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Department of Veterinary Physiology and Pharmacology Texas A & M University College Station, Texas 77843-4466
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(8, 9, 10, 11, 12). Additionally, E2F1 induces apoptosis in
different cancer cell lines; this is separable from effects of this
protein on cell cycle progression and does not require p53 (13, 14, 15, 16).
Studies with E2F1 knockout mice confirm that E2F1 plays an important
role in apoptosis, and increased tumorigenesis in these animals
suggests that E2F1 may also function in some tissues as a tumor
suppressor gene (17, 18). 17ß-Estradiol (E2) stimulates proliferation and DNA synthesis in estrogen receptor (ER)-positive breast cancer cells, and this is accompanied by an increased percentage of cells in S phase and a decreased fraction in G0/G1. Several studies have shown that this hormone-induced response is accompanied by modulation of several genes/proteins that regulate cell cycle progression (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30). For example, treatment of MCF-7 cells with E2 is accompanied by increased cyclin D1 mRNA and protein, cdk7-, cdk2-, and cdk4-dependent kinase activities, cdc25A phosphatase protein, and increased phosphorylation of retinoblastoma (Rb) protein (20, 21, 22, 23, 24, 25). Rb and related proteins physically interact with E2F1 to give a transcriptionally repressed complex (31, 32), and mitogens such as E2 catalyze phosphorylation of Rb and the subsequent release of E2F1 from the protein complex (20, 21, 22). This study shows that treatment of MCF-7 cells with E2 also induces E2F1 protein and gene expression, and deletion analysis of the E2F1 gene promoter has identified a region between -146 to -54 that is required for E2 responsiveness. This sequence contains a GC-rich Sp1-binding site and two CCAAT motifs that bind NF-Y proteins. Previous studies in this laboratory have demonstrated that ER/Sp1 interactions with GC-rich sites are required for E2-mediated transactivation of some genes (33, 34, 35, 36); however, results of this study show that hormone-mediated transactivation of the E2F1 gene requires not only ER/Sp1 binding to GC-rich sites but also interactions with NF-Y proteins bound to downstream CCAAT sites in the E2F1 gene promoter. It has previously been reported that Sp1 and ER physically interact (33), and this study shows that Sp1 also interacts with NF-YA in coimmunoprecipitation experiments, and Sp1 enhanced NF-Y-DNA binding in gel mobility shift assays. These results indicate that DNA-bound Sp1 protein plays a central role in E2-induced E2F1 gene expression by interacting with ER protein, and downstream NF-Y proteins bound to two CCAAT motifs.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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show
that immunoreactive E2F1 protein and mRNA levels are induced after
treatment with E2. These data are consistent with parallel
induction of E2F1-dependent DNA synthesis in this cell line. E2F1
protein levels are maximally induced 12–24 h after treatment with
E2, and E2F1 mRNA levels are significantly increased more
than 4-fold 12 h after addition of E2. This is the
first report showing that estrogen induces E2F1 gene expression.
Immunoreactive Sp1 protein levels were unchanged in MCF-7 cells after
treatment with E2 for 24 or 48 h (data not shown).
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). In contrast,
both basal and hormone inducibility were restored with
pE2F1g (-173/-54) in which the two downstream E2F1-binding
sites were deleted. Loss of basal activity was observed in cells
transiently transfected with constructs (pE2F1e and
pE2F1f) that do not contain the two downstream CCAAT
motifs. These results indicate that the -173 to -54 region
(pE2F1g) of the E2F1 gene promoter exhibited high basal
activity and was E2-responsive. This sequence contains
three Sp1-binding sites flanked by one upstream and two downstream
CCAAT motifs. Deletion of a downstream CCAAT site (pE2F1i)
resulted in loss of hormone responsiveness, whereas E2
induced luciferase activity in MCF-7 cells transfected with
pE2F1h (-169/-54) in which the upstream CCAAT site was
deleted (Fig. 2A). E2 also induced luciferase activity in
ER-negative MDA-MB-231 cells transiently transfected with pE2F1 h and
ER expression plasmid (data not shown). The potential role of the three
GC-rich sites was investigated by mutational analysis (Fig. 2B) of
pE2F1h; individual mutations of the three upstream GC-rich
sites did not markedly affect hormone responsiveness; however, basal
activity was lowest with pE2F1hm3 that is mutated in Sp1
site 3. Mutation of individual CCAAT sites (pE2F1hm4 and
pE2F1hm5) resulted in loss of basal and hormone-induced
luciferase activity. pE2Fj incorporates a single upstream
GC-rich site and both downstream CCAAT sites and represents a potential
minimal E2-responsive region of the E2F1 gene promoter.
Deletion of the upstream GC-rich site (pE2F1jm1) resulted
in decreased basal luciferase activity and loss of hormone
inducibility, and similar results were observed for an Sp1 mutant
construct (data not shown). Mutation of the CCAAT sites
(pE2F1jm2) also resulted in decreased basal activity and
loss of hormone responsiveness, suggesting that interactions of nuclear
factors at the GC-rich and CCAAT sites within the E2F1 gene promoter
are required for high basal activity and E2 responsiveness
in MCF-7 cells.
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illustrate binding of
nuclear extracts from solvent [dimethylsulfoxide (DMSO)] control
(lane 1) or cells treated with 10 nM E2 to
[32P]-146/-54 oligonucleotide containing the GC-rich
and CCAAT sites. Four major high mol wt retarded bands were observed
(A–D), and band intensities were slightly increased using nuclear
extracts from cells treated with E2 (lane 2). Competition
with consensus unlabeled Sp1 oligonucleotide (lane 3) eliminated bands
B, C, and D (lane 3), and competition with unlabeled downstream
CCAAT-containing oligonucleotide (-115/-54) eliminated bands A, C,
and D and decreased the intensity of band B. This downstream sequence
contains GC-rich elements that may compete for protein binding to the
upstream GC-rich site in the -146/-54 oligonucleotide. Competition
with unlabeled estrogen response element (ERE) and mutant
Sp1m oligonucleotides (lanes 5 and 6) slightly decreased
retarded band formation. A high mobility band (unlabeled) was
competitively decreased only by the -115/-54 oligonucleotide. Low
molecular weight proteins were observed in assays using nuclear
extracts; these bands were not supershifted by antibodies and may be
due, in part, to partially degraded proteins. The results suggest that
nuclear extracts from MCF-7 cells express proteins that bind GC-rich
and CCAAT elements within the -146/-54 region of the E2F1 gene
promoter. Based on results of oligonucleotide competition studies, band
A contains only a CCAAT-binding protein, band B contains a protein
binding a GC-rich site, and bands C and D contain proteins that bind
both CCAAT- and GC-rich sequences.
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) and CCAAT-containing downstream
(-122/-54) (Fig. 5) oligonucleotides in
gel electrophoretic mobility shift assays. Incubation of nuclear
extracts from DMSO (solvent control) and E2-treated cells
(lanes 1, 2, and 8) with [32P]-169/-116 gave one major
specifically bound retarded band (bound-DNA) that was decreased by
coincubation with unlabeled -169/-116 and consensus Sp1
oligonucleotides (lanes 3 and 4). Competition with unlabeled ERE
slightly decreased intensity of the retarded band (lane 7), whereas
mutant Sp1 and -115/-54 (E2F1 gene promoter) oligonucleotides (lanes
5 and 6) did not competitively decrease intensity of the bound DNA
complex. A high mobility band was also observed and only wild-type
-169/-116 competitively decreased intensity of this band (lane 3).
Supershift experiments (Fig. 4B) with Sp1 (lanes 9–11) and Sp3 (lane
12–14) antibodies or nonspecific IgG antibody (lane 15) showed that
Sp1 antibodies supershifted the bound DNA complex. In addition, Sp3
antibodies also gave two weak supershifted bands, suggesting that an
Sp3-DNA complex may be part of the Sp1-DNA retarded band. Proteins
binding to the downstream (-122/-54) region of the E2F1 gene promoter
(Fig. 5A) gave one major higher molecular weight retarded band (A), two
less intense bands (B and C), and two additional intense bands (D and
E). Intensity of retarded band A was signfiicantly decreased after
competition with wild-type -122/-54 and NF-Y oligonucleotides (lanes
3 and 5). Wild-type and mutant -122/-54 oligonucleotides also
decreased the intensity of band E. This suggests that additional
nuclear proteins are bound to sites other than the CCAAT motif in this
oligonucleotide. The intensity of this band (E), as well as bands A-D,
were not decreased after competition with consensus nuclear factor
1 (NF-1), CCAAT-enhancer-binding protein (C/EBP), Sp1, or ERE
oligonucleotides. Identities of proteins associated with retarded band
E are unknown. Band D appears to be a nonspecific complex that is not
competitively decreased by any of the unlabeled oligonucleotides.
Supershift experiment with antibodies to NF-YA, NF-1, C/EBP, Sp1,
ER, and nonspecific goat or rabbit IgG (lanes 11–17,
respectively) showed that only NF-YA antibodies formed a ternary
supershifted complex (lane 11); in addition, NF-YB antibody
eliminated bound-DNA complex but no supershift was observed (data not
shown).
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-Sp1 and Sp1-NF-Y Interactions protein with Sp1 protein and GC-rich
oligonucleotides resulted in enhanced Sp1-DNA retarded band intensity
without forming a ternary supershifted complex (33, 34, 35, 36, 42). Results in
Fig. 6A demonstrate
that purified recombinant Sp1 protein binds to the GC-rich
[32P]-169/-116 to form a retarded band (bound-DNA)
(lane 1), and retarded band intensity is enhanced (2- to 3-fold) after
coincubation with recombinant human ER (lanes 2 and 3) but not
recombinant transforming growth factor- (TGF) protein (lane 6)
used as a control; intensity of the enhanced band was markedly
decreased after coincubation with unlabeled consensus Sp1
oligonucleotide and competition with mutant Sp1 oligonucleotide also
decreased intensity of the retarded band (lanes 4 and 5). The intensity
of the Sp1-DNA band formed after coincubation of
[32P]-169/-116 with nuclear extracts (Fig. 4
) is also
enhanced after coincubation with ER (data not shown).
These results are consistent with previous studies that show enhanced
Sp1-DNA binding by ER was associated with an increased
on-rate for this complex, whereas dissociation of the Sp1-DNA complex
was unchanged by ER (33). In contrast, incubation of
in vitro translated NF-YA plus NF-YB did not increase
Sp1-DNA binding in the presence or absence of ER (data
not shown). Coincubation of recombinant human ER (Fig. 6B, lanes 2–4) with nuclear extracts from MCF-7 cells and
[32P]-122/-54 does not enhance intensity of the
NF-Y-DNA retarded band (B2). However, coincubation of recombinant human
Sp1 protein significantly increased intensity of the
NF-Y-[32P]-122/-54 (retarded band) and also resulted in
formation of a higher molecular weight band (B1) (lanes 5–7).
Intensities of both bands were decreased after coincubation with
unlabeled -122/-54 oligonucleotide, suggesting that NF-Y binds both
CCAAT sites in the higher molecular weight complex (B1). Supershift
experiments with NF-YA or Sp1 antibodies indicated that bands B1 and B2
bound NF-YA (data not shown). Further incubation with ER
does not increase the intensity of the Sp1-enhanced NF-Y-DNA complex
(B1 and B2). Incubation of [32P]-122/-54 with
nonspecific TGF protein does not form a retarded band (data not
shown). Interestingly, competition with unlabeled -122/-54 mutated in
both CCAAT sites (lane 11) eliminated band B1 but not B2, suggesting
that this DNA sequence binds other proteins and Sp1 may enhance
formation of this complex. The functional significance of this
interaction is doubtful since mutation of both CCAAT sites to give
pE2F-1jm2 (Fig. 2C) resulted in loss of hormone inducibility. A
high-mobility specifically bound complex was also observed; however,
this band was not affected by coincubation with ER or
Sp1 and was not further investigated. Direct interactions of
ER and Sp1 with NF-YA were investigated in
coimmunoprecipitation assays (Fig. 7).
Sp1 antibodies coimmunoprecipitated [35S]NF-YA after
incubation with Sp1 protein (lane 3). In contrast,
ER antibodies did not precipitate
[35S]NF-Ya protein after coincubation with
ER (lane 5), ER plus Sp1 (lane 6), or
ER plus TGF (lane 7). Interactions of
[35S]NF-YA with chimeric GST-Sp1 fusion proteins were
also investigated (data not shown); repeated experiments were
unsuccessful due to high background binding of NF-YA to the GST-Sp1
chimeric protein.
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summarize the time-dependent effects of
Sp1 and ER proteins on NF-Y binding to
[32P]-122/-54. NF-Y alone rapidly forms a retarded band
within 1 min, and intensity of this complex does not significantly
change over 20 min (Fig. 8A). Coincubation with Sp1 protein also
resulted in rapid (within 1 min) maximal formation of an NF-Y-DNA
complex; however, the intensity of the retarded band was five times
higher than observed in the absence of Sp1. The t1/2 values
for the rate of complex formation were less than 1 min in the
presence or absence of Sp1 protein, and the major effects of Sp1 were
on the Bmax values for NF-Y-DNA complex formation, which
were increased more than 5-fold at all time points. Coincubation with
ER did not significantly affect retarded band formation
(data not shown; Fig. 6B). The rate of complex dissociation (off rate)
was also determined (Fig. 8B), and the t1/2 value for loss
of 50% of the retarded band intensity was approximately 0.9 min after
incubation with nuclear extracts alone (NF-Y) or in combination with
Sp1 protein. In contrast, coincubation of nuclear extracts with Sp1
plus ER proteins significantly increased the
t1/2 (1.9 min) for complex dissociation, suggesting that
ER functions to stabilize the Sp1/NF-Y-DNA complex.
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and 9)
and cotransfection with wild-type expression plasmids for NF-YA or
NF-YB do not significantly affect E2 responsiveness; in
cells cotransfected with NF-YA and NF-YB, there was a slight increase
in the induction response (6.2-fold) (Fig. 9). The 
4YA13m29 construct encodes for
a dominant negative NF-YA mutant protein (45), and 4YA13 does not
exhibit dominant negative activity. In MCF-7 cells cotransfected with
pE2F1j plus 4YA13m29, there was a marked decrease in basal CAT
activity and loss of E2 inducibility, thus confirming the
role of NF-YA in ER-mediated induction of E2F1 gene
expression. The control plasmid (4YA13) did not affect
E2 responsiveness. Thus, ER/Sp1 in
combination with NF-Y proteins are required for induction of E2F1 gene
expression by E2, and these data describe a new
multiprotein complex required for ER action.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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also demonstrate that both E2F1
protein and mRNA levels also significantly increased after treatment of
growth-arrested MCF-7 cells with E2, suggesting that
E2-mediated cell cycle progression is due, in part, to E2F1
gene activation. This study further investigates the molecular basis of
hormone inducibility by analysis of the E2F1 gene promoter.
Maximal basal activity in MCF-7 cells was observed for
pE2F1g (-173/-54), and this includes some overlap with
results in rat embryo fibroblasts showing that the -204 to -122
region of the E2F1 gene promoter was important for basal activity (40).
The results in MCF-7 cells also show that downstream regions of the
promoter (i.e. -122 to -54) are also required for high
basal activity (Fig. 2). The pE2F1g construct contains an
upstream and two downstream CCAAT sites separated by a GC-rich region,
and further deletion of the upstream or downstream CCAAT motif
(pE2F1h and pE2F1i) resulted in a 40% or 94%
decrease in basal activity, suggesting that at least one of the
downstream CCAAT sites plays an important role in basal activity of the
E2F1 gene promoter. This was confirmed by comparing luciferase
activities of cells transfected with pE2F1h (-169/-54) or
pE2F1j (-146 to -54) and several mutant constructs;
deletion of either of the downstream CCAAT motifs resulted in a more
than 75% loss of basal luciferase activity. This high basal activity
conferred by the two CCAAT sites was also dependent on the presence of
at least one upstream GC-rich site. For example, a comparison of
luciferase activities of wild-type and mutant pE2F1h
(-169/-54) and pE2F1j shows that the two CCAAT sites
alone are not sufficient for high basal luciferase activity, and one or
more of the upstream GC-rich sites are also required (Fig. 2, B and C).
These results are consistent with previous studies (40) showing that
the E2F1-luc construct (-122) containing only the
downstream CCAAT motifs exhibited low basal and serum-inducible
reporter gene activity, indicating that Sp1 and NF-Y proteins function
as trans-activators in regulating basal E2F1 gene
expression.
The E2F1 gene promoter does not contain perfect or imperfect
palindromic EREs, and results of deletion analysis (Fig. 2) showed that
promoter regions required for E2 responsiveness were
similar to those observed for basal activity. Maximal activity required
at least one of the three GC-rich sites (-169 to -116) and both
downstream CCAAT motifs (e.g. pE2F1h and mutants
and pE2F1j). Several studies have reported that
E2 responsiveness of constructs derived from the cathepsin
D, retinoic acid receptor 1, and c-fos gene promoters are
associated with ER/Sp1 interactions at GC-rich sites
(34, 35, 36, 42). The upstream GC-rich sequence in the E2F1 gene promoter
exhibits some similarities to these functional enhancer sequences; for
example, Sp1 protein binds [32P]-169/-116 and
ER enhances Sp1-DNA interactions (Fig. 6A) but does not
form a supershifted ternary complex as previously reported for
E2-responsive and nonresponsive GC-rich promoter elements
(33, 34, 35, 36, 42). In contrast, deletion analysis of the E2F1 gene promoter
shows that the upstream GC-rich sites alone are not sufficient for
transactivation in transient transfection studies (Fig. 2). Thus,
hormone responsiveness of the E2F1 gene promoter (-169 or -146/-54)
in transfection studies in MCF-7 cells not only requires
ER/Sp1 binding to GC-rich sites but also interactions
with downstream CCAAT-binding proteins, suggesting possible
interactions between these transcription factors.
Gel mobility shift assays using [32P]-146/-54 and
nuclear extracts from MCF-7 cells gave a complex pattern of at least
four retarded bands that contain Sp1- and CCAAT-binding proteins (Fig. 3), and these interactions could be simplified using
[32P]-labeled GC-rich upstream (-169/-116, Fig. 4) or
CCAAT-downstream (-122/-54, Figs. 5 and 6B) oligonucleotides. Direct
and competitive binding and antibody supershifts show that Sp1 and NF-Y
proteins interact with upstream and downstream sequences, respectively
(Figs. 4 and 5). These results suggest that E2-mediated
transcriptional activation of E2F1 involves binding of Sp1 and NF-Y
proteins to their respective enhancer elements, coupled with
interactions of these proteins with ER protein, that
does not bind promoter DNA. Previous studies have demonstrated that
ER and Sp1 physically interact (33), and
ER enhanced Sp1-DNA binding in gel mobility shift assays
without forming a ternary supershifted complex (33, 34, 35, 36, 42). Similarly,
ER enhanced binding of Sp1 to
[32P]-169/-116 (Fig. 6A), and this type of interaction
has been observed in other studies showing that human T cell leukemia
virus type-1 Tax, sterol-regulatory element-binding protein, and cyclin
D1 enhanced bZIP, Sp1, and ER binding to their respective cognate DNA
enhancer sequences (46, 47, 48).
Interactions between Sp1 and NF-Y or other CCAAT-binding proteins have
been observed on other gene promoters (43, 44, 49, 50, 51), and results in
Fig. 6B demonstrate that the NF-Y-DNA (-122/-54) complex forms a
retarded band that is not affected after coincubation with
ER, whereas Sp1 protein markedly enhanced intensity of
the NF-Y-DNA band. In contrast, NF-Y did not enhance Sp1-DNA
([32P]-169/-116) complex formation or form a ternary
supershifted complex (data not shown). Previous studies showed that
ER-Sp1 physically interacted in coimmunoprecipitation
and GST pull-down assays (33). Direct interactions of ER and NF-YA were
not observed in coimmunoprecipitation assays, whereas Sp1 and NF-YA
proteins were coimmunoprecipitated (Fig. 7). Thus, Sp1 binds both
ER and NF-YA proteins, and this is consistent with their
cooperative interactions on the E2F1 gene promoter observed in this
study.
Stabilities of NF-Y binding to CCAAT regions in the fatty acid
synthetase and major histocompatibility complex class II gene promoters
are enhanced by Sp1 protein, and this has been related to increased
half-lives of NF-Y-DNA complexes (43, 44). Binding of NF-Y to
[32P]-122/-54 in the presence or absence of Sp1 protein
was maximal within 1 min, and differences in on-rate t1/2
values could not be determined (Fig. 8). However, results showed that
the Bmax for binding was increased by more than 5-fold, and
coincubation with ER did not significantly affect the on-rate of
NF-Y-DNA binding (data not shown). In contrast,
dissociation of the NF-Y-DNA complex was not significantly affected by
Sp1, and differences between this and other studies (42, 43) may be
promoter dependent; however, in the presence of Sp1 plus
ER, there was a 2-fold increase in stability of the
DNA-NF-Y complex (Fig. 8). These in vitro results suggest
that transcriptional activation of E2F1 gene expression by
E2 may be due, in part, to stabilization of the
Sp1-NF-Y-DNA complex by ER, and this is consistent with
increased retarded band intensities using extracts from
E2-treated cells compared with solvent controls (
Figs. 3–5). CCAAT-binding sites are frequently observed in TATA-less
promoters (including the E2F1 gene promoter) (49), and
ER, Sp1, and NF-Y can induce conformational changes in
chromatin structure. These effects could result in recruitment of
general transcriptional machinery via both protein-protein and
protein-DNA interactions (Fig. 10).
However, other possible mechanisms may also play a role in ER-dependent
transactivation, and this includes activation of kinases and modulation
of other proteins that affect NF-Y or Sp1 action.
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/Sp1 action
is complex and gene promoter specific. For example, GC-rich motifs in
the c-fos and retinoic acid receptor 1 promoters are
sufficient for E2-induced signaling in MCF-7 cells via
ER/Sp1 interactions (34, 36). In contrast,
ER/Sp1 interactions at GC-rich sites in cathepsin D,
Hsp27, and uteroglobin gene promoters require, in addition, ERE
or ERE half-site motifs for ER-DNA binding (54, 55, 56, 57). Results of this
study demonstrate that ER/Sp1 binding to GC-rich sites
in the E2F1 gene promoter are necessary but not sufficient for
transactivation of E2F1, and cooperative interactions with NF-Y are
required for a hormone-induced response (Fig. 10). Previous studies
have shown that hormone-induced responses can be mediated through
interactions of their receptors with DNA-bound Sp1 protein (33, 34, 35, 36, 42, 58), and the results of this study also show that both
ER and Sp1 proteins are required for transcriptional
activation of E2F1 in MCF-7 cells. However, our results also
demonstrate that interactions with NF-Y are also required for
E2 responsiveness, and ER/Sp1 interactions with NF-Y and
other transcription factors are currently being investigated in this
laboratory. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, NF-1, and C/EBP
antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). NF-YA antibody was purchased from
Rockland, Inc. (Gilbertsville, PA). Luciferase and ß-galactosidase
enzyme assay systems were obtained from Promega Corp. (Madison, WI). All other chemicals and biochemicals
were the highest quality available from commercial sources.
Oligonucleotides were synthesized by the Gene Technology Laboratory,
Texas A&M University (College Station, TX).
Plasmids and Cloning
The human ER expression plasmid was a
generous gift from Dr. Ming-Jer Tsai (Baylor College of Medicine,
Houston, TX). The expression plasmids of wild-type NF-YA and NF-YB and
mutant NF-YA (4YA13m29) and control plasmid (4YA13) were kindly
provided by Dr. Roberto Mantovani (Universita di Milano, Milan, Italy)
(45). Constructs pE2F1a, pE2F1b, and pE2F1d were kindly provided by Dr.
J. R. Nevins, Duke University (Durham, NC). The pE2F1c construct
was made by the RT-PCR method (forward primer:
5'-CCGCCATTGGCCGTACCGCCCC-3'; reverse primer:
5'-GATCTTCCCGGCCACTTTTACGCGCCAAA-3') and inserted into pGL2 basic
vector (Promega Corp.) at SacI and
BglII cloning sites. The remaining E2F1 promoter-reporter
constructs were made by inserting synthetic oligonucleotides into pGL2
basic vector (Promega Corp.) digested with SacI
and BglII enzymes at the cloning sites. Resulting plasmids
were sequenced by the Gene Technology Laboratory, Texas A&M University,
to confirm appropriate insertion of the oligonucleotide inserts. The
sequences of Sp1-binding sites (CCGCCCC) and CCAAT protein-binding
sites in the E2F1 promoter were mutated into CCtttCC and atgcT,
respectively, in all constructs containing mutation in these sites.
Transient Transfection Assay
MCF-7 cells were seeded in 60-mm petri dishes in DME/F12
medium supplemented with 2.5% dextran-coated charcoal FBS and grown
until 70% confluence was reached. Plasmids (2–3 µg) were
transiently cotransfected with ER expression plasmid (2 µg) using the
calcium phosphate method. After transfection, cells were grown
overnight in serum-free medium and treated with DMSO [0.1% (vol/vol)
as control] or 10 nM E2 for 44 h. Cells
were then washed with PBS and harvested with cell lysis buffer
(Promega Corp.). Cell lysates were prepared by
freeze-thawing (one time) followed by centrifugation at 10,000 x
g for 5 min. Luciferase activity was determined in a
luminometer (Parkard Instrument Co., Meriden, CT) with a luciferase
assay kit (Promega Corp.) and normalized to
ß-galactosidase enzyme activity obtained after transfection with a
ß-galactosidase-lacZ plasmid (2.0 µg) obtained from
Invitrogen (Carlsbad, CA).
Preparation of Whole-Cell Extracts
Cells were seeded in 100-mm petri plates and grown in DME/F12
media plus 5% dextran-coated charcoal-FBS. After cells reached
70% confluence, they were synchronized in serum-free medium for 3 days
and then treated with DMSO (0.1% vol/vol as a control) and 10
nM E2 for 6, 12, and 24 h, respectively.
Cell monolayers were then washed once in ice-cold PBS and scraped into
lysis buffer [50 mM HEPES (pH 7.5), 150 mM
sodium chloride, 10% (vol/vol) glycerol, 1% Triton X-100, 1.5
mM magnesium chloride, 1 mM EGTA, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 1 mM
phenylmethylsulfonylfluoride, 200 µM sodium
orthovanadate, 10 mM pyrophosphate, and 100 nM
sodium fluoride]. Cells were incubated for 30 min, and then
centrifuged at 10,000 x g for 5 min. Supernatants were
precleared by addition of 20 µl of protein A-agarose beads for 30 min
followed by centrifugation for 5 min at 10,000 x g.
Lysates used for Western blot analysis were stored at -80 C until
required. All procedures were carried out at 4 C.
Western Immunoblot Analysis
Cell lysates, prepared as described above, were loaded on
SDS polyacrylamide gel. After electrophoresis, proteins were
transferred to a nitrocellulose membrane using an electroblotting
apparatus overnight at 4 C. Membranes were blocked with TBS [10
mM Tris-HCl, (pH, 8.0); 150 mM sodium
chloride] plus 5% milk (blotto bluffer) for 1 h and then
incubated in primary antibody at 0.1 to 1.0 µg/ml in the blotto
buffer for 1–2 h at 20 C. Membranes were then rinsed with water and
washed for 5 min (two times) in TBS buffer. Membranes were incubated in
enhanced chemiluminescence (ECL) reagents (Amersham Pharmacia Biotech, Arlington Heights, IL) for 1 min, excess ECL reagent
was removed by dabbing with a Kimwipe, and the membrane was sealed in
plastic wrap. Membranes were then exposed to ECL hyperfilm for
visualization of immunoreactive bands. E2F1 antibody was purchased from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and the
assay was carried out as described in treatment protocols provided by
the company. Protein levels were quantitated using a Sharp JX-330
densitometer and a Scanalytics Zero D software package (Scanalytics,
Billerica, MA).
Northern Blot Analysis
MCF-7 cells were seeded and grown as described above. Cells were
then treated with DMSO (0.1% ol/vol) or 10 nM
E2 for 0.5, 1.0, 2.0, 4.0, and 12 h, respectively. RNA
was extracted using an RNA extraction kit from Tel-Test
(Friendswood, TX). Twenty-five micrograms of total RNA obtained from
each treatment group were separated by electrophoresis on 1.2% agarose
gel, transferred onto a nylon membrane, bound to the membrane by UV
cross-linking, and baked at 80 C for 2 h. The membrane was then
prehybridized in a solution containing 0.1% BSA, 0.1% Ficoll, 0.1%
polyvinyl pyrolidone, 10% dextran sulfate, 1% SDS, and 5x SSPE (0.75
M sodium chloride, 50 mM
NaH2PO4, 5 mM EDTA) for 18–24 h at
60 C and hybridized in the same buffer for 24 h with the
[32P]-labeled DNA probe (106 cpm/ml). The
E2F1 cDNA probes were labeled with [
-32P]dCTP. Gels
were exposed to film Eastman Kodak Co., Rochester, NY) and
quantitated via densitometry as described above. ß-Actin mRNA was
used as an internal control to standardize E2F1 mRNA levels.
Nuclear Extract Preparation and Gel Mobility Shift Assay
Nuclear extracts were prepared from MCF-7 cells treated with
DMSO (0.1% vol/vol) or 10 nM E2 for 4 h
utilizing cells maintained in serum-free medium for 3 days. Nine
picomoles of oligonucleotides were labeled at the 5'-end using
T4-polynucleotide kinase and [-32P]ATP. Nuclear
extracts from control (DMSO) or E2-treated cells or
recombinant human Sp1 (Promega Corp.) or ER (Pan Vera Co.,
Madison, WI) protein were incubated for 15 min at 0 C in HEGD [2
mM HEPES, 1.5 mM EDTA, 1.0 mM
dithiothreitol, 10% glycerol (vol/vol), pH 7.6] buffer with 1 µg
poly[d(I-C)] to bind nonspecific DNA-binding proteins and 200-fold
excess of unlabeled wild-type or mutant oligonucleotide competitors for
the competition experiments. After addition of
[32P]-labeled DNA, the mixture (final volume, 20 µl)
was incubated for an additional 20 min at 20 C. Reaction mixtures were
loaded onto a 5% polyacrylamide gel and electrophoresed at 200 V in
0.9 M Tris-borate and 2 mM EDTA, pH 8.0, at 4
C. Gels were dried and protein-DNA complexes were visualized by
autoradiography and quantitated using a Sharp JX-330 densitometer and a
Scanalytics Zero-D software package as described above. For gel
supershift experiments, antibodies were added after standard gel
mobility shift assay procedure, and reactions were further incubated
for 20–30 min at 20 C. For the off-rate assay, a 5-fold scale up of
the standard gel shift assay (described above) was carried out and
incubated for 20 min at 20 C. A 60-fold molar excess of the competitor
(NF-Y DNA-binding element) was added to the binding reaction mixtures.
Fifteen micoliters of the reaction samples were loaded onto a
continuously electrophoresing gel at 0, 1, 3, 5.5, 9, and 13 min after
addition of the competitor. For on-rate assay,
[32P]-labeled probes were added to the reaction for 1, 2,
4, 8, 15, and 20 min after incubation with poly[d(I-C)]. The samples
were then loaded onto a 5% polyacrylamide gel. Intensities of the
bound bands were quantitated as described above. Oligonucleotides
(sense strand) used for the competition of CCAAT-binding proteins are
given below:
NF-Y: 5'-GGT AGG AAC CAA TGA AAT GCG AGG TAA-3'
C/EBP: 5'-TGC AGA TTG CGC AAT CTG CA-3'
NF-1: 5'-TTT TGG ATT GAA GCC AAT ATG ATA A-3'
wt-ERE: 5'-GTC CAA AGT CAG GTC ACA GTG ACC TGA TCA AAG TT-3'
mt-ERE: 5'-GTC CAA AGT CAG GaC ACA GTG tCC TGA TCA AAG TT-3'
wt-Sp1: 5'-AGC TTA TTC GAT CGG GGC GGG GCG AGC G-3'
mt-Sp1: 5'-AGC TTA TTC GAT CGa aGC GGG GCG CAG CG-3'
Coimmunoprecipitation Assays
[35S]NF-YA was synthesized using
[35S]methionine and a rabbit reticulocyte lysate kit
(TNT-coupled reticulocyte lysate system; Promega Corp.).
Four microliters of in vitro translated
[35S]NF-YA was incubated with 30 ng Sp1, 30 ng ER, 30 ng
Sp1 plus 30 ng ER, or 30 ng TGF plus 30 ng ER proteins at 4 C for
1 h in the same buffer system used for gel mobility shift assays.
One microgram of anti-NF-YA, anti-Sp1, anti-ER antibodies, or
preimmune serum was added and incubated for 1.5 h at 4 C
with gentle rocking and 15 µl Agarose A/G PLUS beads were then added
and further incubated at 4 C for another 1.5 h. The beads were
washed with cell lysis buffer (8x, as described above), boiled for 5
min in 2x SDS loading buffer, and resolved on 10% SDS-PAGE.
[14C]-Labeled protein molecular weight markers
(Amersham Pharmacia Biotech) were used to determine the
molecular weight of precipitated [35S]NF-YA protein.
Statistics
Results are expressed as means ± SE for at
least three independent (replicate) experiments for each treatment
group. Statistical significance was determined by ANOVA and Student’s
t test, and the levels of probability are noted for each
experiment.
|
FOOTNOTES |
|---|
This work was supported by the Welch Foundation, the NIH (Grants CA-76636 and ES-09106), and the Texas Agricultural Experiment Station. S.S. is a Sid Kyle Professor of Toxicology.
Received for publication March 12, 1999. Revision received April 20, 1999. Accepted for publication May 3, 1999.
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Y. Ge, L. H. Matherly, and J. W. Taub Transcriptional Regulation of Cell-specific Expression of the Human Cystathionine beta -Synthase Gene by Differential Binding of Sp1/Sp3 to the -1b Promoter J. Biol. Chem., November 16, 2001; 276(47): 43570 - 43579. [Abstract] [Full Text] [PDF]
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D. Curtin, S. Jenkins, N. Farmer, A. C. Anderson, D. J. Haisenleder, E. Rissman, E. M. Wilson, and M. A. Shupnik Androgen Suppression of GnRH-Stimulated Rat LH{beta} Gene Transcription Occurs Through Sp1 Sites in the Distal GnRH-Responsive Promoter Region Mol. Endocrinol., November 1, 2001; 15(11): 1906 - 1917. [Abstract] [Full Text] [PDF]
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J. Shimada, Y. Suzuki, S.-J. Kim, P.-C. Wang, M. Matsumura, and S. Kojima Transactivation via RAR/RXR-Sp1 Interaction: Characterization of Binding Between Sp1 and GC Box Motif Mol. Endocrinol., October 1, 2001; 15(10): 1677 - 1692. [Abstract] [Full Text] [PDF]
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C. Li, M. R. Briggs, T. E. Ahlborn, F. B. Kraemer, and J. Liu Requirement of Sp1 and Estrogen Receptor {{alpha}} Interaction in 17{beta}-Estradiol-Mediated Transcriptional Activation of the Low Density Lipoprotein Receptor Gene Expression Endocrinology, April 1, 2001; 142(4): 1546 - 1553. [Abstract] [Full Text]
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I. Samudio, C. Vyhlidal, F. Wang, M. Stoner, I. Chen, M. Kladde, R. Barhoumi, R. Burghardt, and S. Safe Transcriptional Activation of Deoxyribonucleic Acid Polymerase {{alpha}} Gene Expression in MCF-7 Cells by 17{{beta}}-Estradiol Endocrinology, March 1, 2001; 142(3): 1000 - 1008. [Abstract] [Full Text] [PDF]
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J. S. Foster, D. C. Henley, A. Bukovsky, P. Seth, and J. Wimalasena Multifaceted Regulation of Cell Cycle Progression by Estrogen: Regulation of Cdk Inhibitors and Cdc25A Independent of Cyclin D1-Cdk4 Function Mol. Cell. Biol., February 1, 2001; 21(3): 794 - 810. [Abstract] [Full Text]
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W. Xie, R. Duan, I. Chen, I. Samudio, and S. Safe Transcriptional Activation of Thymidylate Synthase by 17{beta}-Estradiol in MCF-7 Human Breast Cancer Cells Endocrinology, July 1, 2000; 141(7): 2439 - 2449. [Abstract] [Full Text] [PDF]
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L. Salvatori, L. Ravenna, M. P. Felli, M. R. Cardillo, M. A. Russo, L. Frati, A. Gulino, and E. Petrangeli Identification of an Estrogen-Mediated Deoxyribonucleic Acid-Binding Independent Transactivation Pathway on the Epidermal Growth Factor Receptor Gene Promoter Endocrinology, June 1, 2000; 141(6): 2266 - 2274. [Abstract] [Full Text] [PDF]
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B. Saville, M. Wormke, F. Wang, T. Nguyen, E. Enmark, G. Kuiper, J.-A. Gustafsson, and S. Safe Ligand-, Cell-, and Estrogen Receptor Subtype (alpha /beta )-dependent Activation at GC-rich (Sp1) Promoter Elements J. Biol. Chem., February 25, 2000; 275(8): 5379 - 5387. [Abstract] [Full Text] [PDF]
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M. Stoner, F. Wang, M. Wormke, T. Nguyen, I. Samudio, C. Vyhlidal, D. Marme, G. Finkenzeller, and S. Safe Inhibition of Vascular Endothelial Growth Factor Expression in HEC1A Endometrial Cancer Cells through Interactions of Estrogen Receptor alpha and Sp3 Proteins J. Biol. Chem., July 21, 2000; 275(30): 22769 - 22779. [Abstract] [Full Text] [PDF]
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E. J. Cram, B. D. Liu, L. F. Bjeldanes, and G. L. Firestone Indole-3-carbinol Inhibits CDK6 Expression in Human MCF-7 Breast Cancer Cells by Disrupting Sp1 Transcription Factor Interactions with a Composite Element in the CDK6 Gene Promoter J. Biol. Chem., June 15, 2001; 276(25): 22332 - 22340. [Abstract] [Full Text] [PDF]
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B. Saville, H. Poukka, M. Wormke, O. A. Janne, J. J. Palvimo, M. Stoner, I. Samudio, and S. Safe Cooperative Coactivation of Estrogen Receptor alpha in ZR-75 Human Breast Cancer Cells by SNURF and TATA-binding Protein J. Biol. Chem., January 18, 2002; 277(4): 2485 - 2497. [Abstract] [Full Text] [PDF]
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M. R. Walters, M. Dutertre, and C. L. Smith SKF-82958 Is a Subtype-selective Estrogen Receptor-alpha (ERalpha ) Agonist That Induces Functional Interactions between ERalpha and AP-1 J. Biol. Chem., January 11, 2002; 277(3): 1669 - 1679. [Abstract] [Full Text] [PDF]
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) or in
combination with Sp1 protein ([
), and the time-dependent formation
of the NF-Y-DNA complex was determined by gel mobility shift assays as
described in Materials and Methods. The results for each
time point are means ± SE for three separate
determinations. Coincubation with Sp1 protein significantly
(P < 0.05) increased the Bmax value
for intensity of the specifically bound retarded band by more than
5-fold at every time point. Coincubation with ER
