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J. Biol. Chem., Vol. 275, Issue 50, 39411-39419, December 15, 2000
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From the Departments of
Received for publication, July 24, 2000
RET finger protein (RFP) belongs to the large
B-box RING finger protein family and is known to become oncogenic by
fusion with RET tyrosine kinase. Although RFP is reported to be a
nuclear protein that is present in the nuclear matrix, its function is largely unknown. Here we show that RFP interacts with Enhancer of
Polycomb (EPC) and strongly represses the gene transcription. Yeast
two-hybrid assays revealed that the coiled-coil domain of RFP was
associated with the EPcA domain and the carboxyl-terminal region of
EPC. In addition, both proteins were co-precipitated from the lysates
of human cells and mostly colocalized in the nucleus. Using the
luciferase reporter-gene assay, we found that they repress the gene
transcription activity independent of the differences of enhancers and
promoters used, although the repressive activity of RFP was much
stronger than that of EPC. The coiled-coil domain of RFP and the
carboxyl-terminal region of EPC were most important for the repressive
activity of each protein, whereas the EPcA domain had the transcription
activating ability that is unique as the Polycomb group protein
function. These results suggested that RFP may be involved in the
epigenetic gene silencing mechanism cooperating with Polycomb group
proteins and that EPC is a unique molecule with both repressive and
transactivating activities.
RET finger protein
(RFP)1 belongs to the large
B-box RING finger protein family, members of which contain a tripartite
motif consisting of a RING finger, a B-box, and a coiled-coil domain with three defined helices (1-5). In addition, it contains a specific
carboxyl-terminal region known as the RFP domain or the B30.2-like
domain (6). RFP mRNA was detected in a variety of human
and rodent tumor cell lines as well as in male germ cells at high
levels (1). It was also shown that RFP is associated with the nuclear
matrix (7) and is expressed in the nuclei of various cells, including
peripheral and central neurons, hepatocytes and adrenal chromaffin
cells (8), and partially colocalizes with PML and int-6 (9, 10).
There are over 200 members of the RING finger protein family reported
to date, including PML, BMI1/Mel-18, RING1, and KAP-1 (11-13). RING
finger proteins are thought to play roles in the formation and
architecture of large protein complexes that contribute to diverse
cellular processes such as oncogenesis, apoptosis, development, and
ubiquitination (11, 14). BMI1/Mel-18 and RING1 belong to Polycomb group
proteins (15, 16), and especially RING1 was reported to form the
Polycomb group protein complex with HPC2, BMI1, and HPH and repress the
gene transcription (16, 17). In addition, PML, BMI1/Mel-18, RING1 and
RFP are all involved in oncogenesis by the formation of oncogenic
fusion proteins (1, 18-22) or by deregulating the expression levels of
certain oncogenes (17, 23, 24).
Polycomb group proteins have been initially identified in
Drosophila as being involved in the maintenance of the
correct expression pattern of homeotic genes. Polycomb group proteins
form the chromatin associated-protein complexes that are involved in
the epigenetic gene silencing and in the maintenance of cell type
specificity (25-27). The homologues of Polycomb group proteins were
also found in other vertebrates and invertebrates such as human, mouse,
chicken, Xenopus, and Caenorhabditis elegans
(28). On the other hand, epigenetic gene activation mechanism is known
to be mediated by distinct protein complexes consisting of trithorax
group proteins.
Enhancer of Polycomb (EPC) is a unique member of the Polycomb group
proteins (29). Although mutations in E(Pc), the
Drosophila homologue of EPC, exhibit no homeotic
transformations, they enhance homeotic mutations by other Polycomb
group genes, such as Pc, Pcl, ph,
Sce, Scm, and sxc (30, 31). Mutations
in E(Pc) also function as the strong suppressors of
position-effect variegation (PEV) that is another epigenetic gene
silencing mechanism and is associated with the heterochromatin
formation (32, 33). Among Polycomb group genes, only E(Pc)
and Enhancer of zeste (E(z)) were shown to function as
Su(var)s (suppressors of PEV) (29). Interestingly, E(z) was
reported to have both transcription activating and repressing functions
and can be classified as both Polycomb and trithorax group proteins
(34). In contrast to most of other Polycomb group proteins, no
interacting proteins of EPC have been found so far (28). These results
suggest that EPC may have a unique role in the gene silencing
mechanisms mediated by Polycomb group proteins.
Here we report that RFP interacts with human EPC and that RFP itself
functions as a strong transcriptional repressor when targeted to
reporter genes. This interaction is mediated by the binding between the
coiled-coil domain of RFP and the conserved EPcA domain or the
carboxyl-terminal region of EPC. Immunofluorescence study revealed that
RFP mostly colocalizes with EPC in the nuclei of human cells,
suggesting cooperative roles of RFP and EPC in gene silencing.
Plasmid Constructs--
The RING finger B box region (amino
acids 2 to 135), the coiled-coil domain region (amino acids 132 to
317), and the RFP domain region (amino acids 316 to 514) (Fig.
2A) were amplified by specific primers with a flanking
EcoRI site on the 5' primer and a BamHI site on
the 3' primer, using the full-length human RFP cDNA as a
template. The resulting PCR products were subcloned into the pGEM-T
vector (Promega) and sequenced to ensure that there were no mutations
due to PCR errors. Each product was subcloned into the
EcoRI/BamHI sites of pAS2-1 vector
(CLONTECH).
The cDNA fragment obtained by yeast two-hybrid screening encoded
amino acids 1 to 252 of human EPC. To obtain the full-length EPC cDNA, we performed 5' and 3' rapid amplification of
cDNA ends from human testis cDNA library by the specific
nested primers using the Marathon cDNA amplification kit
(CLONTECH). Both 5' and 3' rapid amplification of
cDNA ends products were subcloned into the pGEM-T vector (Promega).
Sequence analysis resulted in the identification of an additional
249-bp 5' cDNA fragment and an additional 1887-bp 3' cDNA
fragment that included a termination codon. To generate the full-length
EPC cDNA, an NcoI/XbaI fragment of
pACT2-EPC (amino acids 1-252) vector that was derived from the first
yeast two-hybrid screening was ligated into the
NcoI/XbaI sites of 3' rapid amplification of
cDNA ends product cloned into the pGEM-T vector.
EPcA domain region (A region, amino acids 2 to 285), EPcB domain region
(B region, amino acids 280 to 496), EPcC domain and glutamine-rich
region (CQ region, amino acids 493 to 620), and CQ and
carboxyl-terminal region (CQCT region, amino acids 493 to 836) (Fig.
2B) were amplified by the specific primers with a flanking
EcoRI site on the 5' primer and an XhoI site on
the 3' primer, using the full-length EPC cDNA as a
template. The PCR products were subcloned into the pGEM-T vector
(Promega) and sequenced to ensure that there were no mutations. These
products were cloned into the EcoRI/XhoI sites of
pACT2 vector (CLONTECH).
The pV5-HisC-GAL4BD plasmid was generated by two steps. A
HindIII-EcoRI fragment of the pAS2-1 vector that
contains a GAL4BD site was subcloned into the pcDNA3.1/V5-HisC
vector (Invitrogen) and then the multicloning site between
SfiI and EcoRI was replaced by that of the pACT2
vector. The full-length EPC cDNA or its fragments (A
region, B region, CQ region, and CQCT region) were cloned in-frame into
the pV5-HisC-GAL4BD plasmid. To obtain pV5-HisC-GAL4BD-RFP plasmids, a HindIII-BamHI fragment of pAS2-1
full-length RFP or pAS2-1 RFP domains was cloned
into the same site of the pcDNA3.1/V5-HisC vector.
Yeast Two-hybrid Screening of RFP Interacting Proteins--
The
full-length RFP cDNA was cloned into the pAS2-1 vector
(CLONTECH) and used as bait to screen for its
interacting proteins in a two-hybrid screen. The pAS2-1 RFP
plasmid was co-transfected with human testis cDNA library
(CLONTECH) into the Y190 strain of
Saccharomyces cerevisiae. The transformants were plated
first on selective medium lacking tryptophan and leucine, and then on selective medium lacking histidine, tryptophan, and leucine with 40 mM 3-amino-1,2,4-triazole (3-AT) (histidine
"jump-start" method). From approximately 5 × 106
independent clones, 50 colonies were found to grow on a minimal medium
lacking leucine, tryptophan, and histidine, and positive for
To characterize the interaction between each RFP and EPC domain in the
yeast two-hybrid system, the RFP and EPC domains amplified by PCR were
cloned into pAS2-1 and pACT2, respectively, and transformed into Y190.
Positive interactions meet the two criteria of growth in the presence
of 40 mM 3-AT and the expression of GAL4 Fusion Reporter-gene Targeted Repression Assay--
The
GAL4-binding site was amplified from the pFR-Luc vector (Stratagene) by
PCR and cloned into the KpnI/SacI sites of pGL3 luciferase reporter plasmid (Promega). Serum response element (SRE),
cAMP response element (CRE), and herpes simplex virus thymidine kinase
minimal promoter were cloned from pSRE-SEAP or pCRE-SEAP vectors
(CLONTECH). 293 human embryo kidney cells were
cultured in 24-well tissue culture plates and co-transfected with 30 ng of luciferase reporter plasmid, 30 ng of pRL-TK plasmid (Promega), and
240 ng of pCMV-GAL4 RFP or EPC fusion constructs by the LipofectAMINE method (Life Technologies, Inc.) according to the manufacturer's instructions. The cells were harvested 48 h after transfection, and luciferase assays were performed as described previously (35). Co-transfection with the pRL-TK plasmid was used to normalize all
luciferase values.
Antibodies--
A synthetic peptide corresponding to the
carboxyl-terminal 20 amino acids of EPC was prepared by a solid phase
method and purified by high performance liquid chromatography. Rabbits
were immunized with 100 µg of the synthetic peptide coupled to 500 µg of thyroglobulin in complete Freund's adjuvants. The anti-RFP polyclonal antibody was described previously (8).
Coimmunoprecipitation Studies--
293 cells plated in three
60-mm dishes 24 h before transfection were transfected with 2 µg
of pFLAG-CMV2 EPC expression vector using the LipofectAMINE method.
After 48 h of incubation, cells were washed in phosphate-buffered
saline (PBS) and lysed in lysis buffer (50 mM Tris-HCl, pH
7.4, 150 mM NaCl, 20 mM MgCl2,
0.5% Nonidet P-40, and 1 mM sodium orthovanadate)
containing 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml
leupeptine, 1 µg/ml aprotinin, 5 µg/ml benzamidine, and 1 µg/ml
pepstatin A. 2 µg of rabbit anti-RFP antibody with protein
A-Sepharose (Sigma) or 2 µg of mouse anti-FLAG M2 antibody (Sigma)
with protein G-Sepharose (Sigma) were incubated for 4 h at 4 °C
and then the whole cell lysates were added and incubated for 8 h
at 4 °C. After washing four times in lysis buffer and twice in PBS,
SDS sample buffer was added and boiled for 5 min. Samples were
separated on SDS-7.5% polyacrylamide gel electrophoresis and
transferred to polyvinylidene difluoride filters (Millipore). After
blocking with 5% skim milk in PBS and 0.05% Tween 20, filters were
cut into upper and lower strips and incubated with anti-EPC and
anti-RFP polyclonal antibodies, respectively. Then peroxiase-conjugated goat anti-rabbit IgG antibodies were added and developed by the ECL
Plus Western blotting detection system (Amersham Pharmacia Biotech).
Immunofluorescence and Confocal Microscopy--
SW480 human
colorectal adenocarcinoma cells were grown on coverslips for 24 h
and fixed in 4% paraformaldehyde in PBS for 5 min at room temperature,
incubated twice for 5 min in PBS, and permeabilized for 5 min in PBS
with 0.1% Triton X-100. Cells were blocked with 1% bovine serum
albumin in PBS. For indirect immunofluorescences, the cells were
incubated with primary antibodies diluted 1:1000 and then with
secondary antibodies conjugated to FITC.
In order to perform double labeling of RFP and EPC with two rabbit
polyclonal antibodies, the anti-RFP antibody was conjugated to Alexa
546 using the Alexa 546 antibody labeling kit (Molecular Probes). For
double staining, EPC was stained with the primary polyclonal antibody
and the secondary FITC-conjugated anti-rabbit IgG antibody, followed by
incubation with Alexa 546-labeled anti-RFP antibody.
For the double labeling of RFP or EPC with PML, SW480 cells were
stained with mouse monoclonal anti-PML antibody (PG-M3) (Santa Cruz)
and secondary FITC-conjugated anti-mouse IgG antibody, followed by
incubation with Alexa 546-labeled anti-RFP antibody or anti-EPC polyclonal antibody and secondary rhodamine-conjugated anti-rabbit IgG
antibody. After immunostaining, slides were mounted in PermaFluor (Shandon) and observed with a confocal microscope (MicroRadiance, Bio-Rad).
Cloning and Chromosomal Mapping of Human EPC--
To identify the
interacting proteins with RFP, we performed a yeast two-hybrid
screening. The full-length coding region of RFP was cloned
into the pAS2-1 vector and co-transformed with a human testis
Matchmaker two-hybrid library (CLONTECH) into the yeast Y190 strain. The transformants were plated first on selective medium lacking tryptophan and leucine, and then on selective medium lacking histidine, tryptophan, and leucine with 40 mM 3-AT
(histidine jump-start method). Of approximately 5 × 106 independent clones, 50 clones were His+ and
To obtain the full coding sequence of human EPC, we performed the 3'
and 5' rapid amplification of cDNA ends method. By using the human
testis cDNA library as a template, we identified a 249-bp 5'
cDNA. A total 270-bp untranslated sequence includes a stop codon
57-bp upstream of the first ATG codon with rough conformation to a
Kozak consensus sequence. A 1887-bp 3' cDNA product included conserved EPcB and EPcC domains and a glutamine-rich (Qx) region, followed by a termination codon. The predicted amino acid sequence of
EPC (836 amino acids) is shown in Fig.
1A. The calculated molecular mass of EPC was approximately 92 kDa. EPcA, EPcB, and EPcC domains and
a Qx region are highly conserved in many species, including Drosophila, C. elegans, yeast, mouse, and human
(29). There appear to be two E(Pc) paralogues in mammals, and mouse
paralogues are named Epc1 and Epc2 (29). Although mouse Epc1 and Epc2
are about one-third of the length of the Drosophila E(Pc),
all three domains are conserved (Fig. 1B). Mouse Epc1 and
Epc2 have longer (Epc1-L and Epc2-L) and shorter variants (Epc1-S and
Epc2-S), and the shorter variants contain the EPcA domain only (29)
(Fig. 1B). Alignment of the deduced amino acid sequences
from human EPC and mouse Epc1 clearly shows a high degree of
conservation (81% amino acid identity) (Fig. 1B).
There seem to be at least two human EPC genes named
EPC1 and EPC2 that were mapped to 10p11-12 and
22q13.3, respectively (29). By the radiation hybrid mapping method
(Research Genetics, Inc.), the EPC gene that we cloned was
mapped on 10p11, corresponding to the location of human EPC1
(data not shown).
Identification of Interacting Domains between RFP and EPC--
In
the original two-hybrid screening, the full-length RFP was shown to
interact with the EPcA domain of human EPC. To further clarify their
interacting domains, we cloned the different regions of RFP
and EPC in-frame into pAS2-1 and pACT2 vectors, respectively (Fig. 2, A and B).
Each vector was transformed into yeast Y190, and interactions were
assayed by growth on selective medium lacking histidine, tryptophan,
and leucine with 40 mM 3-AT and the RFP and EPC Coimmunoprecipitate in Vivo--
To identify the RFP
and EPC proteins, we developed polyclonal antibodies against their
carboxyl-terminal regions. RFP and EPC were detected as 58- and 92-kDa
proteins, respectively, in the lysates of 293 human embryo kidney cells
and SW480 human colorectal adenocarcinoma cells by Western blotting
(Fig. 3A). In addition, a
faint 88-kDa band of EPC was present when a longer exposure was carried
out (data not shown).
In order to test whether the interaction between RFP and EPC in
two-hybrid assays signifies an in vivo interaction, we
performed immunoprecipitation experiments. However, the detection of
RFP with EPC in coprecipitaion was impossible because the band of RFP
(58 kDa) that should be recognized by the secondary anti-rabbit immunoglobulin antibody was superimposed by that of anti-EPC rabbit polyclonal antibody used for immunoprecipitation (data not shown). Thus, to overcome this problem, we transiently transfected 293 cells
with the FLAG-tagged form of EPC expression vector (Fig. 3A)
so that EPC can be immunoprecipitated by the anti-FLAG mouse monoclonal
antibody. As shown in Fig. 3B, we found coprecipitation of
RFP with FLAG-EPC by using the anti-FLAG monoclonal antibody. Reciprocally, EPC was coimmunoprecipitated with RFP by the anti-RFP polyclonal antibody (Fig. 3C). These interactions were not
detected when normal mouse or rabbit immunoglobulins were used for
immunoprecipitation (Fig. 3, B and C). From these
results, we concluded that EPC and RFP can form complexes in mammalian cells.
RFP and EPC Colocalize in the Nucleus--
Having confirmed the
association of RFP and EPC in vitro and in vivo,
we next analyzed the subcellular localization of EPC in relation to
that of RFP by immunofluorescence experiments. In order to determine
the subcellular localization of endogenous EPC, SW480 human colorectal
adenocarcinoma cells were stained with the anti-EPC polyclonal antibody
by indirect immunofluorescence. As shown in Fig.
4, B, E, and
H, EPC proteins were found in the nuclei of SW480 cells
throughout the nucleoplasm. In addition, the brightly labeled domains
and the nuclear membrane distribution were apparent in some nuclei. The
size and number of nuclear domains stained with the antibody were
different depending on each nuclei and several domains seemed to be
attached to the nuclear membrane. Endogenous RFP was also stained by
using the anti-RFP polyclonal antibody conjugated with Alexa 546 and
showed the similar distribution pattern in the nuclei (Fig. 4,
A, D, and G). Superimposition of the
two confocal images revealed that EPC and RFP colocalized in brightly
labeled nuclear domains and nuclear membrane as visualized by the
yellow color (Fig. 4, C, F, and I).
Colocalization was also detected in the less-condensed area in the
nuclei. The fine granular pattern was too complex to allow analysis of
any systematic colocalization (Fig. 4, C, F, and
I). Both the homogenous and concentrated distribution
patterns in the nuclei were similar to those of other Polycomb group
protein complexes (16). These results suggest that RFP and EPC
colocalize in the nuclei and form the nuclear structure as observed for
other Polycomb group proteins.
It was previously reported that a portion of RFP colocalizes with PML
within PML nuclear bodies (9). To further characterize the subcellular
localization of RFP and EPC and their association with PML nuclear
bodies, the double staining of PML and RFP or EPC was carried out. Fig.
5 shows that the distribution pattern of
PML detected as multiple concentrated nuclear bodies was distinct from
that of RFP or EPC. Superimposition of the confocal images showed that
colocalization of PML and RFP or PML and EPC seemed to be limited and
most PML bodies did not colocalize with RFP or EPC in the nuclei of
SW480 cells.
RFP and EPC Repress Transcription--
Because of the structural
similarity of RFP to RING1 which can act as a transcription repressor
(16) and the association of RFP with Polycomb group protein EPC, we
tested the possibility that both RFP and EPC work as transcriptional
repressors. Polycomb group proteins have been found to mediate the
transcriptional repression of reporter genes in mammalian cells and
Drosophila embryos when they are targeted as GAL4 or LexA
fusion proteins (16, 26, 36, 37). Thus, we analyzed the ability of the GAL4-EPC or GAL4-RFP fusion protein to repress the gene activity, using
different luciferase reporter constructs (Fig.
6A).
We made four different reporter constructs containing five tandem
repeats of GAL4, binding sites for transcriptional enhancers (SRE or
CRE), and SV40 promoter or herpes simplex virus-thymidine kinase
minimal promoter, immediately upstream of the luciferasse gene (Fig.
6A). After transfection into 293 cells, CRE was enhanced by
100 µM forskolin for 2 h. We found that GAL4-RFP and
GAL4-EPC constructs repressed the luciferase expression by
approximately 75 and 30-40%, respectively, compared with the
construct with the GAL4-binding domain alone (Fig. 6B). In
addition, repressive activities of both RFP and EPC were independent of
the differences of enhancers or promoters used (Fig. 6B),
suggesting that their activities could be epigenetic.
We further evaluated the activities of different RFP and EPC domains on
the gene transcription. The expression vectors that include the fusion
genes encoding the GAL4-binding domain and various regions of
RFP or EPC (Fig. 2, A and
B) were co-transfected with the luciferase reporter vector
containing SRE as an enhancer and SV40 promoter. As shown in Fig.
7A, the coiled-coil and RFP domain regions repressed the luciferase expression by about 80 and
50%, respectively. The repressive activity of the RING finger B-box
region was rather weak (~30%). In the case of EPC, the CQCT and B
regions repressed the luciferase expression by about 55 and 25%,
respectively. On the other hand, the A region strongly activated the
transcription (Fig. 7B). The transactivating capacity of the
EPcA domain is unique among the Polycomb group proteins and this may
provide the reason why the repression by GAL4-full EPC was much weaker
than that by GAL4-RFP (Fig. 6).
Interaction and Colocalization of RFP and EPC in Nuclear
Domains--
The Polycomb group genes were first identified in
Drosophila as the genes that maintain the homeotic gene
repression through a possible chromatin regulatory mechanism. All
Polycomb genes cloned from Drosophila have mammalian
homologues, suggesting the strong conservation of this system in
different species (27, 28, 38). EPC (Enhancer of
Polycomb) is known to be a unique member of the Polycomb group
genes (29). Although mutations in E(Pc),
Drosophila homolog of EPC, exhibit no homeotic
transformations, they enhance homeotic mutations by other Polycomb
group genes (30, 31). Mutations in EPC also function as the
strong suppressors of PEV that is associated with the heterochromatin
formation (33). Polycomb group proteins form multiprotein complexes and
their interacting proteins except for Pho and E(Pc) have been
identified (28). In the present study, we identified RFP as an
interacting protein of EPC and showed their nuclear colocalization by
immunofluorescence experiments. EPC and RFP were detected in discrete
nuclear domains and largely colocalized, suggesting the functional
importance of their interaction. This kind of nuclear distribution is
similar to those of other Polycomb group proteins, such as HPH1, BMI1, hPc2, and RING1 (16).
RFP was reported to associate with PML and partially colocalize in the
PML nuclear bodies (9). PML body is the nuclear matrix-associated
structure that is 250-500 nm in diameter and is present in the nuclei
of most cell lines (39-42). Although the PML nuclear body suspected to
be involved in oncogenesis and viral infection (43, 44), its exact
function remains unknown. These structures may be the sites of storage
of transcription factors, the sites of transcription or the sites of
RNA accumulation. La Morte et al. (45) reported that these
structures correspond to the sites of incorporation of
fluorescein-conjugated uridine triphosphate in nascent RNA polymerase
II transcripts. It was also shown that CBP, transcription
co-activator/histone acetyltransferase, is present in these nuclear
structures (45). On the contrary, Boisvert et al. (46)
reported that PML nuclear body itself does not accumulate RNA and
proposed that these structures may contribute to the formation of the
nuclear environment for the expression of specific genes. Our study
showed that RFP and EPC associate with only a limited subset of PML
nuclear bodies, consistent with the previous observation (9). Thus it
seems likely that RFP is involved in the gene silencing mechanism that
is different from the speculated functions of the PML nuclear body.
Further study will help to determine the roles of RFP-EPC and RFP-PML association in these nuclear complexes.
RFP Acts as a Transcriptional Repressor--
RFP was first
identified as a gene which becomes oncogenic by rearrangement with
RET proto-oncogene (1, 22, 47). RFP is a member of the B-box
RING finger family that possesses a tripartite motif consisting of a
RING finger, a B-box zinc finger, and a coiled-coil domain. RFP also
has a characteristic carboxyl-terminal domain called the RFP domain or
B30.2-like domain (6). The RING finger has been found in a wide variety
of proteins, such as PML, BMI1/Mel18 and RING1. Among them, BMI1/Mel18
is classified as a Polycomb group protein. RING1 interacts with Poycomb
group proteins (HPH, BMI1, and HPC2) and is involved in the formation of Polycomb group protein complex distinct from EED (embryonic ectoderm
development)/EZH (enhancer of zeste) (17, 28, 48). In addition, it was
shown that RING1 acts as a transcriptional repressor (16).
This study identified, for the first time, the strong transcriptional
repression activity of RFP that was dependent on its coiled-coil and
RFP domains. Since the coiled-coil domain and RFP domain are thought to
be involved in protein-protein interactions (9, 10), it is
reasonable to speculate that the function of RFP could depend on the
interaction with other nuclear proteins. In fact, consistent with this
view, we found that the coiled-coil domain is strongly associated with
EPC. Thus, this association may be crucial for the function of RFP as
the transcriptional repressor, although it is possible that nuclear proteins other than EPC are also involved in the RFP-mediated gene
silencing by forming multiprotein complex. Furthermore, it is important
to point out that RFP represses the gene transcription independent of
the enhancer or promoter differences, suggesting that the RFP-mediated
gene silencing may be epigenetic, cooperating with the Polycomb group proteins.
Dual Functions of EPC in Transcription--
EPC is
classified as a Polycomb group gene and supposed to function as a
transcriptional repressor. However, this study indicated that the
repressive activity of EPC is relatively weak and surprisingly, the
conserved EPcA domain functions as a strong transcriptional activator.
EPC was identified in many species and especially the EPcA domain was
known to be as the largest conserved domain of EPC (29).
Another Polycomb group protein, E(z), Drosophila homologue
of human EZH, is also suspected to have a dual function. E(z) cannot only act in gene silencing but also be involved in the maintenance of
transcriptional activity by trithorax group proteins (34). Although
E(z) associates with Esc, the Drosophila homologue of human
EED, and works as the repressor (32, 49, 50), this association seems to
be restricted in blastderm and early gastulation stage embryos in
Drosophila (48, 51). The double heterozygous combination of
recessive loss of function mutation of E(z) and trithorax group gene
ash1 alleles expresses homeotic transformation phenotypes
similar to those expressed by double heterozygous combination of
recessive loss of function trithorax and ash1
alleles in Drosophila (52). E(z) encodes a SET
domain that is also present in trithorax group genes (53). These
evidences suggested that E(z) may be involved in the complex formation
of both the Polycomb group proteins and the trithorax group proteins,
probably depending on the developmental stages. E(Pc) and E(z) are
different from other polycomb group proteins because their mutations
also function as Su(var)s (the suppressor of PEV) that is
associated with the heterochromatin formation (29, 32, 33). These
findings suggest that E(Pc)/EPC and E(z)/EZH have an additional
function different from the function of other Polycomb group proteins.
It is not clarified how the EPcA domain acts as a transactivator. It is
possible to speculate that the EPcA domain itself has the transcription
activating function or this domain binds another transactivator such as
trithorax group proteins. In this respect, it is interesting to note
that mouse homologues, Epc1 and Epc2, have longer (Epc1-L and Epc2-L)
and shorter (Epc1-S and Epc2-S) forms (29). Epc1-L and Epc2-L contain
the EPcA, EPcB, and EPcC domains and the glutamine-rich region, whereas Epc1-S and Epc2-S have the EPcA domain and the alanine-rich region only
(Fig. 1B). Thus, we speculate that Epc1-S and Epc2-S could function primarily as the transcription activator like trithrax group
proteins, whereas Epc1-L and Epc2-L can function as the repressor like
Polycomb group proteins rather than the transactivator. In addition,
weak repressive function of human EPC that we observed may be due to
the presence of the EpcA domain.
In summary, we found that RFP could function as the strong
transcription repressor in mammalian cells. The fact that RFP and EPC
associate and mostly colocalize in the nucleus suggests that EPC may be
involved in the epigenetic gene silencing mediated by RFP. In addition,
EPC appears to have quite different functions depending on each domain,
and especially the conserved EPcA domain acts as the transcriptional
activator. Further study will provide new insights into possible unique
roles of EPC and RFP in the gene silencing as well as the
transactivation that are important for cell growth and/or differentiation.
We are grateful to K. Imaizumi and M. Kozuka
for technical assistance and Y. Sekido and Y. Horio for discussions and
encouragement throughout this work.
*
This work was supported by a grant-in-aid for COE research
from the Ministry of Education, Science, Sports and Culture of Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper for the human
Enhancer of Polycomb gene (EPC) has been deposited in the GenBankTM/EBI Data Bank with accession number
AF277374.
¶
To whom correspondence should be addressed: Dept. of
Pathology, Nagoya University School of Medicine, 65 Tsurumai-cho,
Showa-ku, Nagoya 466-8550, Japan. Tel.: 81-52-744-2093; Fax:
81-52-744-2098; E-mail: mtakaha@med.nagoya-u.ac.jp.
Published, JBC Papers in Press, September 6, 2000, DOI 10.1074/jbc.M006585200
The abbreviations used are:
RFP, RET finger
protein;
EPC, Enhancer of Polycomb;
PEV, position-effect variegation;
E(z), Enhancer of zeste;
Su(var), suppressor of PEV;
GAL4BD, GAL4
DNA-binding domain;
SRE, serum response element;
CRE, cAMP response
element;
PCR, polymerase chain reaction;
bp, base pair(s);
3-AT, 3-amino-1,2,4-triazole;
PBS, phosphate-buffered saline;
FITC, fluorescein isothiocyanate.
RET Finger Protein Is a Transcriptional Repressor and
Interacts with Enhancer of Polycomb That Has Dual Transcriptional
Functions*
§,,¶
Pathology and
§ Internal Medicine I, Nagoya University School of Medicine,
65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase expression. -Galactosidase activity was assayed by
the filter assay method according to the manufacturer's instructions (CLONTECH). After DNA isolation, these clones were
further characterized by sequencing and analyzed for gene homology by
the BLAST data base.
-galactosidase. Measurements were performed using six independent colonies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase positive. Four isolated clones were identical and
their inserts were 777 bp in length. The nucleotide sequence was shown
to be highly homologous to the EPcA domain of Drosophila Enhancer
of Polycomb (E(Pc)) gene by searching the BLAST data
base. E(Pc) was identified in many species and especially the EPcA
domain was the longest conserved region (29).
View larger version (45K):
[in a new window]
Fig. 1.
Amino acid sequence of human EPC and
comparison of EPC homologues in eukaryotes. A, the
deduced amino acid sequence of human EPC. EPcA, EPcB, and EPcC domains
and a glutamine-rich (Qx) region that are conserved in
Drosophila, mice, and human are underlined by
thin, thick, double, and dotted lines,
respectively. B, diagram of the protein structure of EPC
homologues. The diagram is oriented with the amino-terminal on the
left and the conserved EPcA, EPcB, EPcC domains, and
glutamine-rich (Qx) and alanine-rich (Ax) regions are designated. % indicates the amino acid identity of each region.
-galactosidase activity. Each experiment was done by using six independent colonies and the results are shown in Fig. 2C. Strong interaction was
detected between the RFP coiled-coil domain region (CC region) and the EPcA domain region (A region) or the CQCT region that includes the EPcC
domain, glutamine-rich (Qx) region (CC region) and carboxyl-terminal region. Although the CQCT region of EPC interacted with RFP, the CQ
region (C domain and Qx region) of EPC had no interacting activity (Fig. 2C), suggesting that the carboxyl-terminal region of
EPC is important for EPC-RFP binding. In contrast, the RING finger B
box region (RB region) or the RFP domain (RD region) of RFP showed no
or very weak interacting activity in two-hybrid assays. In addition,
the EPcB domain region (B region) had very weak interaction with any
regions of RFP. These results indicate that the coiled-coil domain of
RFP is important for EPC binding, and both the highly conserved EPcA
domain and the carboxyl-terminal region of human EPC could associate
with RFP.
View larger version (14K):
[in a new window]
Fig. 2.
Mapping of binding domains for RFP-EPC
interaction in yeast two-hybrid system. A, schematic
representation of RFP plasmids used to determine the binding
domains in yeast. Plasmids containing GAL4-DNA binding domain
(GAL4BD)-RFP fusion genes were constructed by cloning the
various regions of RFP in-frame into pAS2-1 vector. Each
protein motif of RFP is indicated. B, schematic
representation of EPC plasmids used to determine the binding
domains in yeast. Plasmids containing GAL4-DNA activating domain
(GAL4AD)-EPC fusion genes were constructed by cloning the
various regions of EPC in-frame into pACT2 vector. Each
protein motif of EPC is indicated. C, the result of binding
assays by yeast two-hybrid system. The association is considered positive when the two criteria of
the growth in the presence of 40 mM 3-AT and the
-galactosidase activity were met. The positive association is graded
by strong (++), moderate (+), and weak (±). Absence of the cell growth
or detectable color is considered negative (
). Measurements were
performed using six independent colonies.
View larger version (32K):
[in a new window]
Fig. 3.
Coimmunoprecipitation assay of RFP-EPC
association. A, the whole cell lysates of 293 human
embryo kidney cells and SW480 human colorectal adenocarcinoma cells
were analyzed by Western blotting with anti-RFP antibody or anti-EPC
antibody. The lysate from 293 cells transfected with the FLAG-tagged
EPC expression vector was also analyzed. 58-kDa RFP and 92-kDa EPC
proteins are indicated. An additional 88-kDa band was detected in the
lysate from 293 cells transfected with the FLAG-tagged EPC expression
vector. B, coimmunoprecipitation of RFP and EPC. The plasmid
encoding EPC tagged with FLAG was transfected into 293 cells. The whole
cell lysates were immunoprecipitated with anti-FLAG mouse monoclonal
antibody (FLAG IP) or normal mouse IgG (Mock IP).
The precipitated proteins and the whole cell lysate (Input)
were analyzed by Western blotting with anti-RFP antibody or anti-EPC
antibody. C, reciprocally, the whole cell lysates were
immunoprecipitated with anti-RFP rabbit polyclonal antibody (RFP
IP) or normal rabbit IgG (Mock IP). The precipitated
proteins and the whole cell lysate (Input) were analyzed by
Western blotting with anti-EPC antibody. Although the same sample was
analyzed with anti-RFP antibody, RFP could not be detected because of
superimposition of rabbit polyclonal antibodies used for
immunoprecipitation (data not shown).
View larger version (60K):
[in a new window]
Fig. 4.
RFP colocalizes with EPC in the nuclei of
SW480 cells. Double immunostaining of RFP and EPC was preformed.
The cells were stained with anti-EPC antibody followed by incubation
with FITC-conjugated anti-rabbit IgG antibody, and then stained with
Alexa 546-labeled anti-RFP antibody. The staining patterns for RFP
(A, D, and G) and EPC (B, E, and
H) are shown, corresponding to a single optical section of
the representative cells. The two images were merged digitally and
colocalization of the two staining is indicated by yellow
color (C, F, and I).
View larger version (22K):
[in a new window]
Fig. 5.
RFP and EPC only partially colocalize with
PML nuclear bodies in SW480 cells. Double immunostaining of RFP or
EPC with PML nuclear bodies in SW480 cells. The cells were stained with
the anti-PML mouse monoclonal antibody (PG-M3) followed by incubation
with FITC-conjugated anti-mouse IgG antibody. Then they were stained
with Alexa 546-labeled anti-RFP antibody, or anti-EPC antibody followed
by incubation with secondary anti-rabbit IgG antibody conjugated with
rhodamine. A and B illustrate the staining
patterns for RFP-PML and EPC-PML in the same
cells, respectively. The two images were merged digitally and
colocalization of the two staining yielded the yellow
color.
View larger version (38K):
[in a new window]
Fig. 6.
RFP and EPC repress transcription independent
of enhancer or promoter differences. A, physical map of
each effector and reporter constructs. The effector plasmid was
constructed by ligating GAL4-binding domain (GAL4BD) in-frame with
full-length RFP or EPC cDNA. The reporter
plasmids contain five tandem repeats of GAL4-binding sites (5 × GAL4), followed by SRE or CRE as an enhancer and SV40 promoter or
herpes simplex virus-thymidine kinase minimal promoter
(Tkm). B, repressive activity of RFP and EPC.
Different reporter genes were co-transfected with the GAL4-RFP or
GAL4-EPC effector constructs into 293 cells. After transfection, CRE
was enhanced by 100 µM forskolin for 2 h. Luciferase
activity in cells transfected with the plasmid containing the
GAL4-binding domain alone was set at 100% and luciferase activities of
cells transfected with the designated effector plasmids were expressed
as percentages of control value ± S.E. Each value represents a
result of at least three experiments.
View larger version (28K):
[in a new window]
Fig. 7.
Transcriptional activity of different regions
of RFP and EPC. A, activity of each domain of RFP. The
GAL4-BD effector constructs with different regions of RFP (Fig.
2A) were co-transfected into 293 cells with a luciferase
reporter plasmid containing SRE as an enhancer and SV40 promoter.
B, activity of each domain of EPC. The GAL4 BD effector
constructs with different regions of EPC (Fig. 2B) were
co-transfected with the same luciferase reporter plasmid into 293 cells. Luciferase activity in cells transfected with the plasmid
containing GAL4-binding domain alone was set at 100% and luciferase
activities of cells transfected with the designated effector plasmids
were expressed as percentages of control value ± S.E. Each value
represents a result of at least three experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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