinct foci along the progression of
the S phase, which is more obvious for G9a represented
by red spots (Figure 2A). Most of major distinct foci
of the hUHRF1 and G9a were found to be colocalized
as revealed by yellow spots in the merged images
(Figure 2A). This colocalization study was repeated with
GFP-mUHRF1 (mouse UHRF1) and DsRed-G9a, essen-
tially giving similar results and confirming that both
human and mouse UHRF1 can associate with G9a in
the nucleus (Figure 2B). Furthermore, deletion of N-term-
inal hUHRF1-interacting
NiG9a) substantiallyimpaired
GFP-fused hUHRF1 in COS-7 cells (Figure S1).
region on
colocalization
G9a(DsRed-
with
UHRF1 plays arole in chromatinassociation of G9a
We have previously shown that UHRF1 recruits DNMT1
to chromatin (9). This finding led us to speculate that
UHRF1 may assist in the recruitment of G9a onto chro-
matin by direct physical interaction. To validate this
hypothesis, we first examined the chromatin-loading pat-
terns of hUHRF1 and G9a in HeLa cells after synchro-
nization at G1/S border and release from the arrest for
given hours. Consistent with the colocalization data
(Figure 2A), hUHRF1 and G9a displayed an increase in
chromatin association over time (Figure 3A). The progres-
sive chromatin loading patterns of hUHRF1 and G9a
were followed by the gradual increase in histone H3-K9
dimethylation (H3K9me2) that is the reaction product of
G9a. Next, we investigated if the absence of UHRF1 pro-
tein would affect the overall chromatin association of G9a
by examining the chromatin fractions isolated from mouse
wild-type and mUhrf1-null (?/?) ES cells. The G9a pro-
tein levels in cell extracts were similar in both ES cell lines,
whereas the chromatin fractions from mUhrf1-null ES
cells contained a significantly lower amount of G9a than
those from wild-type cells, along with the corresponding
decrease in H3K9me2 (Figure 3B). These data suggest that
UHRF1 plays a role in chromatin association of G9a.
hUHRF1 cooperates withG9a toenhance the
transcriptional repression
G9a has been shown to be involved in transcriptional
repression in euchromatin (23,25–29). We hypothesized
that hUHRF1 might recruit G9a to specific promoters
in euchromatin to suppress the transcription. To test this
possibility, reporter assays were devised using a Gal4-
driven luciferase construct (pG5-cdc2-luc) with varied
combinationsofGal4DBD-fused
hUHRF1) and GFP-G9a plasmids. The low basal activity
of pG5luc reporter containing a minimal TATA box made
it difficult to detect any distinct decrease in luciferase
activity possibly mediated by hUHRF1. To allow for a
clear detection of transcriptional repression mediated by
hUHRF1(G4-
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hUHRF1 and/or G9a, we engineered the pG5luc reporter
by replacing the intrinsic minimal promoter with a partial
cdc2 promoter fragment containing several Sp1 sites (20),
creating the pG5-cdc2-luc plasmid. Transfection with
increasing amounts of G4-hUHRF1 alone resulted in pro-
gressive repression of the reporter gene expression
(Figure 4A). To determine if hUHRF1 can cooperate
with G9a to enhance the transcriptional inhibition of the
reporter, increasingamountsofGFP-G9awere
cotransfected to COS-7 cells with or without a constant
amount of G4-hUHRF1. In the absence of G4-hUHRF1,
exogenous GFP-G9a had little effects on the repression of
the reporter gene transcription (Figure 4B, lanes 6–8)
while it significantly suppressed the transcription of the
reporter in a dose-dependent manner in the presence of
G4-hUHRF1 (Figure 4B, lanes 3–5), suggesting that the
G9a-mediated transcriptional inhibition is dependent on
the presenceofhUHRF1.To determine whether
A
B
C
D
Figure 1. Physical interaction of hUHRF1 and G9a. (A) Coimmunoprecipitation of hUHRF1 and G9a in cell extracts from COS-7 cells transfected
with GFP control or GFP-G9a. The antibodies used for immunoprecipitation (IP) are indicated at the top of the panel. Western-blot analysis of
immunoprecipitates was performed with antibodies as indicated. The input shows 2% of each lysate. (B) Coimmunoprecipitation of endogenous
hUHRF1 and G9a in HEK293 cell extracts. HEK293 cells were synchronized to late S phase by serum starvation for 20h and the subsequent release
into 10% FBS-containing medium for 15h before cell harvest. Antibodies used for immunoprecipitation and western blot are shown. The input
represents 2% of each lysate. (C) Direct binding of hUHRF1 to G9a and mapping of the G9a-binding region on hUHRF1 using GST fusions of
hUHRF1 fragments. Various domains of hUHRF1 are indicated along with a schematic presentation of the GST fusion constructs marked with
amino acid numbers. The blot from GST pull-down assay was probed with anti-G9a antibody and stained with Ponceau solution to visualize the
transferred proteins. Positions of fusion proteins are marked with asterisks. (D) Mapping of hUHRF1-binding region on G9a. Schematic diagram of
the various GST-fusion constructs of G9a is shown with amino acid numbers. Western blot with anti-hUHRF1 antibody is shown along with the
corresponding Ponceau-stained membrane.
Nucleic Acids Research,2009, Vol. 37,No. 2 497