Article

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

New England Biolabs, Ipswich, MA 01938-2723, USA.
Nucleic Acids Research (Impact Factor: 9.11). 02/2009; 37(2):493-505. DOI: 10.1093/nar/gkn961
Source: PubMed

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.

Full-text

Available from: Pierre-Olivier Estève, Jan 13, 2014
Published online 4 December 2008 Nucleic 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 Kim
1
, Pierre-Olivier Este
`
ve
1
, Steven E. Jacobsen
2
and Sriharsa Pradhan
1,
*
1
New England Biolabs, Ipswich, MA 01938-2723 and
2
Department 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
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 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 suggest that
UHRF1 may serve as a focal point of transcriptional
regulation mediated by G9a and other chromatin
modification enzymes.
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-
matic localization of human UHRF1, and down-
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
*To whom correspondence should be addressed. Tel: +1 978 380 7227; Fax: +1 978 921 1350; Email: pradhan@neb.com
ß 2008 The Author(s)
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Page 1
modifications including methylation, acetylation, phos-
phorylation and ubiquitination (12). Enzymes that
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 changes in chromatin structure occurring
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 of UHRF1
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.
MATERIALS AND METHODS
Cell culture, transfections and RNA 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 55 mM b-mercaptoethanol.
All media (Invitrogen) were supplemented with 2 mM
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 20–100 nM
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 (3 mg/ml) instead of thymidine (2 mM) 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-
NG9a) was previously described (21). The DsRed-
NG9a 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 (BD Biosciences), anti-G9a Ab
(Sigma), anti-human p21 Ab (Cell Signaling
Technology), anti-mouse p21 Ab (Abcam), anti-dimethyl
histone H3 (Lys9) Ab (Millipore), anti-histone H3 Ab
(Cell Signaling Technology), anti-phospho-histone H3
494
Nucleic Acids Research, 2009, Vol. 37, No. 2
Page 2
(Ser10) Ab (Cell Signaling Technology), anti-actin Ab
(Sigma) and anti-DNMT1 Ab (New England Biolabs).
Coimmunoprecipitation and western blot analysis
After 48 h 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
(1.2 mg) were pre-incubated with BSA-blocked protein
G-magnetic beads (New England Biolabs) for 1 h at 48C
to reduce non-specific binding of proteins to the beads.
After brief spin, the precleared cell lysates were incubated
with 2 mg of indicated antibodies for 2 h at 48C before
precipitation of the immune complexes with protein
G-magnetic beads for 1 h. Immunoprecipitates were ana-
lyzed using western blot as described previously (19). For
coimmunoprecipitation of endogenous proteins, HEK293
cells were synchronized by serum starvation for 20 h and
the subsequent release into 10% FBS-containing medium
for 15 h 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 or GFP-mUHRF1 plasmids with
TransPass D2 reagent (New England Biolabs) for 48 h.
Fluorescence microscopy was performed as described pre-
viously (19). Cells were visualized with a Zeiss 200M
microscope with a 63 oil objective lens at 488 nm for
GFP-hUHRF1 and GFP-mUHRF1 proteins, 568 nm for
DsRed-G9a detection and 460 nm for DNA staining with
Hoechst 33342.
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 20 s followed by centrifugation at 4000 g for
5 min. 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 2 h 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 (50 mM Tris–HCl, pH 8.0,
500 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1%
deoxycholate, 0.1% SDS) once. DNA was eluted in 1%
SDS elution buffer (1% SDS, 10 mM EDTA, 50 mM
Tris–HCl, pH 8.0) and de-crosslinked at 658C overnight.
DNA was purified by using QIAquick PCR purification
kit (Qiagen) and eluted in 50 ml 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 48 h 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 5
0
-ATGGA
ACTTCGACTTTGTCACC-3
0
and 5
0
-AGGCACAAGG
GTACAAGACAGT-3
0
(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 10
5
cells 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 24 h incubation,
BrdU labeling was performed for 2 h and determined by
Nucleic Acids Research, 2009, Vol. 37, No. 2 495
Page 3
using cell proliferation ELISA kit (Roche) according to
the manufacturer’s instruction.
Bisulfite sequencing
Bisulfite conversion of genomic DNA (2 mg) 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:
5
0
-TTTTTGTTTGTTAGAGTGGGTTAG-3
0
and 5
0
-AC
AACTACTCACACCTCAACTAA-3
0
. 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 with G9a in vivo and in 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
presence of specific association between G9a and
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.
UHRF1 colocalizes with G9a in vivo
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 0 h 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 region on G9a (DsRed-
NiG9a) substantially impaired colocalization with
GFP-fused hUHRF1 in COS-7 cells (Figure S1).
UHRF1 plays a role in chromatin association 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 with G9a to enhance 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
combinations of Gal4DBD-fused hUHRF1 (G4-
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
496
Nucleic Acids Research, 2009, Vol. 37, No. 2
Page 4
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, increasing amounts of GFP-G9a were
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 presence of hUHRF1. 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 20 h and the subsequent release
into 10% FBS-containing medium for 15 h 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
Page 5
hUHRF1 affects the G9a-mediated transcriptional repres-
sion by additional mechanisms other than physical recruit-
ment of G9a to the promoter, we further examined the
possibility that hUHRF1 may modulate G9a methyltrans-
ferase activity by its interaction with G9a. Using the pur-
ified hUHRF1 and G9a that were used for GST pull-down
assays (Figure 1C and D), G9a methyltransferase activity
was measured in vitro in the presence or absence of
hUHRF1, and no significant difference in activity was
observed (data not shown). These results provide the evi-
dence supporting that hUHRF1 cooperates with G9a to
enhance the transcriptional repression primarily by
recruiting G9a to the target promoters.
hUHRF1 suppresses p21 expression
Several studies demonstrated that hUHRF1 protein levels
are elevated in tumor tissues (8,11,30), and the protein is
required for proliferation in cancer cells (2,8,11). The
hUHRF1 was also shown to bind to the methylated pro-
moter of tumor suppressors such as p14 and p16 (8),
possibly to suppress the expression of these genes in
cancer cells. However, the same study showed that
hUHRF1 expression level did not have any correlation
with the expression of these cell-cycle regulators in prolif-
erating cells, suggesting that there might be additional
hUHRF1 targets involved in cell-cycle regulation to pro-
mote proliferation in cancer cells. Previously, it was shown
that G9a cooperates with CDP/cut and Gfi1 transcrip-
tional regulators to suppress p21 expression (23,28). On
the basis of these prior observations, we hypothesized that
p21 promoter may be one of the in vivo targets to which
hUHRF1 directs G9a to enhance the repression of the
tumor suppressor, thus promoting proliferation in
cancer cells. To test this hypothesis, we examined the
p21 mRNA and protein levels after the individual KD
of hUHRF1 and G9a in HeLa cells by siRNA transfec-
tion. The G9a KD resulted in 60% increase in p21
mRNA level determined by quantitative RT–PCR, com-
pared to the CTL KD (Figure 5A). Although the fold
increase is relatively small (<2-fold), it was a statistically
significant change that was reflected by the roughly
GFP-hUHRF1 DsRed-G9a Merged Nucleus Nucleus/Merged
0
2-4
8-12
>15
A
(hr)
B
DsRed-G9a
>15
GFP-mUHRF1
Merged
2-4
Nucleus Nucleus/Merged
(hr)
Figure 2. Colocalization of UHRF1 and G9a. (A) Subnuclear localization of GFP-hUHRF1 and DsRed-G9a transiently expressed in COS-7 cells.
Cells were synchronized with aphidicolin and released from G1 arrest for a given number of hours through S phase. Nuclei were visualized with
Hoechst stain. (B) Colocalization of GFP-mUHRF1 and DsRed-G9a in COS-7 cells at given hours of release from synchronization.
498 Nucleic Acids Research, 2009, Vol. 37, No. 2
Page 6
corresponding change in the protein levels (Figure 5B). As
expected, hUHRF1 KD significantly induced the expres-
sion of p21 at both mRNA and protein levels (Figure 5A
and B). In addition, dual hUHRF1 and G9a KD resulted
in additive effects on the induction of p21 expression at
both transcript and protein levels (Figure 5A and B). The
p21 is a well-characterized target gene of p53-mediated
transcriptional regulation (31). However, HeLa cells
tested in this study lack functional p53, suggesting that
the p21 induction in siRNA-transfected HeLa cells is a
p53-independent process. To verify this observation, we
performed a similar experiment using COS-7 cell line
where the endogenous p53 is abundantly expressed but
inactivated by SV40 large T antigen. As shown in
Figure 5C, UHRF1 KD did not affect the p53 protein
level, yet substantially elevated the p21 expression,
demonstrating that the UHRF1 KD-mediated p21 eleva-
tion is independent of p53. Then, we further validated the
observation that UHRF1 is involved in the repression of
p21 expression by examining the amounts of p21 protein
A
B
Figure 3. UHRF1 affects chromatin association of G9a. (A) Concurrent loading of hUHRF1 and G9a onto chromatin during S phase. HeLa cells
were synchronized by double aphidicolin/thymidine block and released to regular medium for hours indicated at the top of the panel. The chromatin
fractions at each time point were used for western-blot analysis with antibodies indicated. Anti-H3 and H3 phopho-Ser10 (H3 pSer10) antibodies
were used for a loading control and mitosis marker, respectively. Densitometric scans of hUHRF1, G9a and H3K9me2 levels in each chromatin
fraction are shown after normalization by the corresponding H3 level. (B) Impaired chromatin association of G9a in the absence of mUHRF1. Cell
extracts and chromatin were prepared from wild type (+/+) and mUhrf1-null (/) ES (ES) cells and used to detect the presence of G9a and
dimethylated H3K9 (H3K9me2) with antibodies indicated. The relative G9a level in cell extracts is shown as percentage G9a at the bottom of the
panel by a densitometric analysis after normalization by the actin-loading control. Normalized densitometric scans of G9a and H3K9me2 levels in
chromatin are shown in graphs.
Nucleic Acids Research, 2009, Vol. 37, No. 2 499
Page 7
in cell extracts of mouse wild-type and mUhrf1-null (/)
ES cells. As shown in Figure 5D, the p21 protein level
was found to be elevated in the mUhrf1(/) cells.
Furthermore, mUhrf1(/) ES cells displayed retarded
growth and less BrdU (bromodeoxyuridine) incorporation
compared to the wild-type counterpart although it is not
clear whether these effects directly resulted from the
increased p21 expression in mUhrf1(/) ES cells
(Figure 5E and F).
hUHRF1 cooperates with G9a to enhance the transcriptional
repression of p21 promoter
We also examined whether hUHRF1 is recruited to the
p21 promoter by reporter assays using pGL2–p21. If the
p21 promoter is one of the natural promoters targeted by
hUHRF1, it should be recruited to the naive promoter
without using the Gal4 reporter system that was used in
Figure 4. Indeed, exogenous expression of hUHRF1
inhibited the p21 promoter activity in a dose-dependent
manner, possibly by recruiting the endogenous epigenetic
regulators including G9a to the p21 promoter (Figure 6A).
Cotransfection of increasing amounts of G9a expression
vector with a constant amount of hUHRF1 plasmid
resulted in further repression of the reporter (Figure 6B,
lanes 3–5), compared to the case without the exogenous
hUHRF1 (Figure 6B, lanes 6–8). The dose-dependent
decrease in p21 promoter activity in the absence of exo-
genous hUHRF1 may have been caused by the recruit-
ment of endogenous hUHRF1 to the promoter
(Figure 6B, lanes 6–8). To validate that the UHRF1-
mediated p21 promoter repression resulted from the
direct interaction between G9a and UHRF1, a G9a
mutant lacking the N-terminal UHRF1-interacting
region (21) was used for the identical reporter assay in
parallel with wild-type G9a. As shown in Figure 6C, the
deletion of UHRF1-interaction region on G9a (NiG9a)
impaired UHRF1/G9a-mediated repression of p21 pro-
moter. Taken together, p21 appears to be one of the
in vivo targets whose expression is repressed by coordi-
nated actions of UHRF1/G9a.
hUHRF1 recruits G9a and other chromatin-modifying
enzymes to p21 promoter
To demonstrate that hUHRF1-mediated G9a recruitment
to the endogenous p21 promoter is one of the mechanisms
underlying p21 repression in HeLa cells, we transfected
HeLa cells with hUHRF1 or control siRNAs and per-
formed ChIP with selected antibodies. The hUHRF1
and G9a were found to associate with the proximal
region of the p21 promoter in CTL KD (transfection
with control siRNA), suggesting that these proteins are
recruited to the p21 promoter under the native conditions
(Figure 7B). In agreement with G9a association with the
promoter, dimethylation at histone H3K9 (H3K9me2)
was detected in CTL KD. Furthermore, DNMT1 was
also found to associate with the proximal region of the
promoter. As demonstrated by the previous studies,
the proximal region of the p21 promoter also displayed
the association with HDAC1 (32). None of these enzymes
were found on the distal region of the promoter. Then, we
examined the effects of hUHRF1 on p21 promoter asso-
ciation of G9a and other chromatin modifiers described
earlier. As expected, hUHRF1 KD significantly reduced
the amount of hUHRF1 bound to the proximal region of
A
B
Figure 4. hUHRF1 cooperates with G9a to enhance transcriptional
repression. (A) Transcriptional repression of luciferase reporter gene
by hUHRF1. COS-7 cells were cotransfected with a Gal4-driven luci-
ferase reporter (2 mg) and increasing amounts of Gal4DBD-hUHRF1
(G4-hUHRF1). Luciferase activity was measured 48 h post-transfection
and represented by the means SD of duplicate determinations from
three independent experiments. ( B) Enhanced transcriptional repression
by hUHRF1-mediated G9a recruitment. Luciferase activities were mea-
sured after 48 h of transfection using COS-7 cells cotransfected with the
same reporter in (A), increasing amounts of EGFP-G9a (0.1–1 mg) and
with or without a constant amount (0.1 mg) of G4-hUHRF1. The data
represent the means SD of duplicate determinations from four sepa-
rate experiments.
500 Nucleic Acids Research, 2009, Vol. 37, No. 2
Page 8
the p21 promoter compared to the CTL KD. The
hUHRF1 KD also decreased G9a association with the
p21 promoter resulting in reduced H3K9me2 modification
on it, suggesting that G9a loading to the promoter is
dependent on hUHRF1. We have previously shown that
hUHRF1 KD impairs the recruitment of DNMT1 onto
chromatin at global levels (9). Consistent with this
finding, hUHRF1 KD reduced the DNMT1 association
with the p21 promoter. In addition, HDAC1 association
was also decreased upon hUHRF1 KD (Figure 7B).
A
B
C
D
E
F
Figure 5. hUHRF1 suppresses p21 expression in cooperation with G9a. (A) Quantitative RT–PCR analysis of p21 expression. Total RNA was
isolated from HeLa cells transfected with siRNAs as indicated. The Q-PCR data normalized by GAPDH control are shown as the means SD of
triplicate determinations from four independent experiments. Statistical significance of the differences among the groups was determined by Student’s
t-test.
P < 0.05;

P < 0.01. (B) Western blot analysis of p21 expression in siRNA-mediated KD HeLa cells. After each KD, as indicated at the top
of the panel, cell extracts were used for detection of p21. The densitometric scan of p21 expression is shown at the right. (C) Western blot analysis of
p53 and p21 expression in COS-7 cells after siRNA-mediated KD of hUHRF1. The densitometric scan of p21 expression is shown at the right. (D)
Enhanced p21 expression in mUhrf1/ ES cells. The blot for p21 is shown along with the densitometric analysis. (E) Growth of wild type (+/+)
and mUhrf1-null (/) ES cells. Cell growth was monitored over 3 days after plating, and the data represent the means SD of six replicates. (F)
BrdU incorporation of wild-type (+/+) and mUhrf1-null (/) ES cells. After 24 h incubation, BrdU labeling was performed for 2 h and determined
by a colorimetric assay. The data represent the means SD of three separate experiments. Statistical significance of the difference between the
groups was determined by Student’s t-test.
P < 0.05.
Nucleic Acids Research, 2009, Vol. 37, No. 2 501
Page 9
Next, we investigated whether G9a and its histone methy-
lation can affect the loading of hUHRF1 to the promoter,
because hUHRF1 was recently identified as a methyl K9-
specific histone H3-binding protein (4). Therefore, G9a
protein level was reduced by siRNA transfection and the
relative p21 promoter occupancy of hUHRF1 was exam-
ined by quantitative ChIP analysis. As shown in
Figure 7C, G9a KD moderately impaired the hUHRF1
association with the promoter (20% compared to the
CTL KD), whereas hUHRF1 KD more profoundly
(55%) disrupted the G9a binding to the promoter and
reduced the H3K9me2 to a comparable level to that of
G9a KD. Previously, we have also shown that siRNA-
mediated KD of DNMT1 impairs G9a loading onto
chromatin (19). Moreover, there are several studies
demonstrating the interdependency between histone and
DNA methylation (33–35). To test whether DNMT1 KD
can negatively affect G9a recruitment onto the p21 pro-
moter, HeLa cells were transfected with DNMT1 or con-
trol siRNAs. After DNMT1 KD, the promoter occupancy
A
C
B
Figure 6. hUHRF1 cooperates with G9a to enhance the transcriptional repression of p21 promoter. (A) hUHRF1-mediated transcriptional repres-
sion of the p21 promoter-luciferase reporter. COS-7 cells were cotransfected with the reporter (2 mg) and increasing amounts of EGFP-hUHRF1 as
indicated at the bottom of the panel. (B) Enhanced transcriptional repression by G9a in the presence of exogenous hUHRF1. Luciferase activities
were measured from COS-7 cells cotransfected with the same reporter described earlier and increasing amounts of EGFP-G9a (0.1–1 mg) with or
without a constant amount (0.4 mg) of EGFP-hUHRF1. The luciferase assays were performed as described in Figure 4, and the data represent the
means SD of duplicate determinations from three separate experiments. Western blot analyses of hUHRF1 and G9a expression by anti-GFP
antibody are shown for each cotransfection group. (C) Loss of interaction between UHRF1 and G9a abolishes the UHRF1/G9a-mediated repression
of p21 promoter. Reporter assays were performed as described in (B), using the wild-type G9a plasmid (EGFP-G9a) and its deletion mutant lacking
the N-terminal UHRF1-interacting region (EGFP-NG9a). Expression of hUHRF1 and G9a/NG9a is shown by western blot analyses with anti-
GFP antibody.
502 Nucleic Acids Research, 2009, Vol. 37, No. 2
Page 10
of DNMT1 was substantially decreased, whereas G9a
association and H3K9me2 modification were not affected
much compared to CTL KD (Figure 7D). These results
indicate that DNMT1 makes little contribution to the
physical recruitment of G9a to p21 promoter. In addition,
DNMT1 KD did not appear to affect UHRF1 loading
onto the promoter significantly (Figure 7D). Consistent
with this observation, bisulfite sequencing of the p21 pro-
moter region (398 to +11) revealed that the CpGs
within the sequence is infrequently methylated except for
one CpG dinucleotide at 371 position which was methy-
lated by 50% (Figure S2). Although the functional signif-
icance of DNMT1 recruitment onto the p21 promoter is
not clear given the infrequent methylation patterns on the
p21 promoter, these results suggest that UHRF1 recruit-
ment onto p21 promoter may use additional mechanisms
other than CpG methylation.
DISCUSSION
In mammalian cells, gene expression is regulated via epi-
genetic mechanisms. Some mechanisms involve covalent
modifications on DNA and histone molecules on the chro-
matin to create either a permissive or a repressive tran-
scriptional environment. Given the emerging evidence
supporting the role of UHRF1 as a transcriptional regu-
lator (2,16,17) and its interaction with a histone methyl-
transferase G9a in this study, we hypothesized that
UHRF1 might serve as a transcriptional corepressor
along with G9a that has been shown to be involved in
transcriptional repression in euchromatin (25). Indeed,
UHRF1 alone was capable of repressing reporter gene
transcription in a dose-dependent manner, possibly by
recruiting the endogenous epigenetic regulators to the
target promoters. Consistent with this notion, coexpres-
sion of UHRF1 and G9a enhanced the transcriptional
repression of the reporter genes, suggesting that the
direct physical interaction of UHRF1 and G9a constitutes
an effective silencer complex. Among many possible
endogenous target genes regulated by the cooperation of
UHRF1/G9a, we examined the expression of p21 in HeLa
cells where it is poorly expressed in a p53-independent
manner. The p21 is a cell-cycle regulator by inhibiting
cyclin-dependent kinases and a modulator of apoptosis
by interacting with other proteins involved in the regula-
tion of apoptosis (36). Transcription of p21 gene relies on
the control of multiple different regulators, of which many
are yet to be identified. In this study, UHRF1 appears to
be one of the intrinsic regulators of p21 gene expression.
We have demonstrated that the endogenous p21 pro-
moter displayed the presence of UHRF1, G9a and other
chromatin-modifying enzymes such as DNMT1 and
HDAC1 in HeLa cells by ChIP assays. The siRNA-
mediated UHRF1 KD significantly reduced the p21
promoter occupancy of all three chromatin-modifying
enzymes, placing UHRF1 as a focal point of recruitment
of these enzymes and also suggesting possible coordinated
efforts of UHRF1, G9a, DNMT1 and HDAC1 for efficient
repression of p21. This observation raises an intriguing
question on whether all these proteins form a single macro-
molecular complex or various distinct complexes depend-
ing on the specific promoter architecture and cellular
context. Gel-filtration chromatography of Jurkat cell
lysates containing the endogenously expressed proteins
mentioned earlier has revealed that UHRF1 cofractionates
with G9a, DNMT1 and HDAC1 to a various extent, indi-
cating the possible formation of the common complexes
that are assembled by UHRF1 (data not shown).
A similar coordinated epigenetic repression of p21 was
previously reported, involving a transcriptional regulator,
A
B
C
D
Figure 7. hUHRF1 recruits G9a and other chromatin modification
enzymes to p21 promoter. (A) Linearity of PCR amplification using
primer sets for proximal (385 to 240) and distal (4164 to
3959) regions of p21 promoter with increasing amount of input
DNA. (B) ChIP analysis of p21 promoter after KD of hUHRF1.
HeLa cells were transfected with either control siRNA (CTL KD) or
hUHRF1 siRNA (hUHRF1 KD). Using the chromatin isolated from
the KD cells, ChIP was performed to detect the proteins or histone
modification as indicated at the top of the panel. 5% input is shown.
(C) Quantitative ChIP analysis for relative p21 promoter occupancy of
hUHRF1, G9a and dimethylated H3K9 (H3K9me2) after KD of
hUHRF1 or G9a. Q-PCR data of each group were normalized to its
input as % input. The relative p21 promoter occupancy of hUHRF1 or
G9a KD samples represents the fold change in percentage input over
that of the CTL KD. Error bars indicate standard deviation of three
independent experiments. (D) ChIP analysis of p21 promoter after KD
of DNMT1. HeLa cells were transfected with either control siRNA
(CTL KD) or DNMT1 siRNA (DNMT1 KD). CHIP was performed
as described in (B) and 5% input is shown.
Nucleic Acids Research, 2009, Vol. 37, No. 2 503
Page 11
Gfi1 (28). The Gfi1 recruits G9a and HDAC1 to p21 pro-
moter and represses its expression in HL-60 cells. Both
HDAC1 and G9a were found in the repressive complex
assembled by Gfi1, and the KD of Gfi1 elevated the p21
expression by 2–3-fold. We also observed a similar level of
p21 induction after UHRF1 KD in HeLa and COS-7 cells.
Other repressor proteins can also modulate p21 expression
by epigenetic mechanisms. For example, a transcription
factor CDP/cut was shown to recruit G9a to the human
p21 promoter, and the transcriptional repression function
of CDP/cut is mediated through the activity of its asso-
ciated G9a (23). Polycomb group (PcG) proteins are chro-
matin modifiers that can transcriptionally silence their
target genes and maintain them in the repressed state
through cell divisions during development (37). Recently,
NSPc1, a transcriptional repressor homologous with PcG
protein Bmi-1, was demonstrated to repress p21 expres-
sion via direct binding to the retinoid acid response ele-
ment of its promoter (38).
Furthermore, the murine Uhrf1-null ES cells also dis-
played a higher level of p21 protein, implicating the role of
UHRF1 in transcriptional repression in ES cell environ-
ment. An orphan nuclear receptor TLX recruits a set of
HDACs to its target genes for transcriptional repression in
neural stem cells (39). One of the genes targeted by this
repressive complex is p21, suggesting a role very similar to
that of UHRF1-G9a repressive complex. Both histone
H3K9me2 accumulation via G9a and deacetylation by
HDACs are found on many repressed genes in cell lines.
As siRNA-mediated KD of G9a or inhibition of HDACs
can result in p21 derepression, it is also plausible that
HDACs and G9a crosstalk during p21 repression. In
fact, a recent study on SHP-mediated regulation of
CYP7A1 promoter revealed the functional interplay
between SHP-recruited HDACs and G9a in altering the
chromatin structure of CYP7A1 promoter (29). These
findings suggest multiple mechanisms of p21 gene expres-
sion that is modulated by both histone methylation and
deacetylation. Moreover, as exemplified earlier, the pre-
sence of multiple functional equivalents of UHRF1 that
can recruit similar effectors such as G9a and HDAC1
appears to ensure tight repression of p21 in cancer cells.
Perhaps, this may constitute the basis for the finding that
functional disruption of a single factor in p21 repression
only results in relatively minor p21 derepression (2–3-
fold) as we and other investigators observed.
The UHRF1-mediated p21 repression presents another
intriguing idea of reciprocal regulation occurring between
UHRF1 and p21 expression in response to different extra-
cellular stimuli, because hUHRF1 expression was shown
to be down-regulated by p53/p21-dependent DNA
damage check point signal (40). This observation may
lead to a hypothesis that, in the presence of DNA
damage, the p53/p21-dependent checkpoint response
may outweigh the transcriptional repression effects of
UHRF1 against p21 resulting in down-regulation of
UHRF1 expression, whereas the absence of DNA
damage signal allows UHRF1 to keep p21 in a repressed
state promoting cell proliferation. The reciprocal down-
regulation, involving UHRF1, can be observed in another
case where overexpression of hUHRF1 negatively
regulates pRb expression (16), whereas pRb interacts
with E2F-1 transcription factor and thereby inhibits the
expression of the E2F-1 target genes including UHRF1
(8,30). These findings point to the dynamic regulation
mode for UHRF1 in cancer cells.
In summary, our data demonstrate a new role of
UHRF1 as a transcriptional corepressor in recruitment
of histone methyltransferase G9a and other chromatin-
modifying enzymes to target promoters, and suggest that
UHRF1 acts as a focal point of gene repression mediated
by various chromatin modifiers.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
ACKNOWLEDGEMENTS
We thank Dr Jane B. Trepel for kindly providing the
pGL2–p21 plasmid. We also thank George R. Feehery
and other colleagues of our laboratory for technical assis-
tance and support. We are grateful to Drs D.G. Comb and
Rich Roberts at New England Biolabs, Inc. for their sup-
port and encouragement. S. E. J. is an Investigator of the
Howard Hughes Medical Institute.
FUNDING
Funding for open access charge: New England Biolabs, Inc.
Conflict of interest statement. None declared.
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  • Source
    • "UHRF1 is an oncogenic factor that is overexpressed in numerous cancers252627. UHRF1 is able to read both DNA methylation and histone methylation, physically linking these two epigenetic markers, because UHRF1 possesses several domains (e.g., ubiquitin-like domain, PHD finger, SRA domain, and ring finger) [28]. The ) were transfected with siUHRF1-535, siUHRF1-1453, or scrambled siRNA control and seeded into the upper chamber of a Transwell plate in 0.1 mL media (supplemented with 1 % FBS). "
    Full-text · Article · Feb 2016
  • Source
    • "UHRF1 is an oncogenic factor that is overexpressed in numerous cancers252627. UHRF1 is able to read both DNA methylation and histone methylation, physically linking these two epigenetic markers, because UHRF1 possesses several domains (e.g., ubiquitin-like domain, PHD finger, SRA domain, and ring finger) [28]. The ) were transfected with siUHRF1-535, siUHRF1-1453, or scrambled siRNA control and seeded into the upper chamber of a Transwell plate in 0.1 mL media (supplemented with 1 % FBS). "
    [Show abstract] [Hide abstract] ABSTRACT: Biochemical recurrence (BCR) is widely used to define the treatment success and to make decisions on if or how to initiate a secondary therapy, but uniform criteria to define BCR after radical prostatectomy (RP) is not yet completely assessed. UHRF1 has a unique function in regulating the epigenome by linking DNA methylation with histone marks. The clinical value of UHRF1 in PCa has not been well done. Therefore, we evaluated the prognostic significance of UHRF1. UHRF1 expression in PCa cells was monitored by qRT-PCR and Western blot analyses. UHRF1 expression was knocked down using specific siRNAs, and the effects of knockdown on the proliferation, migration, cell cycle, and apoptosis of PCa cell lines were investigated. UHRF1 protein expression was evaluated in 225 PCa specimens using immunohistochemistry in tissue microarrays. Correlations between UHRF1 expression and the clinical features of PCa were assessed. The results showed that UHRF1 was overexpressed in almost all of the PCa cell lines. In PCa cells, UHRF1 knockdown inhibited cell proliferation and migration, and induced apoptosis. UHRF1 expression levels were correlated with some clinical features of PCa. Multivariate analysis showed that UHRF1 expression was an independent prognostic factor for biochemical recurrence-free survival. UHRF1 functions as an oncogene in prostate cancer and appears to be capable of predicting the risk of biochemical recurrence in PCa patients after radical prostatectomy, and may serve as a potential therapeutic target for PCa.
    Full-text · Article · Feb 2016 · Journal of Experimental & Clinical Cancer Research
  • Source
    • "asis, HER2 status, stage, and carcinoembryonic antigen (CEA) level are important prognostic factors for gastric cancer. [2,18,19] Epigenetic modifications play a central role in gastric carcinogenesis. [20‑22] UHRF‑1, as an epigenetic regulator, has been shown to be overexpressed and to coordinate tumor suppressor gene silencing in several cancers. [7] UHRF‑1 has been suggested to be an important biomarker to discriminate between cervical high‑grade and low‑grade cancer lesions. [23] Another study has highlighted the efficiency of UHRF‑1 as a marker to differentially diagnose pancreatic adenocarcinoma, chronic pancreatitis, and normal pancreas. [24] UHRF‑1 overexpression was also foun"
    [Show abstract] [Hide abstract] ABSTRACT: Background/Aims: This study aimed to examine whether UHRF-1 and p53 overexpression is a prognostic marker for gastric cancer. Patients and Methods: Sixty-four patients with gastric cancer (study group) and 23 patients with gastritis (control group) were evaluated. Immunohistochemistry was used to examine expression of UHRF-1 and p53 in gastric cancers and a control group diagnosed with gastritis. Results: The median age was 63 years (18-83 years) in the study group. UHRF-1 was positive in 15 (23%) patients with gastric cancer and fi ve (21.7%) patients with gastritis (P = 0.559). UHRF1 expression level in gastric cancer is more powerful than in gastritis (P = 0.046). Thirty-seven (61%) patients with gastric cancer and only one patient with gastritis were p53 positive (P < 0.001). After a median follow-up of 12 months (1-110), the 2-year overall survival rates were 55% and 30% in negative and positive p53, respectively (P = 0.084). Also, the 2-year overall survival rates were 45% and 53% in negative and positive UHRF-1, respectively (P = 0.132). Conclusion: According to this study, UHRF-1and p53 were not prognostic factors for gastric cancer, whereas they may have a diagnostic value for differantiating between gastric cancer and gastritis.
    Full-text · Article · Jan 2016 · Saudi Journal of Gastroenterology
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