From the Departments of Cell and Structural Biology
and § Molecular and Integrative Physiology, University of
Illinois and College of Medicine, Urbana, Illinois 61801
Received for publication, October 1, 2002, and in revised form, November 18, 2002
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ABSTRACT |
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We have identified a novel DEAD box RNA helicase
(97 kDa, DP97) from a breast cancer cDNA library that interacts in
a hormone-dependent manner with nuclear receptors and
represses their transcriptional activity. DP97 has
RNA-dependent ATPase activity, and mapping studies localize
the interacting regions of DP97 and nuclear receptors to the C-terminal
region of DP97 and the hormone binding/activation function-2 region of
estrogen receptors (ER), as well as several other nuclear receptors.
Repression by DP97 maps to a small region (amino acids 589-631) that
has homology to a repression domain in the corepressor protein
NCoR2/SMRTe. This region of DP97 is necessary and sufficient for its
intrinsic repression activity. The N-terminal helicase region of DP97
is, however, dispensable for its transcriptional repressor activity.
The knockdown of endogenous cellular DP97 by antisense DP97 or RNA
interference (siRNA for DP97) results in significant enhancement
of the expression of estradiol-ER-stimulated genes and
attenuation of the repression of genes inhibited by the
estradiol-ER. This implies that endogenous DP97 normally dampens
stimulation and intensifies repression of estradiol-ER-regulated
genes. Our findings add to the growing evidence that RNA
helicases can associate with nuclear receptors and function as
coregulators to modulate receptor transcriptional activity.
Nuclear receptors comprise a superfamily of transcription factors
that activate or repress gene transcription in a manner that is
dependent on the nature of the hormonal ligand and coregulator proteins
(coactivators or corepressors) that are recruited to the
ligand-receptor complex (1). Among the steroid hormone receptors,
estrogen receptors (ERs)1
mediate the diverse stimulatory and repressive biological actions of estrogens and antiestrogens. These ligands, which are naturally occurring as well as synthetic, display a spectrum of activities ranging from full agonist to full antagonist that are reflective of
changes in receptor conformation engendered by the ligand and the
distinct coactivator/corepressor proteins recruited. These observations
have led to the designation of some of these ligands as selective ER
modulators (SERMs) (2, 3).
Many coactivator proteins have been identified, and these assemble into
several dynamic multiprotein complexes (1, 4-7). These coactivator
complexes include the SRC/p160 family of proteins, CREB-binding protein
(CBP) and/or p300, and other factors that are recruited in a temporally
ordered fashion (4) and up-regulate nuclear receptor activity, at least
in part, through enhanced histone acetyltransferase activity (5-7).
ATP-dependent chromatin remodeling complexes, such as
BRG1/hBrm, and the TRAP-DRIP-ARC complex, which act sequentially or
combinatorially, also enhance gene transcription by facilitating RNA
polymerase II recruitment to promoters (4).
In contrast to the coactivators, far fewer corepressors are known. Most
fully characterized are NCoR (nuclear receptor corepressor) and SMRT
(silencing mediator of retinoic acid and thyroid hormone receptor),
which function as major negative regulators of several members of the
nuclear receptor family, including thyroid and retinoic acid receptors.
These corepressors exert much of their repressive activities through
recruitment of histone deacetylases to promote a repressive chromatin
state (6-10). The key determinant role of these coregulators in
mediating repression by steroid hormone nuclear receptors, including
the estrogen, progesterone, androgen, glucocorticoid, and
mineralocorticoid receptors, is less clear (11-13). In the case of the
ER, a few additional negative coregulator proteins have been
identified; these are the repressor of estrogen receptor activity (REA)
(14, 15), a repressor of tamoxifen transcriptional activity denoted RTA
(16), and a metastasis-associated protein corepressor, MTA1 (17), which play important roles in determining the pharmacology and inhibitory effectiveness of ER ligands.
To search for other factors that are involved in regulating the
activity of the ER with estrogen agonist and antagonist ligands, we
used 2-hybrid interaction screening with antiestrogen liganded-ER as
bait. Through this screen, we have isolated a novel DEAD box RNA
helicase, DP97, a 97-kDa protein, that acts as a corepressor of the
liganded ER, and also of other nuclear hormone receptor superfamily
members. This protein is a member of DNA/RNA helicase superfamily 2, which includes the DEAD box and DEAH box proteins (18, 19) that have
roles in ribosome biogenesis, mRNA splicing, and transcriptional
regulation (20, 21).
In this study, we have characterized this novel DEAD box protein and
shown that it has several intriguing properties. DP97 has
RNA-dependent ATPase activity, consistent with its being an RNA helicase. DP97 interacts in a ligand-dependent manner
with ERs and with other nuclear receptors, and represses their
transcriptional activity. The transcriptional repression by DP97 maps
to a small region that shows significant homology to a repression
domain in the well-characterized nuclear receptor corepressor,
NCoR2/SMRTe.
Isolation and Cloning of DP97--
A yeast two-hybrid screen
employing a cDNA library from MCF-7 human breast cancer cells (14)
was used to identify a 3' partial length clone of DP97, which
interacted in a ligand-dependent manner with the ligand
binding/activation function 2 (E and F) regions of wild-type ER Plasmids and Constructs--
For creating GST fusion proteins,
the desired cDNA inserts were subcloned into the pGEX-4T-1
expression vector (Amersham Biosciences, Piscataway, NJ) in-frame with
the N-terminal GST protein. For the in vitro
transcription and translation reactions, the plasmids contained a T7
promoter upstream of the translated sequence. pBluSK-ER
For transient transfection reporter gene transactivation experiments,
the expression vectors for the human estrogen receptor (ER),
pCMV5-ER
For the preparation of FLAG-tagged DP97, the entire DP97 coding
sequence (1-865) as well as sequences encoding the truncated DP97
proteins, DP97-(1-412), DP97-(413-865), DP97-(413-656), and DP97-(657-865), were amplified by PCR using pCMV5-DP97 as a template, and with 5' BamHI and 3' XhoI sites engineered
into the primers. Fragments were ligated into
BamHI-XhoI-digested pCMV-Tag 2 vector (Stratagene, La Jolla, CA) so that they are expressed in-frame with the
FLAG tag. The DP97-(1-412) also had an SV40 nuclear localization sequence 5'-PKKKRK-3' (24) incorporated at its C terminus. The pCMV5-Antisense DP97 was generated from the pCMV-Tag 2-DP97 using the
EcoRI and BamHI restriction enzyme sites.
For the mammalian two-hybrid assays, full-length DP97 as well as
DP97-(1-412), DP97-(413-656), DP97-(657-865), DP97-(589-631), and
DP97-(
The mammalian two-hybrid assays also used pCMV5-VP16-ER Generation and Purification of DP97 Rabbit Polyclonal
Antibody--
DP97 antibody was generated against the peptide
YRPKDFDSERGLSISG (amino acids 668-683), which was chosen based on its
hydrophobicity and likely antigenicity. The peptide was conjugated to
MUC-1 keyhole limpet hemocyanin. Rabbits were immunized according to
standard protocol at the Pocono Rabbit Farm and Laboratory Inc.
(Canadensis, PA). ELISA assays were done to measure the titer of serum
response to the conjugated peptide. Serum was purified and tested by
Western blot analysis using MCF-7 whole cell extracts. Pooled serum
from days 139, 146, 153, and 160 was purified on an affinity column against the immunizing peptide.
Overexpression and Purification of Recombinant GST Fusion
Proteins--
All pGEX4T1 (GST-DP97) constructs were transformed into
E. coli BL21(DE3) (Novagen, Madison, WI).
Bacterial culture (500 ml) expressing the recombinant GST fusion
protein was grown at 37 °C to an OD600 of 0.6, then the
temperature was brought to 25 °C. The cells were treated with 0.1 mM isopropyl-b-D-thiogalactopyranoside (IPTG)
at an OD600 of 1.0. After 3.5 h, bacteria were
resuspended in PBST (PBS, 1% Triton, 2 mM EDTA, 0.1%
ATP Hydrolysis Assays--
The methods used were based on those
of Askjaer et al. (26). Reactions containing 250 ng of
GST-DP97 fusion protein in 5 µl of GST dialysis buffer (see above)
were mixed on ice with MgCl2, RNA, and unlabeled ATP and
0.1 µl of [ In Vitro Protein Interaction
Assays--
35S-radiolabeled nuclear receptors were
generated by in vitro transcription and translation using
the TNT Quick kit from Promega. GST, GST-DP97-(1-865), or
GST-DP97-(664-865) was bound to glutathione-agarose and equilibrated
with 1× GST binding buffer (20 mM HEPES, pH 7.9, 100 mM NaCl, 0.1 mM EDTA, 1 mM
dithiothreitol, 10% v/v glycerol, and protease inhibitors (4.0 µg/ml
of aprotinin, 2.0 µg/ml of leupeptin, 1.0 µg/ml of pepstatin A, and
0.2 mM phenylmethylsulfonyl fluoride) for 2 h. Then, 5 µl of [35S]methionine-labeled receptor was added to the
immobilized GST fusion proteins in 100 µl of 1× GST binding buffer
plus the proper ligands. The incubations proceeded for 2 h at
4 °C with rotary shaking. The beads were washed three times with 1×
GST binding buffer (0.5 ml) and twice with 50 mM Tris (pH
8.0) (0.5 ml) buffer. Bound proteins were eluted with 10 mM
reduced glutathione in 50 mM Tris buffer. Eluted proteins
were resolved by SDS-PAGE, dried onto Whatman 3MM paper, and visualized
by autoradiography.
Cell Transfections and Gene Transactivation Assays--
Chinese
hamster ovary (CHO) cells were maintained and transfected as previously
described (27). Cells were plated at a density of 3 × 104 cells/well of a 24-well plate and incubated for 24 h at 37 °C with 5% CO2. Some transfections also
included increasing amounts of pCMV-FLAG-DP97 along with 300 ng of an
internal Cell Studies Employing Short Interfering RNAs (siRNA)and
Quantitative RT-PCR--
siRNA oligonucleotides were designed using
the Oligoengine program (oligoengine.com) and were obtained from
Dharmacon Research (Lafayette, CO) as duplexed 2' unprotected,
desalted, and purified siRNA. The sequence used for DP97 siRNA was
GAAGAAGUCUGGAGGCUUCdTdT. A scramble oligonucleotide was used as a
negative control.
MCF-7 cells were grown to near confluency in 150-cm2 flasks
in Minimal Essential Medium (MEM) containing phenol red supplemented with 5% calf serum and antibiotics. Cells were then split into 24-well
plates at a dilution of 1:500 per well in MEM minus phenol red
supplemented with 5% charcoal-dextran-stripped calf serum, but without
antibiotics. Cells were allowed to adhere overnight and then
transfected with 0.12 nmol/well of either the DP97 siRNA duplex or
scramble siRNA duplex using Oligofectamine (Invitrogen) At 48 h
after transfection, cells were treated with 10 nM
E2, or the 0.1% ethanol vehicle. The cells were harvested
at either 8 or 24 h after E2 treatment. Total RNA was
isolated using Trizol Reagent (Invitrogen) according to the
manufacturer's instruction. 1 µg of total RNA was
reverse-transcribed in a total volume of 20 µl using 200 units of
reverse transcriptase, 50 pmol of random hexamer, and 1 mM
dNTP (PerkinElmer Life Sciences). The resulting cDNA was then
diluted to a total volume of 100 µl with sterile H2O. The
expression of DP97 and of the estrogen-regulated genes pS2,
WISP2, and c-erbB2, were measured by real time
PCR using the SYBR Green PCR System (Applied Biosystems, Foster City,
CA). Each real-time PCR reaction consisted of 1 µl of diluted RT
product, 1× SYBR Green PCR Master Mix, and 50 nM of
forward and reverse primer. The primers used in the real time PCR were
for DP97: DP97 2763f, 5'-AGGAGTTCGCCAGCAAAGC-3' and DP97 2840r,
5'-ACCCAGAACACATGGAAGCAG-3'; for pS2: pS2 255f,
5'-ATACCATCGACGTCCCTCCA-3' and pS2 401r, 5'-AAGCGTGTCTGAGGTGTCCG-3'; for WISP2: WISP2 8f, 5'-GCACACCGAAGACCCACCT-3' and WISP2 94r, 5'-AGGTACATGGTGTCGGGCA-3'; for c-erbB2: c-erbB2 925f,
5'-GGATCCTGCACCCTCGTCT-3' and c-erbB2 1009r,
5'-GCTTGCTGCACTTCTCACACC-3'; and for 36B4: 36B4 574f,
5'-GTGTTCGACAATGGCAGCAT-3' and 36B4 657r,5'-GACACCCTCCAGGAAGCGA-3'. Reactions were carried out in an ABI Prism 7700 Sequence Detection System (Applied Biosystems) for 40 cycles (95 °C for 15 s,
60 °C for 1 min) following an initial 10-min incubation at 95 °C. The fold change in gene expression was calculated using the Immunocytochemical Detection of DP97 and ER Identification and Characterization of an ER-interacting Protein,
DP97--
In order to identify proteins that might repress the
activity of the ER in a ligand-dependent manner, we used
domains E and F of ER
DP97 contains two bipartite nuclear localization signals and three
nuclear receptor boxes (LXXLL motifs) thought to be
important for coactivator interactions with nuclear receptors (35).
There is also a possible CoRNR box (amino acids 245-249), important for corepressor interactions with nuclear receptors (36, 37). Stretches
of glutamate and lysine residues, of unknown significance, also exist
throughout the sequence and the C-terminal region of DP97 is very
basic. A nonredundant standard protein-protein homology BLAST search
reveals DP97 to have a high degree of homology to the DEAD box family
of ATP-dependent RNA helicases. In fact, DP97 has all of
the eight consensus motifs (bold and underlined
in Fig. 1) that are the hallmark of proteins in this family (19). Therefore, DP97 appears to be a novel member of the DEAD box family of
ATP-dependent RNA helicases.
Northern analysis of DP97 expression showed a predominant mRNA
species of 3.1 kb (85%) and another less abundant mRNA of 4.3 kb
(15%) in several human cell lines including MDA-MB-231 breast cancer
cells, HepG2 hepatoma cells, and MCF-7 breast cancer cells (data not
presented). A human tissue mRNA Master Blot
(Clontech, Palo Alto, CA) revealed that DP97
mRNA is expressed in all tissues examined, with the highest level
of expression in the pancreas and lung (data not presented).
Biochemical Characterization of DP97 as a Putative
ATP-dependent RNA Helicase--
We performed
RNA-dependent ATPase assays, measuring ATP hydrolysis to
AMP by purified GST-DP97 fusion protein in the presence of increasing
amounts of MCF-7 total RNA. As seen in Fig.
2A, we observed an RNA
concentration-dependent increase in ATP hydrolysis. In
contrast, poly(U) RNA (Fig. 2A) and RNase-treated MCF-7
total RNA (not shown) did not stimulate the hydrolysis of ATP by
DP97.
We also assessed the ability of other nucleoside triphosphates (at
10-fold excess concentration) to function as competitors in the
hydrolysis of [ In Vitro Interaction of DP97 with Nuclear Receptors--
To
characterize the interaction of DP97 with ER, we performed interaction
assays in the absence of ligand, or in the presence of the estrogen
estradiol (E2) or the antiestrogen TOT. Wild type ER
We then examined which region of ER
The other ER subtype, ER Mammalian Two-hybrid Interaction of DP97 with ER DP97 Represses the Transcriptional Activity of Nuclear
Receptors--
Using transfection and reporter gene transactivation
assays in mammalian cells, we observed that DP97 repressed the
transcriptional activity of ER
To determine whether the interaction between DP97 and various nuclear
receptors has a functional consequence, we examined the effect of DP97
on the transcriptional activity of several nuclear receptors. DP97
repressed by 60-90% the transcriptional activity of ER Characterization of the Region of DP97 Required for the Repression
of ER DP97 Has Intrinsic Transcription Repression Activity and a
Separable Repression Domain with Homology to
NCoR2/SMRTe--
Corepressors can inhibit the activity of
nuclear receptors by several mechanisms. The nuclear receptor
corepressors NCoR and SMRT have been shown to repress the basal
activity of promoters that they are recruited to as Gal4 DNA binding
domain fusion proteins (34, 43). We tested the intrinsic repression
activity of DP97 in this manner and found that the DP97-Gal4 DNA
binding domain fusion protein repressed a constitutively active SV40
promoter with 5 upstream Gal4 binding sites (Fig.
8A). Thus, DP97 has the ability to repress promoters by recruitment alone.
Gal4 DNA binding domain fusions were also made with DP97-(1-412),
DP97-(413-656), and DP97-(657-865) to determine which region(s) retained the intrinsic repression activity of the entire protein. Gal4-DP97-(413-656) was able to repress the Gal4-SV40-Luc reporter, but neither the Gal4-DP97-(1-412) nor the Gal4-DP97-(657-865) affected the reporter gene activity (Fig. 8A). Using a
protein BLAST homology search for short nearly exact matches, we
found a glutamine-proline-glutamate rich sequence (amino acids
589-631) within DP97-(413-656) that bears significant sequence
similarity with two regions in NCoR2/SMRTe (Fig. 8B). The
first is a glutamine-proline-glutamate rich region in the SANT domain
of NCoR2, and the second is a proline-glutamate rich region in
repression domain 2 (RD2) of NCoR2.
We, therefore, examined the role of this 43 amino acid region (amino
acids 589-631) in the transcriptional repression activity of DP97
(Fig. 8C). As seen before in Fig. 8A, the Gal4
DNA binding domain fused to DP97 repressed the SV40 promoter. Of note,
the region with homology to NCoR2/SMRTe within DP97-(589-631) also had
intrinsic repression activity (Fig. 8C). Furthermore,
removal of this small region from DP97-( Reversal of the Effect of DP97 on ER Transcriptional Activity by
Antisense DP97or siRNA for DP97--
Introduction of antisense DP97
into cells resulted in an enhanced stimulation of ER transcriptional
activity in the presence of estradiol, consistent with the hypothesis
that endogenous DP97 recruited to the hormone-occupied ER normally
suppresses the response to estradiol (Fig.
9A).
To further examine the effect of DP97 on the expression of
estrogen-regulated genes, we used MCF-7 breast cancer cells and employed siRNA molecules (44). Transfection of cells with siRNA reduced
DP97 mRNA to 25% of control levels, while the scramble siRNA had
no effect on DP97 RNA level, as monitored by quantitative real-time
RT-PCR (Fig. 9B). Reduction of DP97 levels in MCF-7 cells
enhanced estradiol-stimulated expression of pS2 and WISP2, two genes
that are known to be up-regulated by estrogen in these cells (45-48).
This was seen at 8 h of E2 treatment for the primary response gene pS2, and at both 8 and 24 h of
hormone treatment for the WISP2 gene. Likewise, silencing of
DP97 substantially reduced the magnitude of estradiol repression of
erbB2 gene expression (Fig. 9B), erbB2
being a gene that is down-regulated by the E2-ER complex in
these cells (49). Hence, the findings with DP97 antisense and siRNA
provide strong evidence that endogenous DP97 plays a role in modulating
ER transcriptional activity in cells, normally dampening
estrogen-stimulated gene expression and increasing the effectiveness of
the E2-ER in suppressing down-regulated genes.
DP97 Localizes to the Nucleolus and Nucleoplasm of Cells--
We
generated a polyclonal antibody against a peptide present in the DP97
sequence. In Western blot analysis, this antibody recognizes
predominantly a 97-kDa protein in MCF-7 cell extracts (Fig.
10A). We used this antibody
and the anti-ER In this work, we describe the identification of a novel DEAD box
protein, DP97, that interacts in a hormone-dependent manner with the estrogen receptor and other nuclear receptors and has several
interesting properties. DP97 has RNA-dependent ATPase activity, consistent with its being an RNA helicase; it interacts with
and represses the activity of nuclear receptors; and it has a small
region of sequence homology with NCoR2/SMRTe that is responsible for
its transcription repression activity. Analysis of the repression activity of truncated forms of DP97 shows that the DEAD box helicase motifs are dispensable for DP97 repression activity, indicating that
its RNA helicase and corepressor activities represent distinct, separable functions.
Nuclear receptor corepressors, such as NCoR1 or NCoR2, typically have
separable functional domains for nuclear receptor interaction and for
transcriptional repression (50, 51). This is the case, as well, with
DP97. Its repression activity maps to a small region (589-631), while
a more C-terminal part of DP97-(664-865) is the region that interacts
with the C-terminal portion of nuclear receptors encompassing the
ligand binding/AF-2 regions. Although DP97 contains 3 NR boxes
(LXXLL motifs) and a possible CoRNR box through which many
coregulators interact with nuclear receptors, these receptor interaction motifs are in the N-terminal half of DP97, not in the
C-terminal region that interacts with nuclear receptors, indicating that other sequences in DP97 are responsible for its receptor interaction. This is consistent with findings from peptide phage display and studies with other coregulators (14-16), showing that peptide sequences in addition to LXXLL motifs and CoRNR
boxes can interact with nuclear receptors with high affinity (52, 53).
Comparisons of the Transcription Repression and the
Ligand-dependent Interactions of DP97 and Other
Corepressors--
We have shown that DP97 has a compact intrinsic
transcription repression region (amino acids 589-631). Like DP97,
several other transcriptional repressor proteins have small regions
with active transcriptional repression activity. These include Nab-1, a
direct acting corepressor of the NGFI-A family of zinc finger transcription factors (54), Sim-2, a protein involved in midline development in mice (55), and AREB6, a zinc-finger homeodomain transcription factor (56).
It is of note that the repression region of DP97-(589-631) shows
considerable homology with two regions in the well known corepressor,
NCoR2/SMRTe, amino acids 498-534 and 813-839 (34, 43, 51, 57). The
latter (813-839) is within the second repression domain (RD2) of NCoR2
(34), whereas the former (498-534) encompasses a polyglutamine and
acidic/basic region (43) that is between SANT(SWI3,
ADA2, NCoR, TFIIIB) domains A and
B. Since this former region (498-534) has only been examined as a Gal4
fusion along with other repressive regions of NCoR2, such as the SANT
domains and the region of high similarity between NCoR and NCoR2 (51), it is not known whether NCoR2-(498-534) has intrinsic repression activity on its own. Nevertheless, our observations with DP97 expand
the utilization of these motifs for nuclear receptor regulation beyond
SMRT/NCoR to a new corepressor, DP97.
The ligand requirements for interaction of SMRT and NCoR with nuclear
receptors is quite different from that observed with DP97. SMRT and
NCoR interact preferentially with type II nuclear receptors, including
retinoic acid and thyroid hormone receptors, and interaction with these
receptors is observed primarily in the absence of ligand, with ligand
occupancy resulting in dissociation of SMRT or NCoR. These corepressor
proteins have also been reported to interact with estrogen receptors
and progesterone receptors, but only in the presence of antagonist
ligands (4, 13). In contrast, DP97 interacts with nuclear receptors in
the presence of agonist or antagonist ligand, and shows no interaction
in the absence of ligand. Hence, SMRT/NCoR and DP97 may serve distinct transcriptional regulatory activities that differ in their hormonal requirements, providing potentially multiple combinatorial regulatory mechanisms (11). Indeed, there is already evidence that NCoR and SMRT
preferentially regulate different receptors (36) and that they exhibit
distinct promoter and cell-type specificity.
With the exception of the small repression region (amino acids
589-631) of DP97, DP97 shows no structural resemblance to NCoR or SMRT
or to three other proteins known to be negative coregulators for
nuclear receptors (REA, MTA1, and RTA). Like DP97, REA preferentially interacts with antiestrogen-liganded ER but it also interacts with
estrogen-occupied ER. However, in sharp contrast to DP97, REA is an
ER-selective coregulator (14, 15, 27). RTA (16) and MTA1 (17) bind to
ER as well as other nuclear hormone receptors, as does DP97, but RTA
interacts through the N-terminal region of the receptors in a
ligand-independent manner, whereas DP97 interaction with receptor, like
that of RTA and MTA1, is via the hormone binding/activation function-2
domain and is ligand-regulated. MTA1 is identical to NuRD-70, a
component of nucleosome remodeling complexes (17). Of interest, RTA
contains RNA recognition motifs that are required for its repressor
function, indicating a role for RNA binding in regulation of nuclear
receptor activity (16). For DP97, its repressor activity is physically
and functionally separable from its DEAD box motif-containing region.
DP97 Variants, Subcellular Localization, and Comparisons with other
DEAD Box RNA Helicases and Coregulators Known to Modulate Nuclear
Receptor Activity and RNA Processing--
DP97 may exist in several
splice variant forms in cells. We found that DP97 mRNA was present
as 3.1-kb and 4.3-kb species. The 3.1-kb message corresponds to the
clone shown in Fig. 1, and the 4.3-kb message appears in the
GenBankTM (accession number NM024072.2) as a similar clone,
isolated from human placenta, that contains in addition a 1.2-kb
3'-untranslated region accounting for the 4.3-kb size. No publication
associated with this GenBankTM entry has appeared. The
3.1-kb mRNA is the predominant form, accounting for 85% of the
total cellular DP97 mRNA in the several human cell lines we
examined (MCF-7, MDA-MB-231, and HepG2). Also, in our isolation of a
full-length DP97 clone from a cDNA library, we found an
alternatively spliced form that would encode a protein missing amino
acids 453-533. It is of note that RNA-binding proteins that modulate
ER activity, such as RTA, also exist in multiple alternatively spliced
forms (16).
Two other DEAD box RNA helicase proteins have been shown to be
specifically involved in modulating nuclear receptor transcriptional activity. RNA helicase p68 interacts with the N-terminal A/B region of
ER
Our identification of DP97 localization in nucleoli, as well as
throughout the nucleus, is consistent with the reported intracellular localization of most RNA helicases. Since ER
DEAD-box RNA helicases, as well as certain RNA species themselves, may
be involved more broadly than previously envisioned in the actions of
nuclear receptor superfamily members and in the regulation of
transcription and RNA splicing (63, 64). In addition to the DEAD box
RNA helicase DP103 that represses SF-1 activity (65), and the RNA
helicase p68 that functions as a specific hormone-independent
coactivator of ER
Our findings add to the growing evidence for RNA helicases associating
with distinct activation function regions of nuclear receptors and
serving as coregulators that can either up- or down-modulate the
activity of these transcription factors. Since RNA helicases are known
to be involved in many aspects of RNA metabolism, including RNA
transcription, processing and transport, and ribosome biogenesis, it is
tempting to speculate that there is a linkage (through coregulator proteins such as DP97) between RNA processing and transcriptional activity of the nuclear hormone receptors. Further investigations are
needed to explore this relationship.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. The
entire open reading frame was isolated from a human muscle cDNA
library (Origene Inc., Rockville, MD). Three clones of DP97 were
obtained. One contained the entire open reading frame; another was a
shorter 5' sequence corresponding to amino acids 33-865. A third
clone, missing amino acids 453-533, may be a product of alternative splicing.
(14),
pBluSK-ER
(ABC) (15), and pCR2.1-ER
(22), have been described
earlier. pBluSK-ER
(DEF) and pBluSK-ER
(EF), pBluSK-ER
(DEF), pBluSK-PRb(DEF), and pBluSK-GR(DEF) were generated by PCR using the sequence from the pCMV construct as template (15), and all constructs were confirmed by sequencing.
and pCMV5-ER
, human progesterone receptor (PR), pCMV5-PRb, human glucocorticoid receptor (GR), pRSV-GR, and pCMV-
, the
-galactosidase internal control vector, have been described earlier (14). pRSV-RAR
was a kind gift from Christopher Glass (University of California at San Diego, CA). The reporter plasmid 2ERE-pS2-Luc was created by inserting a PCR-amplified 2ERE-pS2 product,
engineered with flanking restriction sites (MluI): f, 5'-GCTGTTTAACGCGTTATTCGGCCG-3' and (BglII): r,
5'-GGGAATTGGAGATCTGAGCTT-3', into the pGL2-Basic luciferase
vector (Promega, Madison, WI). The other estrogen responsive constructs
TGF
3-CAT and complement 3-Luc have been described (15). 2PRE-TK-Luc
was a gift from Kathryn Horwitz, University of Colorado Health Sciences
Center, Denver, CO. SV-DR5-CAT, a retinoic acid receptor responsive
promoter, was a gift from Ron Evans, Salk Institute, San Diego, CA.
2Gal4-pS2-CAT has previously been described (23). The reporter
pGal4-SV40-Luc was kindly provided by Mitchell Lazar (University of
Pennsylvania, Philadelphia).
589-631) were cloned into the EcoRI and
BamHI pM vector of the Mammalian Matchmaker two-hybrid assay
system (Clontech, Palo Alto, CA), creating fusions
to the Gal4 DNA binding domain. The DP97-(1-412) insert was generated
by PCR so as to incorporate a nuclear localization signal (NLS) at its
C terminus and used forward primer (BamHI): f,
5'-GGAATTCATGGCCCAGTGGAGG-3' and reverse primer (XhoI): r,
5'-AATCTCGAGTCACTTCCGCTTCTTCTTGGGACGGCCCACG-3' with the
SV40 NLS underlined. All constructs were sequenced to check accuracy.
that was
generated earlier (25), and pG5-CAT reporter, pVP16, pM3-VP16 (three
Gal4 DNA binding domains fused to a VP16 transcriptional activation
domain), and pM-p53 from Clontech (Palo Alto, CA).
-mercaptoethanol, and protease inhibitors (4.0 µg/ml of aprotinin,
2.0 µg/ml of leupeptin, 1.0 µg/ml of pepstatin A, and 0.2 mM phenylmethylsulfonyl fluoride), sonicated, and
centrifuged. Soluble extracts were incubated with a glutathione-agarose
matrix (Sigma) for 1 h at 4 °C, washed three times with PBST
and then washed twice with 50 mM Tris-HCl, pH 8.0. The
fusion protein was eluted from the beads with 5 mM
glutathione and dialyzed to remove glutathione using dialysis buffer
(25 mM Tris-HCl, pH 8.0, 125 mM NaCl, 1.25 mM EDTA, 20% (v/v) glycerol, and 20 mg/ml dithiothreitol).
-32P]dATP (PerkinElmer Life Sciences, 3 Ci/µmol) in a final volume of 15 µl. The optimal concentrations for
the hydrolysis of ATP were 600 µM MgCl2, 100 µM dATP, and 100 ng of MCF-7 total RNA. For the
competition assays, a 10-fold excess of various unlabeled nucleoside
triphosphates were added separately to each tube. The samples were
incubated at 37 °C for 60 min and reactions stopped by placing on
ice and adding 1 µl of 0.5 M EDTA. A portion (3.5 µl)
of the reactions was spotted on PEI cellulose F thin layer chromatography plates (E. Merck). The plates were air-dried briefly, prewetted with ethanol, and developed in 0.5 M
KH2P04. The amount of
[
-32P]dATP hydrolysis was determined by PhosphorImager
analysis. Images were quantitated using ImageQuant software (Molecular
Dynamics, Sunnyvale, CA).
-galactosidase reporter gene to correct for transfection
efficiency and empty pCMV-FLAG to 1000 ng. Cells were incubated with
lipofectin (Invitrogen)-transferrin (Sigma)-complexed DNA for 7-8 h in
serum-free medium and then washed with growth medium and given
subsequent ligand treatment in growth medium. Cells for chloramphenicol
acetyltransferase (CAT) assays were harvested 24 h after ligand
treatment and lysed by 3 cycles of freezing on dry ice and thawing at
37 °C.
-galactosidase activity was measured to normalize for
transfection efficiency and CAT assays were performed as described
(28). Cells to be processed for luciferase assays were rinsed once in
PBS and frozen in the presence of 100 µl of lysis buffer (Promega) at
80 °C. Luciferase activity was measured using a Dynex (Chantilly,
VA) MLX luminometer and the Promega luciferase assay system 1.
Ct method with the ribosomal protein 36B4 mRNA (29) as an internal control.
in CHO and MCF-7
Cells--
CHO cells were transfected using the FuGENE 6 transfection
reagent (Roche Pharmaceuticals). MCF-7 cells were not transfected. At
48 h after transfection, cells were rinsed with calcium- and magnesium-free phosphate buffered saline (CMF-PBS) and fixed in 1.6%
formaldehyde (Polysciences Inc., Warrington, PA) at room temperature.
Cells were then washed three times for 5 min each in CMF-PBS and
permeabilized in 0.1% Triton X-100 detergent (Pierce Chemical) for 5 min. Cells were treated with 2.8 ng/µl of affinity-purified
-DP97
rabbit polyclonal antibody as well as 2 ng/µl
-ER
H222 rat
monoclonal antibody. After washes, cells were treated with fluorescein
isothiocyanate-conjugated goat anti-rabbit secondary antibody and Texas
Red goat anti-rat Affinipure IgG (Jackson ImmunoResearch, West Grove,
PA). Cells were again washed and then mounted in anti-fade solution
(Molecular Probes, Eugene, OR). Images of the MCF-7 and CHO cells were
collected on an inverted light microscope (IMT2, Olympus, Success, NY)
with a cooled, slow-scan CCD camera (Photometrics, Tucson, AZ) as
described previously (30). Optical sections of nuclei were collected
and deconvoluted as described (31).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
complexed with the antiestrogen
trans-hydroxytamoxifen (TOT) as bait in a yeast two-hybrid
screen with a cDNA library prepared from MCF-7 human breast cancer
cells. In this manner, we identified a partial 3' clone that interacted
with the hormone-occupied ER. It showed preference for interaction with
TOT-ER over estradiol (E2)-ER, and it did not interact with
the unliganded ER. The entire open reading frame, which was
subsequently isolated, encodes a protein of 97 kDa, consisting of 865 amino acids (DP97, Fig. 1). A Kozak
consensus sequence is found in the 5'-region that would allow
expression of the DP97 protein (32, 33). A shorter variant of this
sequence, also isolated in library screening, has the corresponding
coding sequence with amino acids 453-533 removed, probably due to
alternative splicing. Bioinformatic analysis of the DP97 sequence using
the BLAST search of the human genome demonstrates that the DP97
sequence is located at chromosome 12q22-12q23. Interestingly, the
corepressor NCoR2/SMRTe has been localized to the region 12q24 in the
human genome (34).
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Fig. 1.
Amino acid and nucleotide sequence of human
DP97. Some notable features are the 8 DEAD box RNA helicase motifs
(bold and underlined), 2 bipartite nuclear
localization signals (bold and boxed), and 3 NR
box LXXLL motifs (underlined italics). The
polyadenylation signal in the 3'-untranslated region is also shown
(underlined italics).
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Fig. 2.
Characterization of DP97
RNA-dependent ATPase activity. A, the
ability of GST-DP97 to hydrolyze [ -32P]dATP in the
presence of increasing amounts of MCF-7 total cell RNA (or poly(U) RNA)
was determined. The level of hydrolysis, monitored by thin layer
chromatography and PhosphorImager quantification, is plotted relative
to the level of hydrolysis without RNA present. Values are means ± S.D. from three independent determinations. B,
hydrolysis by DP97 was specific for ATP, as shown by competition of
hydrolysis of [
-32P]dATP with a 10-fold excess of
radioinert ATP or dATP, but not by the other nucleoside triphosphates
tested. Left-most lanes show GST or GST-DP97 with
[
-32P]dATP and no competitor nucleoside
triphosphate.
-32P]dATP by DP97 in the presence of
MCF-7 RNA (Fig. 2B). GST-DP97 caused hydrolysis of
[
-32P]dATP, whereas the GST protein alone, as
expected, did not. Competition by unlabeled nucleoside triphosphates
was only seen with ATP and dATP. Therefore, we conclude that DP97
hydrolyzes ATP in preference to other nucleoside triphosphates. These
findings provide support for DP97 as an ATP-dependent RNA
helicase, suggested by the phylogenetic motifs in its sequence.
interacted with the full-length DP97 only in the presence of ligand,
and more effectively with TOT-ER than with E2-ER (Fig. 3A). To determine the region
of interaction between the ER and DP97, we created GST fusion proteins
with full-length DP97-(1-865) and with the truncated DP97 proteins
DP97-(1-656), DP97-(413-656), and DP97-(664-865). These proteins
were purified over a glutathione-agarose resin and were analyzed by
SDS-PAGE to verify their sizes. GST pull-down experiments revealed that
only the full-length DP97 and the DP97-(664-865) interacted with ER
(Fig. 3A).
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Fig. 3.
Examination of the interaction of DP97 with
nuclear receptors. Interaction of DP97 with ER (panel
A) as well as ER
, progesterone receptor (PRb), and
glucocorticoid receptor (GR) (panel B) was assessed in the
presence of agonist ligand, antagonist ligand, or in the absence of
ligand. Panel A examines interaction of full-length ER
and ER
domains ABC, DEF, and EF with DP97. Panel B
examines interaction of DP97 with ER
as well as DP97-(664-865) with
the DEF regions of ER
, PRb, and GR. In vitro translated
[35S]methionine-labeled receptor proteins were incubated
with purified GST or GST-DP97 fusion proteins and with
glutathione-agarose beads in the presence of 0.1% control ethanol
vehicle, 10
6 M ligand (E2 and TOT
for ER, R5020 and RU486 for PR, and dexamethasone (Dex)
and RU486 for GR). Lanes (left to right) show
20% input; incubation with GST alone; incubation with GST-DP97 fusion
protein and control vehicle (V), or with agonist ligand, or
with antagonist ligand.
interacted with DP97 using
in vitro translated products: ER
-(domains ABC) containing the receptor N-terminal A/B activation function-1 and DNA-binding domains, ER
-(DEF) containing the hinge and
hormone-binding/activation function-2 regions, and ER
-(EF)
containing the hormone binding/activation function 2 domain and
C-terminal F domain (Fig. 3A). DP97 did not interact with
ER
-(ABC), but it did interact with ER
-(DEF) and ER
-(EF) (Fig.
3A). Interaction did not require domain F nor E domain helix
12, as the ER ligand binding domain E truncated at amino acid 530 interacted as well as did ER
-(EF) (data not shown). We also see a
preference for DP97 interaction with full-length ER
and ER
-(EF)
in the presence of TOT, and no interaction when ER is unliganded (Fig.
3A). This result is consistent with the original yeast
two-hybrid screen where the ER
-(EF) bait interacted with the C
terminus of DP97 with increased affinity in the presence of TOT.
, and its DEF region also interacted with
GST-DP97 in the presence of E2 or TOT (Fig. 3B).
The DEF regions of the progesterone receptor (PRb) and the
glucocorticoid receptor (GR) also interacted with DP97 in the presence
of agonist and antagonist ligand, but not in the absence of ligand
(Fig. 3B).
--
The
interaction between DP97 and ER
was also confirmed in cells using a
mammalian two-hybrid system. DP97 was expressed as a fusion protein
with the Gal4 DNA binding domain and ER
was expressed as a fusion
protein with the VP16 activation domain (AD) in CHO cells. A robust
activation of the Gal4-regulated reporter gene indicated that the two
proteins interact in the presence of E2 and TOT, but not in
the absence of ligand (Fig. 4), which is
consistent with the GST pull-down interaction data.
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Fig. 4.
Mammalian two-hybrid interaction of
ER and DP97. Interaction between DP97
expressed as a fusion protein with the Gal4 DNA binding domain and
ER
expressed as a fusion with the VP16 activation domain was
assessed in CHO cells in the absence of hormone and in the presence of
10
8 M E2 or 10
8
M TOT using a reporter construct containing 5 Gal4 response
elements upstream of the CAT gene. All transfections
contained a
-galactosidase internal control reporter to normalize
for transfection efficiency. Values are means with S.D. from four
independent determinations.
on a variety of promoters having
different estrogen responsive regions. This included the pS2 promoter
with two consensus estrogen response elements (EREs) (Fig.
5A); the complement component 3 (C3) promoter, which contains a mix of consensus and nonconsensus EREs (Fig. 5B), and the TGF
3 promoter, which has an
estrogen-responsive region very different from an ERE and where ER
works by tethering to other DNA-bound protein factors (Fig.
5C). These data demonstrate that DP97 can repress ER
stimulation at diverse estrogen-regulated gene sites. In order to
determine if DP97 inhibits ER activity by interfering with the DNA
binding function of ER, we performed a promoter interference assay in
MDA-MB-231 cells (38). This revealed that DP97 did not inhibit the
ability of ER
to bind to estrogen response elements (data not
shown).
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Fig. 5.
DP97 suppresses the transcriptional activity
of ER when ER is acting as a direct
DNA-binding transcription factor as well as when it is tethered via
other transcription factors at non-estrogen response element DNA
sites. CHO cells were transfected with an expression vector for
ER
and the estrogen-responsive reporter gene construct indicated in
the absence of DP97 or with increasing amounts of DP97 expression
plasmid (10, 100, 500, or 1000 ng). Cells were (+) or were not (
)
exposed to 10
8 M E2. In
A, the reporter plasmid was 2ERE-pS2-Luc; in B,
complement component 3-Luc (C3,
1030 to +58); in C,
transforming growth factor
3-CAT (TGF
3-CAT). All transfections
contained a
-galactosidase internal control reporter to normalize
for transfection efficiency. Values are means with S.D. from four
independent determinations.
(Fig.
6A), PRb (Fig. 6B),
GR (Fig. 6C), and retinoic acid receptor
(RAR
) (Fig.
6D). Therefore, DP97 repressed both type I (steroid) as well
as type II (retinoic acid) nuclear hormone receptors. DP97; however,
did not repress all transcription factors, as it failed to repress the
activity of p53 (Fig. 5E) and the viral protein VP16 (Fig.
5F). Differences in the extent of transcriptional repression
by DP97 among different nuclear receptors might reflect differences in
affinities for the nuclear receptors, or differences in the potency of
DP97 in repression of different promoter-response element gene
constructs by the different nuclear receptor-ligand complexes. There is
substantial evidence for different efficacies of coregulators being
dependent on promoter and cell context (39-42).
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Fig. 6.
DP97 is able to repress the transcriptional
activity of various nuclear receptors but not p53 or VP16. CHO
cells were transfected with plasmids expressing either ER (A), progesterone receptor b (B), glucocorticoid
receptor (C), retinoic acid receptor
(D), p53
(E), or the VP16 transcriptional activator (F);
and the reporter gene construct: ER
, 2ERE-pS2-Luc; PR-b,
2PRE-TK-Luc; GR, 2PRE-TK-Luc; RAR
, DR5-RARE-CAT; p53, 2Gal4-pS2-CAT;
VP16, 2Gal4-pS2-CAT. Transfections also included increasing amounts of
DP97 plasmid (10, 100, 500, or 1000 ng). All transfections contained a
-galactosidase internal control reporter to normalize for
transfection efficiency. Values are means with S.D. from four
independent determinations.
--
To identify the region of DP97 responsible for its
repression, we analyzed the ability of truncated DP97 proteins to
repress the activity of ER
(Fig. 7).
First, we confirmed that FLAG-DP97-(1-865) repressed ER
to a
similar extent as did DP97 without the FLAG tag (not shown). We found
that FLAG-DP97-(1-412), which contains all of the DEAD box motifs, did
not affect the transcriptional activity of the ER. However, the
FLAG-DP97-(413-865) construct repressed ER
even slightly better
than the full-length DP97. Since DP97-(657-865) interacted with ER
,
we divided DP97-(413-865) into two portions. Neither portion,
FLAG-DP97-(413-656) nor FLAG-DP97-(657-865), could repress ER
on
its own. Thus, both the region of DP97 that interacts with ER
(amino
acids 657-865) and the region of DP97 from 413-656 were required for
transcriptional repression of ER
.
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Fig. 7.
Effect of truncated DP97 proteins on
transcriptional activity of ER . A, schematic of DP97
constructs tested. NLS, nuclear localization signals. Since
DP97-(1-412) lacked the NLS, we cloned an NLS (indicated in
black) at the C terminus of this construct. B,
full-length DP97 and DP97-(413-865) repressed ER
transcriptional
activity, while DP97-(1-412), DP97-(413-656), or DP97-(657-865) did
not. Transfections were performed in CHO cells with a 2ERE-pS2-Luc
reporter gene construct and ER
expression plasmid and increasing
amounts of each DP97 construct. Cells were treated with
10
8 M E2. All transfections
included a
-galactosidase internal control reporter to normalize for
transfection efficiency. Values are means with S.D. from four
independent determinations.
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Fig. 8.
Identification of intrinsic transcription
repression activity of DP97 and a region of DP97 with repression
activity that shows homology to the corepressor NCoR2/SMRTe.
A, Gal4 DNA binding domain-DP97 fusion proteins were tested
for their ability to repress the 5-Gal4 DNA binding
sites-SV40-luciferase reporter gene. DP97-(413-656) repressed the SV40
promoter as well as the full-length protein. B, protein
BLAST analysis of DP97-(413-656) reveals significant sequence
similarity of DP97 amino acid region 589-631 with two regions in
NCoR2. *, same amino acid in DP97 and both regions of NCoR2/SMRTe; ,
same amino acid in DP97 and one of the regions in NCoR2/SMRTe.
RD, repression domain; ID, receptor interaction
domain. C, Gal4-DP97-(589-631) maintains the intrinsic
repression activity of the Gal4-DP97 protein, and Gal4-DP97 deleted of
these 42 amino acids (
589-631) loses intrinsic repression activity.
All transfections contained a
-galactosidase internal control
reporter to normalize for transfection efficiency. Values are means
with S.D. from four independent determinations.
589-631) abolished the
ability of DP97 to repress the constitutively active SV40 promoter
(Fig. 8C) and also to repress the transcriptional activity
of the ER (data not shown). Therefore, the region encompassing amino
acids 589-631 functions as the transcriptional repression domain of DP97.
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Fig. 9.
The effect of antisense DP97 or siRNA for
DP97 on estrogen-regulated gene expression. A, an antisense
DP97 plasmid was transfected along with ER expression plasmid into
MDA-MB-231 breast cancer cells and after 48 h, cells received
10
8 M E2 or 0.1% ethanol vehicle
for 8 h. The activity of ER
on a 2ERE-pS2-Luc reporter gene
construct was then monitored. All transfections contained a
-galactosidase internal control plasmid to normalize for
transfection efficiency. Values are means with S.D. from four
independent determinations. B, MCF-7 cells were exposed for
48 h to either a scramble (control, open
bars) siRNA or to DP97 siRNA (filled bars) to reduce
the levels of endogenous DP97. The cells were then treated with either
0.1% ethanol vehicle (
E2) or 10
8
M estradiol (+E2) for 8 or 24 h and the
levels of mRNA for DP97, pS2, WISP2, or c-erbB2 were monitored by
quantitative real-time RT-PCR. S and DP denote
scramble or DP97 siRNA exposure, respectively.
monoclonal antibody H222 to determine the
localization of endogenous DP97 and ER
in MCF-7 cells, and of DP97
and ER
in CHO cells that had been transfected with plasmids
expressing DP97 and ER
(Fig. 10B). Untransfected CHO
cells do not contain ER and if they contain endogenous DP97, it is not
detected with our antibody to human DP97, perhaps because of the
species difference of the cells. In both types of cells, DP97 was
localized in the nucleus; it was present throughout the nucleoplasm and
was concentrated at the nucleoli. As expected, ER
was nuclear
and was present throughout the nucleoplasm but little was nucleolar.
There was overlap of DP97 and ER in speckled structures in the
nucleoplasm, structures that may be associated with RNA processing
events. Hence, the interaction between ER and DP97 may occur at
the edge of the nucleoli and/or with the portion of DP97 that exists in
the nucleoplasm (see "Discussion").
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Fig. 10.
Cellular localization of DP97.
A, antibody to DP97 detects primarily a single protein of
the correct size on SDS-PAGE gels of MCF-7 cell extracts. The gel
marker kDa sizes, run in a lane adjacent to the cell extract, are
indicated. B, endogenous DP97 is localized to the nucleoli
and nucleoplasm in MCF-7 cells, and transfected DP97 is localized
similarly in CHO cells. CHO cells were transfected with DP97 and ER
expression plasmids. MCF-7 cells were not transfected. Cells were
incubated with an antibody to DP97 (anti-DP97 rabbit polyclonal), an
antibody to ER
(H222 monoclonal rat antibody) followed by secondary
antibody conjugated to flourescein or Texas Red, respectively, and DAPI
(to stain nuclear DNA). Cell images shown are from cells without
hormone treatment. However, the distributions of DP97 and ER were not
changed upon exposure to estrogen or antiestrogen.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and selectively potentiates the activity of ER
; this
coactivator does not interact with or serve as a coactivator of either
ER
or other nuclear receptors (58, 59). Another DEAD box RNA helicase, DP103, interacts with the orphan nuclear receptor,
steroidogenic factor-1 (SF-1), and potentiates its transcriptional
repression (23). In contrast, DP97 interacts with the C-terminal
hormone binding region of ER
, ER
and other nuclear receptors, and
functions more broadly as a corepressor of these nuclear hormone
receptors. Our findings with DP97 antisense and siRNA provide evidence
that endogenous DP97 normally dampens the stimulation and intensifies the repression of estradiol-ER-regulated genes, such that the knockdown
of DP97 enables greater estrogen stimulation of up-regulated genes and
attenuates the repression of genes that are normally inhibited by the
E2-ER complex.
has been previously shown to be nuclear and present in the nucleoplasm, but largely excluded from nucleoli, the interaction between ER
and DP97 may occur either at the edge of the nucleoli and/or with the portion of
DP97 that exists within the nucleoplasm. Many nucleolar proteins are
dynamic, and constantly cycle between the nucleolus and nucleoplasm (60), and this may be the case for DP97. The presence of DP97 in the
nucleolus suggests that it may have regulatory roles, possibly in RNA
and ribosome biosynthesis and processing, in addition to its role in
regulation of nuclear hormone receptor activity. In this regard, it is
of note that the coregulator PGC1, originally identified as a
coregulator of PPAR
-mediated transcriptional activity, but shown
more recently to also serve as a coregulator of the ER (61), exerts
dual regulation in that, in addition to its transcriptional regulatory
role, it also mediates mRNA splicing (62). Such dual regulatory
functions may prove to be a common theme among nuclear receptor coregulators.
(66), steroid receptor activator (SRA), itself a
novel RNA species, has been shown to function as a coactivator for
steroid receptors (59, 67); and SHARP, an RNA-binding corepressor
protein can bind to SRA and regulate nuclear receptor activity (68).
Three proteins that associate with thyroid hormone receptors (TLS, PSF,
and NonO/p54nrb) each contain RNA recognition motifs and
may play possible roles in RNA processing (69, 70). One of these, PSF,
functions as a transcriptional repressor. These findings, plus
observations that CBP, which is often a component of nuclear receptor
transcriptional complexes, binds RNA helicase A and that the tumor
suppressor protein BRCA1 is linked to the RNA polymerase II holoenzyme
complex via RNA helicase A (71), imply that RNA helicases and
additional proteins that bind RNA may play crucial roles in the actions
of nuclear receptor superfamily members and in the regulation of transcription in normal and cancer cells (1).
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FOOTNOTES |
---|
* 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 has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY148094.
¶ To whom correspondence should be addressed: Dept. of Molecular and Integrative Physiology, University of Illinois, 407 S. Goodwin Ave., 524 Burrill Hall, Urbana, Illinois 61801-3704. Tel.: 217-333-9769; Fax: 217-244-9906; E-mail: katzenel@life.uiuc.edu.
Published, JBC Papers in Press, December 3, 2002, DOI 10.1074/jbc.M210066200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ERs, estrogen receptors; CAT, chloramphenicol acetyltransferase; DP97, DEAD box protein 97; E2, estradiol; Luc, luciferase; NCoR, nuclear receptor corepressor; siRNA, short interfering RNA; SMRT, silencing mediator of retinoic acid and thyroid hormone receptor; SMRTe, N-terminally extended SMRT; GST, glutathione S-transferase; PBS, phosphate-buffered saline; ERE, estrogen response element; TOT, trans-hydroxytamoxifen; REA, repressor of estrogen receptor activity; RTA, repressor of tamoxifen transcriptional activity; MTA1, metastasis-associated protein corepressor; NLS, nuclear localization signal; CHO, Chinese hamster ovary; NR, nuclear receptor; RT-PCR, real time PCR.
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REFERENCES |
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