Nuclear Factor I-mediated Repression of the Mouse Mammary
Tumor Virus Promoter Is Abrogated by the Coactivators p300/CBP and
SRC-1*
Ali Z.
Chaudhry
,
Alfredo D.
Vitullo§, and
Richard M.
Gronostajski
¶
From the Department of Cancer Biology, Lerner Research Institute,
Cleveland Clinic Foundation, Cleveland, Ohio 44195 and the
Department of Biochemistry, Case Western Reserve
University, Cleveland, Ohio 44106
 |
ABSTRACT |
To better understand the function of nuclear
factor I (NFI) proteins in transcription, we have used transient
transfection assays to assess transcriptional modulation by NFI
proteins on the NFI-dependent mouse mammary tumor virus
(MMTV) promoter. Expression of NFI-C or NFI-X, but not NFI-A or NFI-B
proteins, represses glucocorticoid induction of the MMTV promoter in
HeLa cells. Repression is DNA binding-independent as a deletion
construct expressing the NH2-terminal 160 residues of
NFI-C represses but does not bind DNA. Repression by NFI-C is cell
type-dependent and occurs in HeLa and COS-1 cells but not
293 or JEG-3 cells. NFI-C does not repress progesterone induction of
the MMTV promoter in HeLa cells, suggesting that progesterone induction
of the promoter differs mechanistically from glucocorticoid induction.
NFI-C-mediated repression is alleviated by overexpression of
glucocorticoid receptor (GR), suggesting that NFI-C represses the MMTV
promoter by preventing GR function. However, repression by NFI-C occurs
with only a subset of glucocorticoid-responsive promoters, as the
chimeric NFIGRE
-gal promoter that is activated by GR is not
repressed by NFI-C. Since the coactivator proteins p300/CBP, SRC-1A,
and RAC3 had previously been shown to function at steroid
hormone-responsive promoters, we asked whether they could influence
NFI-C-mediated repression of MMTV expression. Expression of p300/CBP or
SRC-1A alleviates repression by NFI-C, whereas RAC3 has no effect. This
abrogation of NFI-C-mediated repression by p300/CBP and SRC-1A suggests
that repression by NFI-C may occur by interference with coactivator function at the MMTV promoter.
 |
INTRODUCTION |
Nuclear factor I (NFI)1
was initially identified as a host-encoded protein required for the
efficient initiation of adenovirus (Ad) replication in vitro
(1) and was later shown to be required for the expression of many
cellular and viral genes. NFI-binding sites occur both in genes
expressed in multiple tissues (2) and in genes expressed solely in
brain (3), muscle (4), liver (5), mammary gland (6), and other
differentiated cell types (7). NFI-binding sites are also found in the
promoter regions of several viruses including the neurotropic JC virus (8), human papilloma virus type 16 (9), and the mouse mammary tumor
virus (10). Mutational analysis indicates that these NFI sites are
required for the proper expression of many tissue-specific and
developmentally regulated genes (11, 12).
Cloning of cDNAs encoding NFI proteins from several species
(13-15) has identified a family of four genes (NFI-A, NFI-B,
NFI-C, and NFI-X) that are highly conserved from
chickens to humans. NFI proteins contain a highly conserved but
gene-specific NH2-terminal 200 amino acid domain that
mediates DNA binding, dimerization, and the initiation of Ad
replication (16, 17). NFI proteins bind to DNA as both homo- and
heterodimers and recognize the consensus binding site,
TTGGC(N5)GCCAA with the same apparent affinity (18, 19).
However, considerable variation occurs within the COOH-terminal domains
of the NFI proteins that likely encode distinct transcription modulation domains. Additional variation between NFI proteins is
generated through differential splicing of transcripts from each of the
four genes (20).
The existence of four conserved NFI genes in vertebrates that generate
multiple alternatively spliced polypeptides argues in favor of diverse
functions of individual NFI gene products. We have previously shown
that NFI proteins representing each of the four murine NFI genes
exhibit promoter-specific differences in their maximal transcriptional
activation potentials due to differences in their COOH-terminal regions
(21, 22). The molecular basis for such differences in the
transcriptional activation properties of NFI proteins is unknown.
However, direct interactions between a human NFI-C isoform (CTF1) and
components of the basal transcriptional machinery have been reported,
with interactions dependent on a sequence motif related to the
COOH-terminal heptapeptide repeat (CTD) of RNA polymerase II (23). This
is unlikely to be the only mechanism by which NFI proteins activate
transcription as some NFI proteins lacking a CTD repeat are potent
activators in both yeast (24) and mammalian cells (21).
While it is widely accepted that NFI proteins differ in their
activation potentials, their ability to repress transcription is poorly
understood. Here, we show that NFI proteins exhibit cell type- and
promoter-specific differences in their repression properties, with
NFI-C and -X repressing the MMTV promoter in HeLa cells, while NFI-A
and -B do not. In contrast to earlier studies, we show that this
repression domain of NFI-C is contained within the
NH2-terminal DNA binding domain, but repression does not
require DNA binding activity. We also demonstrate that overexpression of p300/CBP or SRC-1A abrogates repression of MMTV expression, suggesting that NFI-C-mediated repression may occur by interference with coactivator function at the MMTV promoter.
 |
MATERIALS AND METHODS |
Plasmid Constructs--
The reporter plasmid pMMTV
-gal
contains the mouse mammary tumor virus-long terminal repeat promoter
expressing the bacterial
-galactosidase gene and has been described
previously (21). Reporter plasmids pNFI
-gal and pNFIGRE-
-gal are
derivatives of the pB series of vectors that were described previously
(22, 25) and contain a single NFI-binding site cloned immediately upstream of the AdMLP (
51 to +33) promoter element. pNFIGRE
-gal contains four glucocorticoid response elements (GREs) adjacent to the
NFI-binding site which render it glucocorticoid-responsive. NFI
effector plasmids pCHNFI-A, pCHNFI-B, pCHNFI-C, and pCHNFI-X have been
described previously (21) and express HA epitope-tagged (26) murine
proteins homologous to chicken NFI-A1.1, -B2, -C2, and human NFI-X2
(20). Plasmid pCHNFI-C-240, which expresses the DNA binding domain of
the murine NFI-C isoform, was created by digesting pCHNFI-C with
BstEII/BglII, repair with Klenow polymerase, and
religation. Alternately, pCHNFI-C was digested with
DraIII/BglII, AspI/BglII,
or MscI/BglII, repair with Klenow polymerase, and religation to create pCHNFI-C1-160, pCHNFI-C1-130, and pCHNFI-C1-75, respectively. pNFI-X-210 and pNFI-B-235 which express the DNA binding
domain of the NFI-X and NFI-B proteins were made by digesting pNFI-X
with AflII/BglII and pNFI-B with
BstEII/BglII, repair with Klenow, and religation.
Sequence, orientation, and correct reading frame of all plasmids was
verified by automated sequencing (CCF Molecular Biotechnology Core
Facility). Plasmids expressing human glucocorticoid receptor (hGR) and
human progesterone receptor (hPR) were gifts from Drs. Ron Evans and
Bert O'Malley, respectively. phGR was created by polymerase chain
reaction amplification and subcloning a fragment containing the human
glucocorticoid receptor coding sequence into a CMV expression vector.
Expression plasmids for coactivators p300 (27), CBP (28), SRC-1A (29),
and RAC3 (30) were gifts from Drs. D. Livingston, R. Goodman, B. O'Malley, and J. Chen, respectively, and contain the CMV
promoter-driving expression of each coactivator.
Cell Culture Transfection and Assays--
The cell lines HeLa,
COS-1, 293, and JEG-3 cells (American Type Culture Collection) were
cultured in
-minimum Eagle's medium (Mediatech) containing 10%
fetal bovine serum. Twenty-four hours prior to transfection, 2 × 105 cells were plated onto 60-mm dishes and then
transfected using calcium phosphate coprecipitation as described (31)
Typically, each coprecipitation consisted of an
NFI-dependent
-gal reporter construct (5 µg),
SV-40-luciferase (pGL2-Control, Promega), internal control (2.5 µg),
and 2.5 µg of various NFI effector constructs. When indicated, 0.5 µg of phGR or phPR were cotransfected with pMMTV
-gal and
pNFIGRE
-gal. Carrier DNA (pBSIIKS+) was added to a total of 15 µg
of transfected DNA per plate. Cells were incubated with
CaPO4/DNA precipitate for 12-16 h, washed with PBS, and
incubated for 24 h in culture media with or without 0.1 µM dexamethasone or 10 nM progesterone
(Sigma). Cells were harvested in 300 µl/plate of Reporter Lysis
buffer (Promega) with luciferase and
-gal assays performed as
described (21). Transfections were performed in quadruplicate using
duplicate precipitates for each point, and all results were confirmed
by multiple independent experiments using at least two different
CsCl-purified preparations of plasmid DNA.
Gel Mobility Shift Assays and HA Antibody Supershifts--
For
whole cell extracts cell monolayers were washed twice with ice-cold PBS
and scraped into a 1.5-ml Eppendorf tube. Cells were pelleted by
spinning at 1000 × g for 5 min at 4 °C; the
supernatant was removed; and the pellet was resuspended in Lysis buffer
(100 mM Tris, pH 7.4, 350 mM NaCl, 10%
glycerol, 1% Nonidet P-40, 1 mM EDTA, 1 mM
dithiothreitol, 1 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml
pepstatin, 10 µg/ml aprotinin, and 1 mM
phenylmethylsulfonyl fluoride) and incubated on ice for 15 min with
occasional swirling. Cell debris was pelleted by spinning at
16,000 × g for 10 min, with the supernatant removed
and stored at
80 °C. Gel mobility shift analysis were performed
using the 32P-labeled FIB-2.6 oligonucleotide as described
previously (18). For antibody supershift analysis with the C125A
anti-HA antibody (Boehringer Mannheim), binding reactions were carried
out for 30 min on ice, followed by the addition of 1 µl of the
anti-HA antibody (200 µg/ml) and further incubation for 20 min at
room temperature. As a control, binding reactions not incubated with antibody were treated with 1 µl of PBS and incubated for 20 min at
room temperature. The DNA-protein complexes were resolved on a 6.5%
polyacrylamide, 0.25% TBE gel, and analyzed using a Molecular Dynamics
model 400 PhosphorImager.
Immunocytochemistry--
To detect the intracellular location of
the HA-tagged NFI proteins, HeLa cells were grown on coverslips,
transfected with vectors expressing the NFI protein or control vectors,
cultured for 48 h, and fixed in chilled methanol at
20 °C for
15 min. Fixed cells were blocked for 1 h at room temperature with
3% bovine serum albumin, 0.1% Tween 20 in PBS (PBST), incubated with
2-3 µg/ml anti-HA antibody in PBST for 45 min, washed in PBS,
incubated for 45 min with fluorescein isothiocyanate-conjugated
secondary antibody, and washed in PBS; the coverslips were mounted with Vectashield (Vector Laboratories); the cells were examined using a
Nikon fluorescence microscope; and the images were captured with an
Oncor imaging system (Cleveland Clinic Fluorescent Microscopy Core).
For control staining, the specific antibody solution was replaced by
PBST alone.
Western Blot Analysis--
Cells transfected with the NFI
effector plasmids and hormone receptor expression plasmids were
pelleted, lysed directly in 1× Laemmli buffer (32), boiled for 10 min,
separated on a SDS-polyacrylamide gel, and electroblotted onto
Immobilon-P membranes (Millipore). The blots were blocked and
hybridized with either anti-HA antibody (Boehringer Mannheim) or
anti-GR PA-510 antibody (Affinity Bioreagents) as per manufacturer's
instructions. Proteins were detected by chemiluminescence (ECL,
Amersham Pharmacia Biotech).
 |
RESULTS |
Overexpression of NFI-C and NFI-X Proteins Represses Glucocorticoid
Induction of the MMTV-Long Terminal Repeat Promoter in HeLa
Cells--
We previously reported the cloning of cDNAs
representing each of the four murine NFI genes, analyzed their
embryonic and postnatal expression patterns, and demonstrated
promoter-specific differences in their relative activation potentials
(21, 22). Our previous studies showed that NFI proteins representing
each of the four NFI genes differentially activated the MMTV promoter
in human JEG-3 choriocarcinoma cells, with NFI-B activating expression ~12-fold, NFI-X activating ~10-fold, NFI-C ~6-fold, and NFI-A only ~2-fold. To determine whether the maximum activation potentials of these NFI isoforms differed in different cell types, we first examined their ability to modulate the MMTV promoter in human HeLa
cells. HeLa cells had previously been used to demonstrate the
requirement for an NFI-binding site for glucocorticoid induction of the
MMTV promoter (33). Surprisingly, coexpression of NFI-C or NFI-X
completely repressed glucocorticoid induction of the MMTV promoter in
HeLa cells (Fig. 1A, lanes 8 and 10 versus lane 2). In contrast,
glucocorticoid induction of the MMTV promoter was not repressed in
cells transfected with either control CMV vector (lane 2) or
vectors expressing NFI-A (lane 4) or NFI-B (lane
6). As expected, expression of the MMTV promoter was completely dependent on the addition of dexamethasone (Fig. 1A, lane 2 versus lane 1) and coexpression of human glucocorticoid
receptor (hGR, Fig. 1B, lane 6 versus lane
2). Repression of MMTV expression by NFI-C and NFI-X appeared to
be promoter-specific, since expression from the chimeric NFI
-gal
promoter, which contains a single NFI-binding site upstream of the Ad
major late promoter, was not repressed by coexpression of any of the
NFI proteins (Fig. 1B, lanes 10-13 versus lane 9). Previous studies had shown that
this promoter requires NFI binding for maximal expression (22, 25).
Differences in repression by the four NFI gene products were not due to
differences in expression since all four proteins were expressed at
similar levels, as assessed by Western blot analysis of the HA-tagged proteins (Fig. 1C).

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Fig. 1.
Promoter-specific differences in the
transcriptional modulation properties of NFI proteins.
A, HeLa cells were transfected with pMMTV -gal (5.0 µg),
phGR (0.5 µg), the internal control SV-40 luciferase vector (pGL2), and 2.5 µg of CMV control
vector (lanes 1 and 2) or CMV vectors expressing
NFI-A (lanes 3 and 4), NFI-B (lanes
5 and 6), NFI-C (lanes 7 and
8), or NFI-X (lanes 9 and 10).
Transfected cells were cultured for 24 h in media in the absence
or presence of 0.1 µM dexamethasone ( or
+Dex). -Galactosidase activity was normalized to
luciferase activity levels, with the bars representing the
mean and range of four measurements from duplicate transfections.
B, HeLa cells were transfected with pMMTV -gal (5 µg,
lanes 1-8) or pNFI -gal (10 µg, lanes
9-13), phGR (0.5 µg, lanes 5-8), and with 2.5 µg
of CMV control vector (lanes 1, 2, 5, 6, and 9)
or CMV vectors expressing NFI-A (lane 10), NFI-B (lane
11), NFI-C (lanes 3, 4, 7, 8, and 12), or
NFI-X (lane 13). Cells were cultured for 24 h in the
absence or presence of 0.1 µM dexamethasone ( or
+Dex). -Galactosidase activity was normalized to
luciferase and is plotted as in A. C, cells were
mock-transfected (lane 1) or transfected with 2.5 µg of
NFI cDNA vectors expressing NFI-A (lane 2), NFI-B
(lane 3), NFI-C (lane 4), or NFI-X (lane
5). Whole cell extracts were analyzed on a 7.5%
SDS-polyacrylamide gel transferred to Immobilon-P membrane and probed
with anti-HA antibodies. Numbers at left indicate
size markers (Mr × 10 3), and
arrows on right indicate HA-tagged NFI
polypeptides. Nonspecific bands are shown by the asterisk on
the right.
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NFI-C Repression Domain Is Contained within the
NH2-terminal 160 Amino Acids and Is DNA
Binding-independent--
To determine which domain(s) of the NFI-C and
-X proteins mediated repressor function, we made COOH-terminal deletion
constructs (Fig. 2A). As NFI-C
and NFI-X both repressed the MMTV promoter in HeLa cells and NFI-A and
NFI-B did not, deletion analysis was performed on the NFI-B and NFI-C
proteins as examples of the two classes. The COOH-terminal deletion
constructs express NFI-C proteins of 75 aa (NFI-C-75), 135 aa
(NFI-C-130), 160 aa (NFI-C-160), and 240 aa (NFI-C-240). A plasmid
containing the DNA binding domain NH2-terminal 235 residues
of the NFI-B isoform (NFI-B-235) was used as a negative control. As
expected, cotransfection of either the full-length NFI-B or NFI-B-235
expression plasmids failed to repress dexamethasone induction of the
MMTV promoter in HeLa cells (Fig. 2B, lanes 14 and
16 versus lane 2). As shown above, glucocorticoid induction of the MMTV reporter was completely repressed upon cotransfection with the full-length NFI-C (NFI-C-439) vector (Fig.
2B, lane 4 versus lane 2). Repression
of the MMTV promoter was also observed with cotransfection with the
NFI-C-240 and NFI-C-160 vectors (Fig. 2B, lanes 6 and
8). Cotransfection of either pNFI-C-130 or pNFI-C-75 failed
to repress dexamethasone induction of the MMTV promoter (Fig. 2B,
lanes 10 and 12 versus lanes 4, 6, and 8), localizing the repression domain to a region
within the NH2-terminal 160 residues of NFI-C.

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Fig. 2.
Repression of MMTV expression requires only
the NH2-terminal 160 residues of NFI-C. A,
diagram of NFI-C and NFI-B HA epitope-tagged COOH-terminal deletion
constructs used to characterize the NFI-C repression domain.
B, HeLa cells were transfected with pMMTV -gal (0.5 µg),
phGR (0.5 µg), pGL2 (2.5 µg), and CMV control vector (2.5 µg,
lanes 1 and 2), or CMV vectors expressing NFI-C
(2.5 µg, lanes 3 and 4), NFI-C-240 (2.5 µg,
lanes 5 and 6), NFI-C-160 (2.5 µg, lanes
7 and 8), NFI-C-130 (2.5 µg, lanes 9 and
10), NFI-C-75 (2.5 µg, lanes 11 and
12), NFI-B (2.5 µg, lanes 13 and
14), or NFI-B-240 (2.5 µg, lanes 15 and 16). Cells were cultured for 24 h in the absence or
presence of 0.1 µM dexamethasone ( or +Dex).
-Galactosidase activity was normalized to luciferase and plotted as
in Fig. 1 with the bars representing the mean and range of
four measurements from duplicate precipitates. C, gel
mobility shift analyses were performed as described under "Materials
and Methods" with an NFI-binding site containing 32P-FIB
2.6 oligonucleotide. Extracts of HeLa cells transfected with 2.5 µg
of CMV control vector (lanes 1 and 2), pCHNFI-B
(lanes 3 and 4), or pCHNFI-C-160 (lanes
5 and 6) were incubated with the oligonucleotide in the
presence (lanes 2, 4, and 6) or absence
(lanes 1, 3 and 5) of the anti-HA antibody.
Arrow A indicates anti-HA antibody-NFI-DNA supershifted
complexes; arrow B indicates NFI·DNA complexes, and
arrow C shows the oligonucleotide DNA trailing edge.
D, cells were mock-transfected (lane 1) or
transfected with 2.5 µg of vectors expressing NFI-B (lane
2) or NFI-C-160 (lane 3). Whole cell extracts were
analyzed as in Fig. 1C. Numbers at
left indicate size markers (Mr × 10 3), and arrows on the right
indicate HA-tagged NFI polypeptides. E, vectors expressing
various HA-tagged NFI proteins were transfected into HeLa cells, the
cells were fixed and stained with anti-HA monoclonal antibodies and
fluorescein isothiocyanate-conjugated antisera, and the cells were
examined by fluorescent microscopy. The panels show cells
expressing NFI-C (panel A), NFI-C-160
(panel B), NFI-C-130 (panel
C), or NFI-C-160 cells stained with secondary antibody alone
(panel D).
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Previous studies showed that ~200 NH2-terminal residues
of NFI proteins are required for DNA binding and dimerization (16, 19,
34). Since HeLa cells contain endogenous NFI proteins (18), anti-HA
antibody supershifts were used to assess the DNA binding ability of the
various transfected HA-tagged NFI proteins. As seen previously (18),
multiple NFI·DNA complexes were formed on the FIB 2.6 oligonucleotide
in extracts of mock-transfected HeLa cells (Fig. 2C, lanes 1 and 2, depicted by bracket B). None of these
complexes were supershifted by anti-HA antibody (Fig. 2C, lane
2 versus lane 1). In contrast, a robust
anti-HA antibody supershift was observed in nuclear extracts from cells
transfected with the NFI-B expression vector (Fig. 2C, lane
4 versus lane 3, arrow A).
Analogous anti-HA-supershifted complexes were detected in extracts of
cells transfected with the NFI-C-439, NFI-C-240, and NFI-B-235 vectors
(data not shown (22)). However, no anti-HA-supershifted complexes were
detected in extracts of cells transfected with the NFI-C-160 vector
(Fig. 2C, lane 6 versus lane 5),
indicating that the NFI-C-160 protein lacks DNA binding activity. The
lack of DNA binding activity is not due to poor expression of
NFI-C-160, since Western blots show that similar levels of NFI-B and
NFI-C-160 proteins are expressed (Fig. 2D). Lack of DNA
binding by NFI-C-160 was confirmed by transfection of the NFI-C-160
vector into JEG-3 cells which lack endogenous NFI proteins (data not
shown). These data, together with previous studies showing that ~200
NH2-terminal residues of NFI proteins are needed for DNA
binding and dimerization (16, 19, 34), indicate that repression of the
MMTV promoter by the NFI-C-160 protein occurs in the absence of NFI DNA
binding or dimerization activity.
To assess whether the differences in repression by the NFI-C
COOH-terminal deletion proteins were due to differences in cellular localization, we examined the intracellular location of the NFI-C proteins by immuno-histochemistry. Mock-transfected cells stained with
the anti-HA antibodies or cells stained with secondary antibody alone
showed no fluorescence (Fig. 2E, panel D, and
data not shown). Although both NFI-C-439 and NFIC-160 exhibited strong
nuclear fluorescence (Fig. 2, panel A versus panel B), cells
expressing NFI-C1-160 exhibited some diffuse cytoplasmic staining not
seen with NFI-C-439 (compare Fig. 2, panel A
versus panel B). In contrast, cells expressing
NFI-C-130 stained weakly in the nucleus but showed strong cytoplasmic
staining with a punctate distribution (compare Fig. 2, panel C
versus panels A and B), suggesting the presence of a nuclear
localization or retention signal in residues 130-160 of NFI-C.
To determine if the lack of repression observed with the NFI-C-130
construct was due to differences in nuclear localization, we made a
chimeric construct containing residues 1-130 of NFI-C fused to
residues 131-235 of NFI-B. Expression and nuclear localization of the
chimeric protein was verified by DNA binding studies (data not shown).
When cotransfected with the MMTV promoter in HeLa cells, the chimeric
NFI construct failed to repress glucocorticoid induction (data not
shown) suggesting that the differences in the repression properties of
the NFI-C-160 and NFI-C-130 proteins are not due solely to differences
in nuclear localization.
Overexpression of Glucocorticoid Receptor Alleviates NFI-C-mediated
Repression in HeLa Cells--
The ability of NFI-C to repress
glucocorticoid induction of the MMTV promoter but not the chimeric
NFI
-gal reporter indicated that NFI-C might repress by inhibition of
glucocorticoid receptor function. Since NFI-C-mediated repression is
DNA binding-independent, it is unlikely that repression occurs by NFI
and GR competing for binding to the MMTV promoter. Thus, repression
likely involves some form of protein-protein interactions, either
through direct interactions between NFI-C and GR or by NFI-C and GR
competing for a limiting coactivator protein required for MMTV
transcription (see "Discussion"). Either model would predict that
repression by NFI-C might be overcome by overexpressing GR. To test
this, we cotransfected increasing amounts of the hGR vector with
pMMTV
-gal, with or without the NFI-C expression plasmid (Fig.
3A). As seen above, HeLa cells
cotransfected with 0.5 µg of phGR, and pMMTV
-gal exhibited strong
dexamethasone-dependent MMTV promoter activity (Fig.
3A, lane 2 versus lane 1). In the
absence of coexpressed NFI-C, increasing amounts of cotransfected phGR
increased dexamethasone-dependent MMTV activity ~2-fold
(lane 4 versus lane 2) and ~2.5-fold
(lane 6 versus lane 2). MMTV promoter
activity in cells transfected with 5.0 µg of the hGR expression
plasmid appeared to represent maximal promoter activation as further
increasing the amount of phGR DNA transfected did not increase
expression (data not shown). As seen above, repression of MMTV
expression was observed when NFI-C was coexpressed together with either
0.5 or 1.5 µg of phGR DNA (lanes 8 and 10 versus lanes 2 and 4, respectively).
However, repression by NFI-C was partially abrogated when 5.0 µg of
phGR was cotransfected (lane 12 versus
lanes 10 and 8). This ability of NFI-C to repress
glucocorticoid induction of the MMTV promoter when sub-saturating
amounts of the GR expression vector are used appears not to be due to
NFI inhibiting GR expression, since Western blot analysis shows that
glucocorticoid receptor levels are unaffected by expression of NFI-C
(Fig. 3B, lane 3 versus lane 2, lane 5 versus lane 4, and lane 7 versus lane 6).

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Fig. 3.
NFI-C repression of the MMTV promoter is
abrogated by overexpression of hGR, but NFI-C expression does not
affect hGR levels. A, cells were transfected with
pMMTV -gal (5.0 µg), pGL2 (2.5 µg), and CMV control vector (2.5 µg, lanes 1-6), or a CMV vector expressing NFI-C (2.5 µg, lanes 7-12) with the indicated amounts of phGR. Cells
were cultured in the absence or presence of 0.1 µM
dexamethasone ( or +Dex) for 24 h. -Galactosidase
values were normalized to luciferase values and plotted as in Fig. 1.
B, HeLa cells were mock-transfected (lane 1) or
transfected with the indicated amounts of phGR and CMV control vector
(2.5 µg, lanes 2, 4, and 6), or the NFI-C
expression vector (2.5 µg, lanes 3, 5, and 7).
Whole cell extracts were analyzed on a 5% SDS-polyacrylamide gel
transferred to Immobilon-P membrane and probed with an anti-GR antibody
(PA510, Affinity Bioreagents). Numbers indicate size markers
(Mr × 10 3); the arrow
indicates glucocorticoid receptor, and the asterisk
indicates a proteolytic fragment of GR.
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Progesterone Stimulation of the MMTV Promoter Is Not Repressed by
NFI-C--
Previous studies have shown that in addition to being
activated by glucocorticoids, MMTV promoter expression can also be
induced by progesterone in the presence of progesterone receptor (35). To test if NFI-C-mediated repression was specific to the glucocorticoid response of the MMTV promoter, we transfected HeLa cells with pMMTV
-gal and vectors expressing either progesterone or
glucocorticoid receptor (Fig. 4). In the
absence of cotransfected hPR, no progesterone-dependent MMTV expression was seen (Fig. 4, lane 2 versus
lane 1 and lane 4 versus lane
3). Transfection of increasing amounts of phPR DNA increased
progesterone-dependent MMTV expression (Fig. 4, lane 10 versus lane 6 and lane 14 versus lanes 10 and 6), with maximal activity approaching ~40% of dexamethasone-dependent
activity (lane 14 versus lane 18).
However, unlike NFI-C-mediated repression of
glucocorticoid-dependent MMTV expression (lane 20 versus lane 18), progesterone induction of the
MMTV promoter was not repressed by NFI-C at all levels of hPR DNA used
(lane 8 versus lane 6, lane 12 versus
lane 10, and lane 16 versus lane
14).

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Fig. 4.
NFI-C does not repress progesterone induction
of the MMTV promoter. Cells were transfected with pMMTV -gal (5 µg), pGL2 (2.5 µg), the indicated amounts of phPR (lanes
5-16) or phGR (0.5 µg, lanes 17-20), and CMV
control vector (2.5 µg, lanes 1, 2, 5, 6, 9, 10, 13, 14, 17, and 18) or the NFI-C expression vector (lanes
3, 4, 7, 8, 11, 12, 15, 16, 19, and 20). Cells were
cultured for 24 h in the absence or presence of 10 nM
progesterone ( or + Pro) or 0.1 µM
dexamethasone ( or +Dex). -Galactosidase activity was
normalized to luciferase levels and plotted as in Fig. 1.
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Expression of NFI-C Does Not Repress pNFIGRE
-gal in HeLa
Cells--
Since expression of NFI-C repressed glucocorticoid
induction of the MMTV promoter but had no effect on progesterone
induction, we tested whether repression occurred with other
glucocorticoid-dependent promoters. pNFIGRE
-gal was made
by the addition of two glucocorticoid response elements (GREs)
immediately upstream of the NFI-binding site in pNFI
-gal (22). We
compared the activity of this promoter with both the MMTV promoter and
the NFI promoter lacking GREs. As seen previously, MMTV expression was
repressed by NFI-C at low GR levels but not at high GR levels (Fig.
5, lane 8 versus lane 4), whereas pNFI expression was unaffected by
NFI-C (lane 12 versus lane
10). In the absence of cotransfected hGR DNA, the NFIGRE promoter
showed a low level of induction by glucocorticoids, and this activity
was not repressed by NFI-C (lane 16 versus lane 14). With increasing amounts of transfected hGR DNA, NFIGRE
activity increased by ~2-fold (0.5 µg, lane 18) and
~4-fold (5.0 µg, lane 22). However, unlike the MMTV
promoter, NFIGRE activity was not repressed by NFI-C at any level of GR
expression (lane 24 versus lane 22,
lane 20 versus lane 18, and lane
16 versus lane 14). This lack of
NFI-C-mediated repression, even in the absence of any coexpressed GR,
indicates that NFI-C expression likely only affects a subset of
GR-responsive promoters. The low level of GR-induced activity of NFIGRE
promoter in the absence of coexpressed GR (Fig. 5, lane 14)
differs from the absolute requirement for GR coexpression for MMTV
activity (Fig. 1B, lane 6 versus lane 2), suggesting that the NFIGRE promoter is more sensitive
than the MMTV promoter to low levels of endogenous GR present in HeLa cells.

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Fig. 5.
NFI-C does not repress glucocorticoid
induction of pNFIGRE -gal. HeLa cells were
transfected with pMMTV -gal (5.0 µg, lanes 1-8),
pNFI -gal (10.0 µg, lanes 9-12), pNFIGRE -gal (5.0 µg, lanes 13-24), pGL2 (2.5 µg), the indicated amounts
of phGR, and cotransfected with the CMV expression vectors (2.5 µg)
indicated below the figure. -Galactosidase and luciferase activity
of extracts prepared from cells cultured in the absence or presence of
0.1 µM dexamethasone ( or +Dex) for 24 h is plotted as in Fig. 1.
|
|
NFI-C-mediated Repression Is Cell Type-specific--
We recently
reported that NFI-C activates the MMTV promoter in JEG-3 cells (22).
Here, we have shown that NFI-C represses glucocorticoid-dependent MMTV expression in HeLa cells. To
determine if NFI-C-mediated repression of the MMTV promoter was unique
to HeLa cells, we performed transient transfections in both COS-1 and
293 cells (Fig. 6A). As with
HeLa cells, cotransfection of phGR was required to detect
dexamethasone-dependent MMTV activation in all cell types
(data not shown). As reported previously, in JEG-3 cells (22),
dexamethasone-dependent activation of the MMTV promoter in the
absence of coexpressed NFI-C is weak (lane 14), which likely
reflects low levels of endogenous NFI in these cells. Expression of
NFI-C in JEG-3 cells results in an ~7-fold increase in
dexamethasone-induced MMTV promoter activity (lane 16 versus lane 14) showing that NFI-C functions as
an activator in these cells. In contrast, in both HeLa and COS-1 cells,
dexamethasone induction of the MMTV promoter was completely repressed
by cotransfection of the NFI-C expression vector (Fig. 6A, lane
4 versus lane 2 and lane 8 versus lane 6, respectively). However,
cotransfection of the NFI-C expression vector failed to repress
glucocorticoid induction of the MMTV promoter in 293 cells (lane
12 versus lane 10), demonstrating that
repression of the MMTV promoter by NFI-C is cell type-specific.
Differences in the transcription modulation properties of the NFI-C
protein in these cell lines were not due to differences in NFI-C
levels, as Western blot analysis showed similar levels of HA-tagged
NFI-C proteins expressed in each cell line (Fig. 6B, arrow).
To ensure that the lack of repression in 293 cells was not due to
higher hGR levels in these cells, we titrated the amount of hGR vector
transfected and saw no repression by NFI-C, even at clearly submaximal
hGR levels (Fig. 6C).

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Fig. 6.
Repression of MMTV promoter activity by NFI-C
is cell type-specific. A, cell lines (indicated above
the figure) were transfected with pMMTV -gal (5 µg), phGR (0.5 µg), pGL2 (2.5 µg) internal control plasmid, and 2.5 µg of the
CMV expression vectors (Control CMV, lanes 1 and
2, 5 and 6, 9 and 10, and
13 and 14; NFI-C, lanes 3 and 4, 7 and 8, 11 and 12, and 15 and
16). -Galactosidase and luciferase activity of extracts
prepared from each cell line cultured with or without 0.1 µM dexamethasone (+ or Dex) for 24 h was plotted as in Fig. 1 with the
bars representing the mean and range of four measurements
from duplicate transfections. B, Western blot analysis was
performed on whole cells extracts prepared from HeLa cells (lane
1 and 2), COS-1 cells (lane 3), 293 cells
(lane 4), and JEG-3 cells (lane 5) that were
mock-transfected (lane 1) or transfected with 2.5 µg of a
NFI-C expression vector (lanes 2-5). Whole cell extracts
were analyzed on a 7.5% SDS-polyacrylamide gel, transferred to
Immobilon-P membrane, and probed with anti-HA antibodies.
Numbers indicate size markers (Mr × 10 3), and the arrow indicates the HA-tagged
NFI-C polypeptide. Nonspecific bands are shown by the
asterisk on the right. C, 293 cells
were transfected with pMMTV -gal (5 µg), pGL2 (2.5 µg), various
amounts of phGR (indicated below the figure), and 2.5 µg of CMV
control vector (lanes 1-6) or the NFI-C expression vector
(lanes 7-12). -Galactosidase and luciferase activity of
extracts prepared from each cell line cultured with or without 0.1 µM dexamethasone (+ or Dex) for 24 h was plotted
as in Fig. 1.
|
|
Coactivators p300/CBP and SRC-1A, but Not RAC3, Overcome
NFI-C-mediated Repression of the MMTV Promoter in HeLa Cells--
The
inability of NFI-C to repress glucocorticoid induction of
pNFIGRE
-gal (Fig. 5), together with the cell type-specific repression by NFI-C (Fig. 6), argues against repression by direct interaction of NFI-C with glucocorticoid receptor. An alternate model
would have NFI-C and GR competing for a limiting cofactor (in HeLa and
COS-1 cells) which is required for dexamethasone induction of the MMTV
promoter. To test this model we overexpressed various steroid receptor
coactivator proteins with NFI-C and pMMTV
-gal to ask if they could
overcome NFI-C-mediated repression (Fig. 7). Cotransfection of the RAC3 expression
plasmid had no effect on MMTV expression in either the absence or
presence of NFI-C (Fig. 7, lane 4 versus lane
2 and lane 12 versus lane
10, respectively). However, expression of either p300 or SRC-1A
increased MMTV promoter activity slightly (<2-fold) in the absence of
NFI-C (Fig. 7, lanes 6 and 8 versus
lane 2) and abrogated the repression of MMTV activity by
NFI-C (Fig. 7, lanes 14 and 16 versus lane
10). Alleviation of NFI-C-mediated repression was also seen with
coexpression of CBP (not shown). This alleviation of NFI-C-mediated
repression by both p300/CBP and SRC-1A suggests several models for how
these proteins may function with NFI-C at the MMTV promoter (see
"Discussion").

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Fig. 7.
Expression of coactivators p300 and SRC-1
abrogate NFI-C-mediated repression. Cells were transfected with
pMMTV -gal (5.0 µg), phGR (0.5 µg), pGL2 (2.5 µg), and CMV
control vector (2.5 µg, lanes 1-8) or a CMV vector
expressing NFI-C (2.5 µg, lanes 9-16) and cotransfected
with CMV vectors expressing RAC3 (2.5 µg, lanes 3, 4, 11 and 12), p300 (2.5 µg, lanes 5, 6, 13, and
14), or SRC-1A (2.5 µg, lanes 7, 8, 15, and
16). Cells were cultured in the absence or presence of 0.1 µM dexamethasone ( or +Dex) for 24 h.
-Galactosidase values were normalized to luciferase values and
plotted as in Fig. 1.
|
|
 |
DISCUSSION |
We have used the NFI-dependent MMTV promoter to assess
the transcription modulation properties of NFI proteins from each of the four murine NFI genes (Nfi-a, -b, -c, and
-x). In transiently transfected HeLa cells, expression of
NFI-C or NFI-X represses glucocorticoid induction of the MMTV promoter,
whereas expression of NFI-A or NFI-B does not (Fig. 1A). In
contrast to all previously described functions of NFI proteins, this
repression is DNA binding-independent, as expression of the non-DNA
binding NH2-terminal 160 residues of NFI-C represses (Fig.
2B). Repression by NFI-C is overcome by overexpression of
the glucocorticoid receptor (Fig. 3A), suggesting that
repression may occur through interference with GR function. However,
the chimeric pNFIGRE
-gal reporter, which is also dependent on both
NFI and GR for expression, is not repressed (Fig. 5), showing that
repression occurs in only a subset of GR-dependent promoters. Repression of MMTV is cell type-specific, occurring in HeLa
and COS-1 cells but not JEG-3 or 293 cells (Fig. 6A), suggesting cell type specificity in either NFI function or in the
mechanism of GR activation of the MMTV promoter. Surprisingly, progesterone-dependent activation of the MMTV promoter is
not repressed by NFI-C (Fig. 4), indicating that the mechanism of activation by progestins may differ from that of glucocorticoids. In
addition, NFI-C-mediated repression is abrogated by coexpression of the
receptor coactivators CBP/p300 and SRC-1A (Fig. 7), suggesting that
repression may occur by interference with coactivator function at the
MMTV promoter.
Previous studies showed that NFI proteins possess highly homologous
NH2-terminal DNA binding/dimerization domains and more divergent COOH-terminal transactivation or repression domains (14, 16,
36, 37). For example, in JEG-3 choriocarcinoma cells, NFI proteins
representing each of the four NFI genes differentially activate the
MMTV promoter, and these differences in transactivation are mediated
solely by their COOH-terminal domains (22). Here we report that in
transfected HeLa cells, NFI proteins differentially repress the MMTV
promoter, with NFI-C or NFI-X repressing glucocorticoid induction while
NFI-A or NFI-B have no effect. Although transcriptional repression by
NFI proteins has previously been reported (38, 39), repression was
dependent on residues within the COOH-terminal domains of NFI proteins
and required DNA binding by NFI or NFI fusion proteins. In contrast,
the repression of the MMTV promoter by NFI-C (and NFI-X) seen here is
mediated by a 160-residue subdomain of the NH2-terminal
domain that is incapable of binding DNA. The DNA binding independence
of the repression suggests that the mechanism of this repression likely
differs from that seen previously by COOH-terminal regions of NFI
proteins. Within this 160-residue repression domain there are 10 residues that differ between NFI-C and the non-repressing NFI-A and
NFI-B proteins, and further studies are needed to identify the specific
residues within NFI-C essential for this repression.
The MMTV promoter is one of the best studied models of steroid
hormone-induced gene expression. Glucocorticoid induction of the MMTV
promoter is dependent on binding sites for glucocorticoid receptor,
NFI- and octamer-binding proteins (33, 40). However, the NFI-binding
site appears dispensable for progesterone induction of the MMTV
promoter, since mutation of the NFI-binding site, which abolished
glucocorticoid induction, did not affect progesterone induction of the
promoter (41). Also, differences between the activation potentials of
GR and PR on episomal and transiently transfected MMTV promoter
constructs have been reported (42). These findings, together with our
observed lack of NFI-C-mediated repression of PR activation of the MMTV
promoter (Fig. 4), lend strong support to the hypothesis that
hormone-dependent activation of the MMTV promoter occurs
through at least two different pathways (43, 44). Moreover, our
observation that the GR-dependent NFIGRE promoter is not
repressed by NFI-C suggests that glucocorticoid induction itself may
occur through two different pathways, only one of which is sensitive to
repression by NFI-C.
One difference between the MMTV and NFIGRE promoters that could affect
repression by NFI-C may be their ability to form phased nucleosomal
arrays in vivo. The MMTV promoter contains precisely phased
nucleosomes when present as autonomously replicating episomal DNA (45).
The spatial distribution of these nucleosomes was found to allow access
of GR to HREs in the MMTV promoter, but to preclude access of NFI to
its binding site (46). One highly regarded model for activation of MMTV
transcription is that GR binds to the promoter in the presence of the
phased nucleosomes and directly, or by recruitment of other proteins,
initiates a chromatin remodeling event needed for NFI binding to the
promoter and activation of transcription (47, 48). However, nucleosome phasing has not been seen on transiently transfected MMTV templates. For example, the NFI-binding site on transiently transfected MMTV plasmids is occupied even in the absence of hormone, suggesting that
the bulk fraction of transiently transfected plasmids does not contain
properly positioned nucleosomes that can repress NFI binding (47).
However, it is unclear what fraction of transiently transfected
templates is actively transcribed in vivo, and thus measurements on the bulk population may not reflect the state of the
transcriptionally active templates. Indeed, the findings that occupancy
of the NFI site correlates directly with glucocorticoid induction of
the MMTV promoter on episomal templates (47), together with the
apparent requirement of the NFI site for glucocorticoid induction on
both episomal and transiently transfected templates (33, 47), suggests
that NFI may function in a similar manner in GR induction on both types
of template. Further studies of NFI-C repression of both episomal and
transiently transfected templates will be needed to address this issue.
As noted above, the difference in the ability of NFI-C to repress
glucocorticoid induction of the MMTV and NFIGRE promoters in HeLa cells
could result from differences in chromatin structure between the two
promoters. Consistent with this hypothesis is the apparent difference
in sensitivity to intracellular GR levels of the MMTV and NFIGRE
promoters. NFIGRE is partially activated by dexamethasone in the
absence of cotransfected phGR (Fig. 4), whereas MMTV appears completely
repressed (Fig. 5 versus Fig. 1B). Moreover, the
basal expression level of NFIGRE in the absence of dexamethasone is
higher than that of MMTV (Fig. 5), suggesting that the NFIGRE promoter
may be more accessible to transcription factor binding even under basal
conditions. Similarly, the apparent cell type-specific repression of
MMTV by NFI-C may be due to differences in the extent or state of
nucleosomal organization of the MMTV promoter between cell lines that
are repressed (HeLa and COS-1) versus cell lines that are
not repressed (JEG-3 and 293) (Fig. 6). Cell type-specific differences
in NFI accessibility to DNA have previously been reported in human
breast T47D and mouse C127 cells containing stably integrated copies of
MMTV. The NFI-binding site of the MMTV promoter was constitutively
occupied by NFI in the absence of dexamethasone in T47 cells, whereas
in C127 cells the NFI-binding site was only occupied in the presence of
dexamethasone (49). Studies using stably integrated or episomal MMTV
templates may be needed to determine the role of chromatin structure in cell type-specific repression of the MMTV promoter by NFI-C.
The two-step model of activation of the MMTV promoter where GR binding
initiates chromatin remodeling, followed by NFI binding, predicts that
proteins that affect nucleosome structure likely play an integral role
at the promoter. One class of proteins that can modify chromatin
structure is the coactivator proteins that have histone
acetyltransferase activity. Hyperacetylation of core histones is a
characteristic of gene activation, and histone deacetylation is
associated with transcriptional repression (reviewed in Refs. 50 and
51). Biochemical and genetic studies have identified a number of
histone acetyltransferase proteins including p300 (52), CBP (52),
SRC-1A (53), P/CAF (54), and ACTR (55). Coexpression of ACTR and GR has
been reported to enhance dexamethasone-dependent activation
of a transiently transfected MMTV reporter ~2-fold in HeLa cells
(55). This finding, together with our observations that coexpression of
p300/CBP or SRC-1A enhances GR-dependent MMTV expression
~2-fold (Fig. 7), suggests that these histone acetyltransferase
proteins may function in the glucocorticoid induction of the MMTV
promoter in HeLa cells. It is important to note that while p300/CBP and
SRC-1A acetylate histones in vitro and effect expression in
our assay system, whether histones are the most important target for
MMTV activation in vivo is not clear. Among the reasons for
this uncertainty are that other targets for p300/CBP acetylation are
known (56, 57) and that p300/CBP and SRC-1A possess both direct
transcriptional activation and protein binding domains that map outside
the domains encoding histone acetyltransferase activity (52, 53, 58).
Knowing which of these functions are required for abrogation of NFI-C repression will be important in determining the precise target of
NFI-C-mediated repression.
Although the molecular basis for repression of the MMTV promoter by the
NFI-C subdomain is unknown, our data are consistent with at least the
following two models: 1) NFI-C interacts with GR and establishes or
stabilizes a repression complex composed of GR, NFI-C, and known or
unknown corepressor proteins, or 2) NFI-C interacts directly with p300,
SRC-1A, or another factor that associates with the known p300·SRC-1A
complex, and represses transcription by preventing the ability of GR to
recruit or activate these proteins at the promoter. Since NFI-C
represses GR-induced expression on only a subset of
GR-dependent promoters, models invoking a direct inhibition
of either DNA binding or transactivation function of GR by NFI-C appear
less likely. Corepressor molecules, including N-Cor, SMRT and others
are known to function at some steroid hormone-dependent
promoters and play an essential role in the repression of thyroid
hormone and retinoid-dependent transcription (59, 60). In
the thyroid hormone system, unliganded RXR·TR heterodimers recruit
multiprotein corepressor complexes that contain N-Cor, SMRT and thyroid
hormone receptors, and histone deacetylases to the promoter and repress
transcription (59, 61). Although these corepressors are not known to
function on glucocorticoid-dependent promoters, it is
possible that binding of the NFI-C subdomain to GR could mimic the
structure of unliganded retinoic acid receptor/thyroid hormone receptor
dimers and mediate active repression of the MMTV promoter. However, we
have failed to see direct interaction of GR with NFI-C in DNA binding
assays or by immunoprecipitation (data not shown), reducing the
likelihood of this model.
A model where NFI-C interacts directly with a component of a p300/CBP
coactivator complex and prevents its recruitment or function at the
MMTV promoter is appealing for several reasons. Previous studies have
demonstrated that microinjection of CBP-inactivating antibodies into
HeLa cells represses glucocorticoid induction of the MMTV promoter in a
dose-responsive manner (62), suggesting an integral role for CBP in
MMTV expression. Our observation that coexpression of p300/CBP or
SRC-1A abrogates NFI-C-mediated MMTV repression is consistent with a
model in which repression by NFI-C occurs through interference with
coactivator function. A requirement for coactivator proteins in MMTV
activation is also supported by the identification of
GR·p300·SRC-1A complexes in vivo (63). However, whereas
expression of SRC-1A or p300/CBP enhanced GR-dependent activity of the MMTV promoter in HeLa cells ~2-fold, the activity of
NFIGRE was not affected (data not shown), suggesting that these coactivator proteins may function (or be rate-limiting) at only a
subset of GR-dependent promoters. Although we favor a model in which NFI-C represses MMTV expression by interacting with a component of the p300/CBP·SRC-1 complex, the direct target of NFI-C
repression is unknown. Thus, it would be of great interest to assess
the ability to abrogate NFI-C-mediated repression of p300/CBP and SRC-1
mutant proteins that are deficient in histone acetyltransferase
activity, transcriptional activation capability, or fail to interact
with GR or other proteins, thereby determining the detailed molecular
mechanism by which NFI-C can repress the MMTV promoter.
 |
ACKNOWLEDGEMENTS |
We thank R. Goodman, B. O'Malley, D. Livingston, and J. Chen for CBP, SRC-1A, RAC3, p300, and hPR
expression plasmids. We are also grateful to D. Luse, D. Driscoll, and
R. Padgett for helpful discussion; C. Campbell for critical reading of
the manuscript; and to J. Lang for photographic services.
 |
FOOTNOTES |
*
This study was supported in part by the Lerner Research
Institute of the Cleveland Clinic Foundation and National Institutes of
Health Grant HD34908 (to R. M. G.).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.
§
Present address: CEFYBO-CONICET, Serrano 669, 1414-Buenos Aires, Argentina.
¶
To whom correspondence should be addressed: Lerner Research
Institute, Dept. of Cancer Biology NB40, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-6629; Fax: 216-445-6269; E-mail: gronosr{at}cesmtp.ccf.org.
 |
ABBREVIATIONS |
The abbreviations used are:
NFI, nuclear factor
I;
MMTV, mouse mammary tumor virus;
GR, glucocorticoid receptor;
CBP, CREB-binding protein;
SRC-1A, steroid receptor coactivator 1A;
RAC3, receptor associated coactivator 3;
Ad, adenovirus;
CTD, carboxyl-terminal domain;
-gal,
-galactosidase;
PR, progesterone
receptor;
CMV, cytomegalovirus;
HA, hemagglutinin;
dexamethasone, 9
-Fluoro-16
-methyl-11
,17
,21-trihydroxy-1,4-pregnadiene-3,20-dione;
progesterone, 4-Pregnene-3,20-dione3-(O-carboxymethyl)oxime;
GRE, glucocorticoid response element;
PBS, phosphate-buffered saline;
h, human;
aa, amino acids.
 |
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