Upstream Stimulatory Factors Mediate Estrogen Receptor Activation of the Cathepsin D Promoter
Weirong Xing and
Trevor K. Archer
Departments of Obstetrics and Gynecology, Biochemistry, and
Oncology The University of Western Ontario London Regional
Cancer Centre London, Ontario, Canada N6A 4L6
 |
ABSTRACT
|
---|
Overexpression of cathepsin D (CD), a ubiquitous
lysosomal protease, is closely associated with a poor clinical outcome
for patients with breast cancer. Estrogen greatly induces transcription
of the CD gene in estrogen receptor (ER)-positive breast cancer cells.
In this report, we transiently introduced a human CD
promoter/chloramphenicol acetyltransferase reporter gene into
human MCF-7 breast cancer cells to study the mechanisms by which the ER
activates the promoter. Using an in vivo Exonuclease III
footprinting assay, we found that estrogen stimulation of MCF-7 cells
induced loading of a transcription factor(s) to a portion of the
promoter (-124 to -104) that is homologous to the adenovirus major
late promoter element. Subsequent gel mobility shift assays with a
21-bp CD -124/-104 probe and nuclear extracts prepared from naive and
estrogen-stimulated cells detected a single sequence-specific
protein-DNA complex. Southwestern and UV cross-linking experiments
detected two proteins of 44 kDa and 43 kDa that were specifically bound
to the 21-bp fragment of the promoter. Gel super-shift assays with
upstream stimulatory factor 1 (USF-1) and USF-2 antibodies demonstrated
that USF-1 and USF-2 bound to the E box probe. Sequence specific
binding was abolished by a 2-bp change shown previously to prevent the
binding of USF to the E box. Incorporation of a mutant E box into the
wild-type CD promoter/chloramphenicol acetyltransferase gene abolished
USF binding and reduced the levels of both basal and
estrogen-stimulated transcription. These results suggest that the ER
targeting of USF-1 and USF-2 is a critical step in hormone activation
of CD gene transcription in human breast cancer cells.
 |
INTRODUCTION
|
---|
The cathepsin D (CD) gene, an estrogen-regulated gene in human
breast cancer cells, encodes a ubiquitous lysosomal aspartyl protease
that is initially synthesized as inactive procathepsin D. The proenzyme
is subsequently converted by posttranslational cleavage to its
enzymatically active form (1, 2). In estrogen receptor (ER)-positive
breast cancer cells, overexpression of procathepsin D has been
correlated with lymphatic metastasis, relapse, and short disease
survival in several retrospective clinical studies (3, 4, 5). The precise
role CD plays in metastasis is not well defined but may involve
proteolytic activity and/or interaction with the mannose
6-phosphate/insulin-like growth factor receptor (6, 7). In
normal breast cells, after its synthesis, CD is transported into the
lysosomal compartment. In tumor cells CD is secreted into the
surrounding medium. This results in delivery of CD to the plasma
membrane rather than to the lysosome. The secreted CD can disrupt the
adhesive interactions holding tumor cells in place and may open a
channel through the basement membrane and interstitial stroma,
resulting in invasion and metastasis (8, 9, 10).
Procathepsin D is constitutively expressed in ER-negative breast cancer
cells but is greatly induced by estrogen and tamoxifen, a partial
estrogen agonist, in ER-positive breast cancer cells (11, 12).
Transcription from the CD gene is initiated at five sites spanning 52
bp and mapping at -20, -44, -51, -60, and -72 bp from the first
base of the initiation codon (13). Of the five start sites, estrogen
stimulates transcription only from the site located downstream of the
TATA box at -20 (13). Molecular analysis of the CD promoter has
revealed a cluster of transcription factor-binding sites that include
potential sites for the ER, activator protein 2, and Simian virus-40
protein-1 (SP1) (12). Transient transfection experiments
demonstrated that a maximal induction of transcription could be
obtained with the proximal 356 bp of the CD promoter (12). This
observation was attributed to a cluster of three estrogen-responsive
element (ERE)-like half-palindromes located in the truncated promoter
(12). It has been suggested that the ER stabilizes the TATA-controlled
preinitiation complex, and that estrogen induction of CD is mediated by
interaction of the ER with a nonconsensus ERE that requires synergy
with other elements located upstream and/or downstream of the central
ERE (13, 14). In particular, much attention has focused on potential
interactions of the ER with the ubiquitous transcription factor SP1
(14). Gel shift experiments with an oligonucleotide bearing a putative
ER-SP1-like sequence (-199 to -165 of the CD promoter) suggest that
both the ER and SP1 bind to this site (14). However, other experiments
have suggested that the major site of ER activity is a nonconsensus
ERE, located at -261 in the CD promoter (12). In addition, a fourth
ERE half-palindrome is located in a site homologous to the adenovirus
major late promoter element (CD -124/-104) (12). Recent studies have
shown that this short region of promoter (-119 to -10) is also
responsive to estrogen stimulation (15). These studies suggest that the
role of the ER in regulating cathepsin gene expression is complex and
may proceed via multiple EREs located within the proximal promoter
(15).
The ER is a member of the nuclear hormone receptor superfamily. These
transcription factors share a similar structure, consisting of an
amino-terminal region that modulates the transcriptional activity of
the receptor, a central DNA-binding domain, and a carboxyl-terminal
ligand-binding domain (16, 17, 18). The binding of agonist to the receptor
initiates a conformational change in the ligand-binding domain that
enables recruitment of coactivators, which are thought to enhance
receptor/basal transcription machinery interactions to efficiently
activate transcription (19, 20, 21, 22, 23, 24, 25). Of the steroid receptors, the
glucocorticoid receptor (GR) is, perhaps, the most extensively studied
with respect to transcriptional activation within chromatin (26, 27, 28, 29).
In particular, studies of the glucocorticoid stimulation of the mouse
mammary tumor virus (MMTV) have demonstrated that the receptor performs
multiple functions in activating the promoter (30). Not only does the
GR remodel chromatin to allow ubiquitous transcription factors, such as
nuclear factor 1 (NF1), to establish a preinitiation complex, but it is
also directly involved in the recruitment of the basal machinery to the
promoter (31, 32). Studies with the MMTV promoter help to establish the
fundamental role of chromatin structure in the hormonal activation of
transcription in vivo (23, 26).
In the present study, we have used the human CD proximal promoter
as a model system to investigate the mechanisms by which the ER
stimulates transcription in MCF-7 cells. By analyzing transcription
factor-DNA interactions on transiently transfected promoters, we
observed estrogen-induced protein binding at the promoter in
vivo. Subsequent in vitro DNA binding experiments
confirmed that the estrogen-induced binding in vivo did not
result from changes in expression of the protein. Southwestern and UV
cross-linking studies with a short promoter probe, CD -124/-104,
demonstrated that two proteins of 44 kDa and 43 kDa were bound
specifically. Further experiments revealed that the proteins were
upstream stimulatory factors 1 and 2, (USF-1 and USF-2). Point
mutations within the USF-binding site in the CD promoter abolished USF
binding and reduced the levels of both basal transcription and
estrogen-stimulated transcription. These data suggest that USF-1 and
USF-2 play important roles in mediating estrogen-inducible
transcription of the CD gene in human breast cancer cells.
 |
RESULTS
|
---|
Estrogen Enhances CD Promoter Activity by the Recruitment of
Transcription Factors
Within a small fragment of the proximal human CD promoter (-365
to -10) that is sufficient to mediate estrogen responsiveness, there
are multiple potential transcription factor-binding sites (11, 12). To
dissect the mechanisms by which this promoter fragment allows estrogen
regulation, we have used a MCF-7 cell line as a model system. In
preliminary experiments we used RT-PCR to quantify estrogen activation
of the endogenous CD promoter. Cells treated with 10 nM
E2 for 24 h exhibited a 6-fold increase in CD mRNA
levels compared with untreated cells (Fig. 1A
). In contrast, within the same cells
ß2-microglobulin mRNA levels were unaffected by
E2 treatment (Fig. 1A
). Analysis of a transiently
introduced proximal human CD promoter/CAT reporter plasmid into MCF-7
cells confirmed E2 activation of the endogenous gene, with
a 6-fold induction of chloramphenicol acetyltransferase (CAT) activity
(Fig. 1B
). The E2 induction was specific for the CD
promoter as the CAT reporter plasmid without the promoter was
unresponsive to E2 (Fig. 1B
).

View larger version (19K):
[in this window]
[in a new window]
|
Figure 1. Estrogen Activation of a Transiently Transfected
Cathepsin D Reporter Gene Is Equivalent to Induction of the Endogenous
Cathepsin D Gene
A, E2 induction of endogenous CD mRNA accumulation. mRNA
was isolated from MCF-7 cells untreated or treated with E2
for 24 h. Twenty micrograms of total RNA were converted into cDNA
with poly(dT)12-18. An aliquot of cDNA corresponding to 18
µg total RNA was used in PCR reactions with CD-specific primers to
generate a product of 178 bp in length. cDNA corresponding to 2 µg
total RNA was used with ß-microglobulin specific primers as an
internal control. PCR products were analyzed on a 5% polyacrylamide
denaturing gel, followed by autoradiaography. Lane 1, Control cells;
lane 2, cells treated with 10 nM E2. B,
E2 activation of transiently transfected CD promoter/CAT
reporter gene. MCF-7 cells were transiently transfected with 10 µg
pCD/355 and 5 µg HEO (human ER expression vector). After 24 h of
transfection, the cells were stimulated with 10 nM
E2 for an additional 24 h. Cell lysates were prepared
and analyzed for relative CAT activity. The results were the means
± SE of three independent experiments.
|
|
Transient transfections represent a powerful approach to characterize
cis- regulatory sequences. More recently we have
demonstrated that it is possible to use transient transfection assays
to characterize proteins that mediate the transcriptional response
(31). To investigate the transcription factor loading on the CD
promoter, we cotransfected MCF-7 cells with the CAT reporter and an ER
expression vector. In vivo footprinting with Exonuclease III
detected a single major estrogen-inducible stop indicative of a bound
protein (Fig. 2B
). This 5'-boundary is
consistent with a protein or proteins bound at or adjacent to the E box
sequence within the CD promoter (Fig. 2A
), a region protected by
deoxyribonuclease I (DNase I) footprinting in vitro (12, 15). Those experiments indicated that the E2-enhanced
transcription from the CD promoter is consistent with the enhanced
transcription factor loading on the promoter in vivo.

View larger version (22K):
[in this window]
[in a new window]
|
Figure 2. Estrogen-Dependent Transcription Factor Loading
in Vivo
A, Schematic diagram of protein-binding sites within the proximal human
CD promoter. Binding sites for proteins are labeled and given a
distinctive fill, and the transcription start sites are indicated by
arrowheads. In ER-positive breast cancer cells, estrogen
only induces a TATA box-dependent transcription from -20. B,
E2-induced transcription factor loading on the CD promoter
in vivo. MCF-7 cells were transiently cotransfected with
pCD/355 and HEO and treated or untreated with E2 for
24 h before harvesting. Isolated nuclei were partially digested
with HindIII and Exonuclease III to detect specific
stops corresponding to 5'-boundaries of transcription factors bound to
the CD promoter. After purification, each sample was analyzed using
linear Taq polymerase amplification with a
32P-labeled single-stranded primer specific for the
bacterial CAT gene. Purified products were analyzed on a 5%
polyacrylamide denaturing gel, followed by autoradiography. Lane 1,
x 174 size marker; lanes 2 and 3,
E2-treated and untreated sample, respectively.
|
|
Identification of Nuclear Factors That Bind the E Box of the CD
Promoter
To biochemically characterize E2-dependent protein
binding, we synthesized oligonuleotides that encompassed sequences from
-124 to -104 of the CD promoter (CD -124/-104). The CD -124/-104
sequence contains a GC island, a half-ERE (5'-GTACC), and an E box
(5'-CACGTG) as potential transcription factor binding sites. These
include transcription factors such as SP1, ER, and the basic
helix-loop-helix (bHLH) transcription factors, c-myc, Myo D,
USF-1, and USF-2 (33, 34, 35, 36, 37, 38). Our gel mobility shift assays detected a
single retarded protein-DNA complex with the nuclear extract from cells
that were untreated or treated with E2 (Fig. 3
, compare lanes 13). Interestingly,
and in contrast to what was seen with the in vivo
footprinting assay (Fig. 2B
), there was no significant increase in
binding with extracts prepared from E2-treated cells
compared with untreated cells (Fig. 3
, compare lanes 2 and 3). This
band appeared to be specific as it was completely competed away by the
unlabeled CD -124/-104 probe, but not by the unlabeled SP1 DNA (Fig. 3
, compare lanes 3, 4, and 6). However, the CD -124/-104 binding was
partially competed away by a 250-fold excess of an unlabeled consensus
ERE probe (Fig. 3
, compare lanes 3, 4, and 5). Inspection of the two
probes revealed that the ERE and CD -124/-104 share a sequence that
is identical at 9 of 10 bases and may suggest that either ER and/or E
box binding protein(s) bind to CD -124/-104 sequence in this
electrophoretic mobility shift assay (EMSA) (Fig. 3
).

View larger version (52K):
[in this window]
[in a new window]
|
Figure 3. Sequence-Specific Binding of Proteins to a CD
-124/-104 Sequence
EMSA was conducted using nuclear extracts (2 µg) from MCF-7 cells
with a 32P-labeled CD -124/-104 probe. The binding
reactions were analyzed on a 5% polyacrylamide nondenaturing gel,
followed by autoradiography. Lane 1, Probe control without nuclear
extract; lanes 2 and 3, probe with nuclear extract from
E2-untreated cells and treated cells, respectively; lanes
4, 5, and 6, probe with nuclear extract from E2-treated
cells, the reaction containing 250-fold molar excess unlabeled
double-stranded DNA CD -124/-104 probe, ERE, and SP1
competitor, respectively. The sequences of the probes are given
below the panel.
|
|
The protein-DNA complex detected in the EMSA may contain one or more
proteins bound to the DNA probe. To characterize the nuclear protein(s)
that bind the CD -124/-104 element, we turned to Southwestern blot
analysis. The CD -124/-104 probe specifically detected two proteins
with apparent molecular mass of
43 kDa and 105 kDa in nuclear
extract but not to a BSA control (Fig. 4
, compare lanes 13). In agreement with results seen with the gel
mobility shift assay, there were no significant differences in the
levels of nuclear proteins detected in extracts prepared from control
and E2-treated cells (Fig. 4
, compare lanes 1 and 2). To
directly compare the protein binding seen in the gel mobility shift
assay and the Southwestern assays we performed UV cross-linking
experiments. The advantage of the UV cross-linking assay is that it
measures the DNA-protein interactions in solution under nondenaturing
conditions. Consistent with the Southwestern experiments, we were able
to cross-link the CD -124/-104 probe with polypeptides of 43 kDa and
44 kDa as well as diffuse protein(s) of
105 kDa (Fig. 5
, compare lanes 17). As seen in the
gel mobility shift assay, the interaction of the CD -124/-104 probe
with the 43-kDa and 44-kDa proteins was competed away completely by an
excess of unlabeled CD -124/-104 probe and partially competed by an
equivalent excess of unlabeled ERE probe (Fig. 5
., compare lanes 9 and
10). In addition, binding of the labeled probe to the 44-kDa and 43-kDa
proteins was unaffected by 250-fold molar excess of unlabeled SP1
oligonucleotide (Fig. 5
, lane 11). In contrast, the binding of
105
kDa protein(s) was not competed by a 250-fold molar excess of unlabeled
CD -124/-104 probe, suggesting that the interactions with the CD
-124/-104 probe might be nonspecific. Interestingly, the interaction
of the CD -124/-104 probe with the
105 kDa protein(s) was
partially competed away by a 250-fold molar excess of unlabeled SP1
probe (Fig. 5
, lane 11). Further, proteinase K digestion completely
eliminated all DNA protein binding (Fig. 5
, compare lanes 2, 7, and 8).
Taken together, the results indicated that the 44-kDa and 43-kDa
proteins were responsible for the sequence-specific interaction with
the CD -124/-104 DNA sequence seen in the biochemical assays (
Figs. 35

).

View larger version (49K):
[in this window]
[in a new window]
|
Figure 4. Southwestern Analysis of CD -124/-104
Probe-Binding Proteins
An aliquot (40 µg) of nuclear extract from MCF-7 cells was separated
on a 7.5% SDS-PAGE gel. The protein was transferred to a
nitrocellulose membrane and probed with 32P-labeled CD
-124/-104 probe, followed by autoradiography. Lane 1, Nuclear extract
from E2-treated cells; lane 2, nuclear extract from
E2-untreated cells; lane 3, BSA as a negative control.
|
|

View larger version (43K):
[in this window]
[in a new window]
|
Figure 5. UV Cross-Linking Analysis of CD -124/-104
Probe-Binding Proteins
An aliquot (5 µg) of nuclear extract was incubated with a
32P-labeled CD -124/-104 probe at room temperature for 20
min, and then irradiated for varying times. The bound proteins were
analyzed by 10% SDS-PAGE, before autoradiagraphy. Lane 1, Protein size
marker; lane 2, probe control without nuclear extract; lane 3; complete
binding reaction control without UV irradiation; lanes 4, 5, 6, and 7,
complete binding reaction with 5-, 15-, 30-, and 60-min UV irradiation,
respectively; lane 8, complete binding reaction with 60-min UV
irradiation, followed by 30-min proteinase K digestion; lanes 9, 10,
and 11, 60-min UV irradiation of complete binding reaction plus
250-fold molar excess of unlabeled CD -124/-104 probe, ERE, and SP1
probe, respectively.
|
|
The specific interactions of the 43-kDa and 44-kDa proteins with a
fragment of the CD promoter that contains an E Box immediately
suggested the bHLH transcription factors USF-1 and USF-2 as candidate
proteins (39, 40). To test this possibility, we compared a purified
USF-1 protein with the nuclear extract by gel mobility shift assays
(Fig. 6
). The EMSA with purified USF-1
protein displayed a similar migration of the DNA-protein complex as in
MCF-7 nuclear extracts (Fig. 6A
, lanes 2 and 4). We confirmed that the
proteins in the nuclear extract bound to the CD -124/-104 probe were,
in fact, USF-1 and USF-2 by gel supershift assays with anti-USF-1 and
anti-USF-2 antibodies (Fig. 6B
, lanes 2, 4, and 6). The DNA-nuclear
factor complex was supershifted in the presence of either USF-1 or
USF-2 antibodies but was unaffected by ER or GR antibodies (Fig. 6B
, compare lanes 36). This would be consistent with a USF-1/USF-2
heterodimer at the promoter, as both antibodies shift essentially the
total DNA-protein complex (Fig. 6B
, lanes 2, 4, and 6). Control
experiments confirm that the interactions in the nuclear extracts
include USF-1 and USF-2 by demonstrating that both antibodies shift the
complex with extracts, but only the USF-1 antibody shifts the complex
with purified USF-1 (Fig. 6B
, compare lanes 4 and 6 with 8 and 9).
These experiments are consistent with the idea of USF-1 and USF-2 as
the E2-dependent factor(s) detected by in vivo
footprinting (Fig. 2B
).

View larger version (43K):
[in this window]
[in a new window]
|
Figure 6. Sequence-Specific Binding of USF-1 and USF-2 to the
Cathepsin D Promoter
A, Presence of USF in the complex formed with the CD -124/-104 probe.
A, Purified USF-1 binds to CD -124/-104 probe. An aliquot (2 µg) of
nuclear extract from MCF-7 cells or various amounts of purified
recombinant USF-1 were used for the EMSA as described in Fig. 3 . Lane
1, Probe control without protein; lane 2, probe with the nuclear
extract; lanes 3, 4, and 5: probes with 0.4, 4, and 40 ng of purified
USF-1, respectively. B, USF binding to the CD -124/-104 sequence as a
heterodimer in nuclear extracts. An aliquot (2 µg) of nuclear extract
from MCF-7 cells or purified USF-1 (4 ng) was used for a gel supershift
assay using the same procedures as those in Fig. 3 , except another
20-min incubation was added after addition of antibodies (1 µg). Lane
1, Probe with nuclear extract from E2-untreated cells; lane
2, probe with nuclear extract from E2-treated cells; lanes
3, 4, 5, and 6, probe with nuclear extract from E2-treated
cells, the reaction containing rabbit polyclonal ER antibody, rabbit
polyclonal USF-1 antibody, mouse monoclonal GR antibody, and rabbit
polyclonal USF-2 antibody, respectively; lane 7, probe with purified
USF-1; lanes 8 and 9, probe with purified USF-1, the reaction
containing rabbit antibody against human USF-1, and rabbit antibody
against mouse USF-2, respectively.
|
|
E Box-Mutated CD Promoter Activity and Transcription Factor
Binding
Previous studies have found that an E box has the consensus
sequence CANNTG and is the binding site for homo- or heterodimers of
bHLH proteins (34, 41). In the E box enhancer sequence
(CACGTG), the cental C and G bases, shown in
bold, are the most important nucleotides for USF binding
(34). Mutation of this core sequence will abolish USF binding in
vitro (41). To understand the contributions that the E box makes
to E2 regulation of CD transcription, we mutated the
central C and G of the E box core to A and T (leaving the E box
consensus sequence, CAatTG, intact) and used the mutated CD -124/-104
probe to test for USF binding. The CDmut -124/-104 probe was not
bound by either recombinant USF-1 or nuclear proteins (Fig. 7A
, compare lanes 911). In addition,
the mutated probe failed to inhibit binding to the wild-type probe,
even at 250 molar excess (Fig. 7A
, compare lanes, 2, 4, 5, and 7).
These experiments strongly suggest that the proteins bound to the E-box
in the CD promoter are USF-1/2.

View larger version (54K):
[in this window]
[in a new window]
|
Figure 7. Mutation of the Cathepsin D E Box Prevents USF-1
Binding in Vitro and in Vivo and Blocks
Estrogen Activation of the Cathespin D Promoter
A, USF-1 fails to bind to the CDmut -124/-104 probe in
vitro. An EMSA was performed as described in Fig. 3 . A
competition experiment was conducted by adding a 250-fold molar excess
of unlabeled probe to the binding reaction. Lane 1, CD -124/-104
probe; lane 2: CD -124/-104 probe with the purified USF-1; lanes 3
and 4: CD -124/-104 probe with the purified USF-1 and a 250-fold
excess of unlabeled CD -124/-104 and CDmut -124/-104 probes,
respectively; lane 5, CD -124/-104 probe with the nuclear extract;
lanes 6, 7, and 8: CD -124/-104 probe with the nuclear extract and a
250-fold excess of unlabeled CD -124/-104, CDmut -124/-104 and SP1
probes, respectively; lane 9, CDmut -124/-104 probe; lanes 10 and 11,
CDmut -124/-104 probe with the purified USF-1 and the nuclear
extract, respectively. B, Disruption of USF binding to the mutated CD
promoter in vivo. An in vivo footprinting
assay was performed in transiently transfected MCF-7 cells as described
in Fig. 2 , using a site-directed mutated construct (pMCD/355) as well
as a wild-type construct (pCD/355). Lane 1, x 175 DNA
size marker; lanes 25, four DNA sequence tracks with mutated
template; lanes 6 and 7, mutated templates, the cells treated and
untreated with E2, respectively; lanes 8 and 9, wild-type
templates, the cells untreated and treated with E2,
respectively; lanes 1013, four DNA sequence tracks with wild-type
template. C, E2 activation is reduced with a mutated CD
promoter. A CAT assay was carried out as described in Fig. 1B , using
the wild-type construct pCD/355 and mutated construct pMCD/355
transfected into MCF-7 cells that were either untreated or treated with
10 nM E2.
|
|
To test the contribution of the USF-binding site to promoter activity
in vivo, we incorporated the same mutations as the CD
-124/-104 oligonucleotides into the CD promoter/CAT gene construct
pCD/355 (15). In vivo footprinting assays failed to detect
the estrogen-induced USF binding to the mutated E box promoter
template, while the E2 treatment effectively stimulated USF
loading on the wild-type promoter (Fig. 7B
, compare lanes 6 and 9).
Close inspection of the footprinting assays revealed the appearance of
a second E2- enhanced transcription factor adjacent to the
fourth half-ERE in the mutated CD promoter (Fig. 7B
, compare lanes 6
and 7). The identity of this protein is not known at present, and it
does not appear to be significantly enhanced by E2 at the
corresponding site on the wild-type promoter. However, it may be
related to the residual induction by E2 seen in the absence
of an intact E box.
To confirm a functional role for USF in E2 induction of CD
gene expression, MCF-7 cells were transiently transfected with either
the wild-type or mutated CD-CAT reporter plasmids (Fig. 7C
). In
agreement with the USF loading experiments, CAT assays demonstrated
that the E2 induction was reduced in pMCD/355 transfected
cells by 90% relative to the wild-type construct (Fig. 7C
). Protein
binding at the E box appeared to contribute both to the induced and
basal levels of expression as the basal transcription from the mutated
construct is reduced by 80% relative to the wild-type promoter (Fig. 7C
). Finally, while the E2 response from the mutated
promoter is significantly less than seen with the wild type, it does
represent a 4-fold elevation over the reduced basal level of expression
seen with the mutated promoter (Fig. 7C
). This E2 induction
may be related to the hormone-dependent protein binding downstream of
the E box and serves to highlight the importance of the USF/E box
interaction in providing a robust activation of the CD promoter
in vivo.
 |
DISCUSSION
|
---|
CD is an important protease that is expressed in human breast
cancer cells (1, 2). Transcription from this gene occurs with multiple
initiation start sites, one of which is stimulated with estradiol (13).
This effect of E2 is of particular interest as the ER
status of breast tumors is an important prognostic indicator for the
clinical outcome in breast cancer (3, 4, 5). In our studies, we have begun
to examine the mechanisms by which the ER activates the CD promoter.
Our analyses demonstrate that the hormonal induction of the promoter
seen in the endogenous gene is recapitulated with a minimal promoter
element (Figs. 1
and 2
). Further, in vivo footprinting
experiments revealed E2-dependent binding of a protein(s)
at an E box (CACGTG) element within the proximal promoter (12, 42)
(Fig. 2
). Subsequent biochemical experiments demonstrated that these
proteins correspond to the ubiquitous transcription factors USF-1 and
USF-2 (39, 40) (
Figs. 36


). The enhanced binding of USF-1/2 on the
promoter occurs in the absence of detectable posttranslational
modifications or changes in the levels of the ER or USF-1/2 (Figs. 4
and 5
, and data not shown). The contribution that USF-1/2 binding makes
to CD activation by the ER was demonstrated by experiments in which
mutation of the E box prevented USF-1/2 binding both in
vitro and in vivo (Fig. 7
, A and B). Incorporation of
the same mutations into the promoter blocked ER-mediated activation of
transcription in vivo. These experiments suggest a model for
ER stimulation of the CD promoter in which recruitment of USF-1/2 to
the promoter is required for activation of transcription.
The ER-mediated recruitment of USF-1/2 to the CD promoter is
reminiscent of observations obtained with the MMTV system (26, 30). In
these studies the GR mediates the recruitment of NF1 and octamer
transcription factors to the promoter to stimulate gene
expression (31, 32). These analyses have suggested the model for
steroid hormone action whereby the GR initiates a cascade of events
that leads to the remodeling of MMTV chromatin to allow access by
ubiquitous nuclear proteins. As the chromatin structure of the CD gene
promoter has not been described, it is not possible to draw a detailed
comparison with the MMTV promoter. However, while these activation
profiles are similar, there are distinct differences that can be
examined. For example, the recruitment of NF1 and octamer transcription
factor at the MMTV promoter, predicated upon the remodeling of MMTV
chromatin, has not been observed when the promoter was transiently
transfected into cells (32). In contrast, the ER-mediated loading of
USF-1/2 to the CD promoter is hormone dependent on
transient-transfected templates (Figs. 2B
and 7B
). Indeed, the USF-1/2
recruitment is more reminiscent of hormone-dependent recruitment of the
TATA binding protein on both stable and transient templates (31, 32).
Consequently, it would be of great interest to ascertain the chromatin
structure of the CD promoter and to determine whether the ER-dependent
binding of USF-1/2 is maintained on the chromatin copy of the gene.
The mutation of the E box in the context of the proximal promoter
results in a diminution of the basal and ER-stimulated transcription
(Fig. 7
). However, the mutated promoter retains a limited degree of
responsiveness to E2 that is mirrored by an enhanced
hormone-stimulated binding of a protein(s) to the ERE half-site on the
CD promoter containing a mutated USF site. Previous in vivo
footprinting experiments had been interpreted as indicating that the
major E2 enhanced binding was attributed to the ER (15).
The mutation of the E box establishes that it a USF-1/2 complex that is
the major protein entity on the promoter that forms as part of ER
stimulation of transcription (Fig. 7B
). The identity of this protein
with a boundary at -111 of the CD promoter is unknown at present.
However, its interaction at or adjacent to the functional ERE half-site
makes the ER a strong candidate (15). The enhanced binding at this
-111 site on the mutated promoter may result from a number of
possibilities. First, it may simply result from the fact that the
mutation, within the E box, by blocking the binding of USF-1/2,
which is upstream of the new protein boundary, the exonuclease proceeds
through until it encounters the bound protein(s). Alternatively, the
failure of USF-1/2 to bind the mutated site may alleviate potential
stearic considerations, thereby allowing for better binding at the
site. Further in vitro footprinting experiments with
purified USF and ER proteins will be necessary to allow us to decide
between these possibilities.
Alternatively, the fact that the promoter exhibits a diminished
but consistent E2 response, when the USF-1/2 site is
mutated, may reflect the contributions of more distal
estrogen-responsive regions of the promoter described earlier (12).
These include nonconsensus EREs at -362, -261, -199, and -87 of the
promoter as defined by gel shift and transient transfection assays (12, 14). Indeed the site at -261 was shown to require sequences that
include the sequences defined by our studies for full function (12).
Interestingly our in vivo footprinting assays do not detect
protein-DNA interactions at these sites, which makes a direct
comparison with our studies of the -124 region more difficult (see
below).
Previous experiments demonstrated a synergistic interaction
between the ER and SP1 at the heat shock protein 27 and CD promoters
(14, 43). For the CD promoter these studies have focused on an ERE
half-site (-199 to -165) that is distal to the region of the promoter
examined here (-124 to -104). Biochemical and transfection evidence
suggest that there is a functional interaction of the ER with SP1 that
mediates the estrogen response (14). In the CD promoter there are
potential SP1- and ER- binding sites adjacent to the E box (Fig. 2A
)
(15). Our biochemical assays find no evidence for interaction of the ER
with USF-1/2 on the E box (
Figs. 36


). The results with respect to SP1
are a bit more complicated. In EMSA competition experiments and
antibody supershift studies with anti-SP1, we find no evidence for SP1
interacting with this short region (-124 to -104) of the CD promoter
(W. Xing and T. K. Archer, data not shown). However, both
Southwestern blotting and UV cross-linking experiments leave open the
possibility of an interaction. Southwestern blotting detects a protein
of approximately 105 kDa, and in UV cross-linking experiments an
unlabeled SP1 probe competes this band away without affecting the
labeled USF-1/2 probe interactions with USF-1 and USF-2. Thus, one
interpretation is that this putative SP1 site is a relatively weak site
such that interactions are not stable to gel shift conditions but are
maintained under the liquid-solid phase interaction obtained with
Southwestern blotting experiments or stabilized by UV
cross-linking.
The ability to detect proteins on transiently transfected templates
provides an important tool for ascertaining those proteins that
interact with a promoter from potential binding sites identified
through computer-based analysis of the sequences (44, 45). Indeed, our
experiments suggest that the presence of a number of binding sites, as
indicated on the CD promoter, does not necessarily mean that these
sites, and the proteins that can potentially bind to them, play a
significant role in activation of transcription. In the future, it will
be important to determine the precise role or potential role of
SP1-USF-1/2 interactions on this promoter. Similarly, while the binding
of USF-1/2 at the promoter is hormone dependent, the precise mechanism
by which this occurs in the absence of identifiable changes in USF-1/2
levels is not known. Thus, it will be important to determine whether
the recruitment of USF-1/2 results from direct or indirect interactions
with the ER and the mechanism(s) by which this results in the formation
of an active preinitiation complex on the CD promoter in
vivo.
 |
MATERIALS AND METHODS
|
---|
Oligonucleotides
The following oligonucleotides were synthesized on an Applied
Biosystems (Foster City, CA) DNA synthesizer 321.
CAT 5'-GCT CCT GAA AAT CTC GCC AAG
5CD: 5'-GCT GCA CAA GTT CAC GTC CAT
3CD: 5'-TGC CAA TCT CCC CGT AGT ACT G
5ßM: 5'-ACC CCC ACT GAA AAA GAT GA
3ßM: 5'-ATC TTC AAA CCT CCA TGA TG
5CDmut: 5'-CCG GCC GCG CCC Aat TGA CCG GTC CGG G-3'
3CDmut: 5'-C CCG GAC CGG TCA atT GGG CGC GGC CGG-3'
ERE: 5'-CAA AGT CAG GTC ACA GTG AGG TGA TCA AAG A
3'-GTT TCA GTC CAG TGT CAC TGG ACT AGT TTC T
Sp1: 5'-GAT GCG GTC CCG CCC TCA GC-3'
3'-CTA CGC CAG GGC GGG AGT CG-5'
CD -124/-104: 5'-GGC CGC GCC CAC GTG ACC GGT
3'-CCG GCG CGG GTG CAC TGG CCA
CDmut -124/-104: 5'-GGC CGC GCC CAatTG ACC GGT CC- 3'
3'-CCG GCG CGG GTtaAC TGG CCA GG-5'
Plasmids and Bacterial Strains
Plasmids pCD/355, proximal CD promoter from -365 to -10, and
HEO, human ER expression vector, were kindly provided by Dr. S. Safe
(Texas A & M University, College Station, TX). Escherichia
coli strain DH5
was used for routine transformation
experiments. Plasmid preparations for transfection were by alkaline
lysis followed by cesium chloride gradient centrifugation (46).
Cell Line and Cell Culture
Human breast cancer cells (MCF-7) were routinely maintained in a
humidified 37 C incubator with 5% CO2 and cultured in
Modified Eagle Medium (MEM) containing 10% FBS (GIBCO BRL,
Gaithersburg, MD), 2 mM L-glutamine, 100 U/ml
penicillin, and 100 µg/ml steptomycin.
mRNA Isolation and RT-PCR
mRNA was isolated with Trizol reagent (GIBCO BRL) according to
the manufacturers instructions. Twenty micrograms of total RNA were
annealed to 750 ng of poly (T)12-18 at 65 C for 5 min,
after which the first-strand cDNAs were synthesized using a
first-strand cDNA synthesis kit (Pharmacia Biotech, Piscataway, NJ).
After a 10-min incubation at 75 C, an aliquot (90%) of total
synthesized first-strand cDNAs was amplified using
32P-labeled primers (5CD and 3CD) by PCR, and the remainder
(10%) was amplified utilizing 32P-labeled human
ß2-microglobulin primers (5ßM and 3 ßM) as an
internal standard. The RT-PCR products were resolved on a 8%
polyacrylamide denaturing gel and visualized by autoradiography.
Transient Transfections
MCF-7 cells were seeded into 150-mm tissue culture plates
(106 cells per plate) in phenol red-free MEM plus 5%
double dextran charcoal-stripped FBS. The following day, medium was
removed and replaced with fresh estrogen-free medium. Transient
transfections were carried out with 10 µg of pCD/355 or pMCD/355 and
5 µg of HEO for 18 h, using calcium phosphate coprecipitation
(31). The following day, fresh media, with or without 10
nM estradiol (E2, Sigma Chemical Co., St.
Louis, MO), was added for an additional 24 h before
harvesting.
Chloramphenicol Acetyltransferase (CAT) Assay
CAT assays were performed as described previously (47). CAT
activity was assayed by mixing 200 µg cell extract with 250 µl
reaction buffer (0.25 M Tris-HCl, pH 7.8, 1.25
mM chloramphenicol, 0.1 µCi of 3H-labeled
acetyl coenzyme A). The results represent the average of three
independent experiments.
In Vivo Exonuclease III Footprinting Assays
MCF-7 cells were transfected as described above, and nuclei were
isolated and partially digested with HindIII and exonuclease
III to detect the 5'-boundaries of transcription factors bound to the
CD promoter as described previously (44). After purification, DNA was
completely digested with HindIII to provide an internal
standard, and 10 µg of each sample were analyzed using linear
Taq polymerase amplification with a 32P-labeled
oligonucleotide (32). Purified extended products, together with DNA
sequencing tracks, were analyzed on a 5% polyacrylamide denaturing gel
and visualized by autoradiography.
Preparation of Nuclear Extracts
Nuclear extracts were prepared as described previously (48).
MCF-7 cells were washed in PBS and lysed on ice for 5 min in Buffer E
[0.3% Nonidet P-40 (NP-40), 10 mM Tris-HCl (pH 8.0), 60
mM NaCl, 1 mM EDTA, 1 mM
dithiothreitol (DTT), 1 mM phenylmethylsulfonylfluoride].
Nuclei were pelleted by spinning for 5 min at 2500 rpm at 4 C. The
pellet was washed in an Buffer E lacking NP-40 and resuspended in 100
µl of Buffer C (20 mM HEPES, pH 7.9, 0.75 mM
spermidine, 0.15 mM spermine, 0.2 mM EDTA, 2
mM EGTA, 2 mM DTT, 20% glycerol, 1
mM phenylmethylsulfonylfluoride). NaCl was added to a final
concentration of 0.4 M, and the nuclei were gently shaken
for 20 min at 4 C, and then spun for 10 min at 12,000 rpm at 4 C.
Nuclear extracts were stored at -80 C.
EMSA
EMSAs were as described previously (49). Briefly, double-strand
oligonucleotides (ERE, SP1, CD -124/-104, and CDmut -124/-104)
were labeled at the 5'-end using T4 polynucleotide kinase and
[
-32P] ATP. Nuclear extracts from MCF-7 cells treated
or untreated with 10 nM E2 for 24 h or
purified recombinant USF-1 protein (kindly provided by D. Steger and J.
Workman, Pennsylvania State University, University Park, PA),
were incubated in a binding buffer containing 10 mM
Tris-HCl (pH 7.5), 50 mM NaCl, 3 mM DTT, 10%
glycerol, 0.05% NP-40, 0.1 mM ZnCl2, 50
µg/ml poly(deoxyinosinic-deoxycytidylic)acid, and 0.02 pmol of
labeled DNA probe. After addition of radiolabeled probe, the mixture
was incubated at room temperature for another 15 min. Excess unlabeled
DNA competitor was added 5 min before the addition of radiolabeled DNA
probe. The reaction mixture was analyzed using a 5% nondenaturing
polyacrylamide gel in 1 x Tris-borate-EDTA buffer. For the
gel supershift assay, 1 µg of antibody was added into the reaction
mixture and incubated at room temperature for an additional 15 min.
Gels were dried and were visualized by autoradiography.
Southwestern Analysis
The Southwesten analyses were performed as described previously
(50). Briefly, 50 µg nuclear extracts were loaded onto a SDS-PAGE
denaturing gel, and electrophoresis was carried out at 15 mA for 5
h. Proteins were then transferred to Hybond-ECL nitrocellulose
(Amersham, Arlington Heights, IL) and membranes were incubated, at room
temperature for 60 min, in a buffer containing 5% dry skim milk, 25
mM NaCl, 5 mM MgCl2, 25
mM HEPES (pH 7.9), and 0.5 mM DTT. The binding
reaction was performed in the same buffer with 106 cpm/ml
of 32P-labeled CD -124/-104 probe, in the presence of 5
µg/ml of salmon sperm DNA, at room temperature for 18 h. Before
autoradiography, blots were washed four times at room temperature
utilizing the same buffer.
UV Cross-Linking
The UV cross-linking protocol was carried out as described
previously (51). Protein DNA binding was at room temperature for 20 min
with 5 µg nuclear extract in EMSA binding buffer. The vials were then
sealed, and a GS Gene Linker (Bio-Rad Laboratories, Richmond, CA), set
at 125 mJ, was used for cross-linking. Subsequently, samples were
electrophoresed as described above, and then the gel was dried before
autoradiography.
Site-Directed Mutagenesis
Mutagenesis was performed using the QuikChange Site-Directed
Mutagenesis Kit (Stratagene, La Jolla, CA). In brief, a supercoiled,
double-stranded pCD/355 plasmid DNA with the proximal CD promoter
(-365 to -10) was used as a template. The two synthetic
oligonucleotides of 5CDmut and 3CDmut containing a mutated E box
(CAatTG) were extended during the temperature cycling by
means of Pfu DNA polymerase. The extension product was
treated with 10 U of DpnI endonuclease at 37 C for 1 h
to digest the parental supercoiled double-stranded DNA template.
The nicked vector DNA incorporating a mutated E box was then
transformed into Epicurian coli XL1-Blue
Supercompetent Cells. Individual clones were isolated, and the DNA was
purified and sequenced to confirm which clones had the correct
mutational changes.
DNA Sequence Analysis
The procedures were performed according to instructions provided
by the manufacturer (Pharmacia Biotech). In brief, 4 µg of
double-stranded plasmid DNA were denatured with 0.4 M
sodium hydroxide for 10 min, neutralized with 0.45 M sodium
acetate, and precipitated in 70% ethanol. The purified DNA templates
were annealed to the CAT oligo and sequenced using the dideoxy chain
termination method with [
-35S ]dATP. Chain
elongation-termination reaction products were resolved on a 7%
polyacrylamide denaturing gel. The gel was dried before autoradiography
at room temperature with Kodak X-OMAT AR 5 film (Eastman Kodak,
Rochester, NY).
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. S. Safe (Texas A & M University, College Station,
TX) for plasmids pCD/355 and HEO; Drs. D. Steger and J. Workman
(Pennsylvania State University, State College, PA) for purified USF-1
protein; and Dr. F Wang and L. Dong (Texas A & M University) and Dr. D.
Rodenhiser and D. Mancini (University of Western Ontario, London,
Ontario, Canada) for helpful technical suggestions and help with DNA
sequencing. We are especially grateful to B. Deroo and K.
McAllister for helpful comments on the manuscript, D. Powers for its
preparation, and Dr. J. Mymryk for help with the figures.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Trevor K. Archer, London Regional Cancer Centre, 790 Commissioners Road East, London, Ontario, Canada N6A 4L6. E-mail: tarcher{at}julian.uwo.ca
This work was supported by grants from the National Cancer Institute of
Canada (NCIC) and the Canadian Breast Cancer Research Initiative to
T.K.A. T.K.A. is a Scientist of the NCIC and supported by funds
from the Canadian Cancer Society.
Received for publication March 30, 1998.
Revision received May 7, 1998.
Accepted for publication May 12, 1998.
 |
REFERENCES
|
---|
-
Westley BR, May FE 1996 Cathepsin D and breast cancer.
Eur J Cancer 32A:1524
-
Rochefort H 1990 Cathepsin D in breast cancer. Breast Cancer
Res Treat 16:313[Medline]
-
Iwase H, Itoh Y, Kuzushima T, Yamashita H, Iwata H 1997 Simultaneous quantitative analyses of c-erbB-2 protein, epidermal
growth factor receptor, cathepsin D, and hormone receptors in breast
cancer. Cancer Detect Prev 21:2935[Medline]
-
Brouillet JP, Dufour F, Lemamy G, Garcia M, Schlup N, Grenier
J, Mani JC, Rochefort H 1997 Increased cathepsin D level in the serum
of patients with metastatic breast carcinoma detected with a specific
pro-cathepsin D immunoassay. Cancer 79:21322136[CrossRef][Medline]
-
Rochefort H 1996 The prognostic value of cathepsin D in
breast cancer. A long road to the clinic. Eur J Cancer 32A:78
-
Garcia M, Platet N, Liaudet E, Laurent V, Derocq D, Brouillet
JP, Rochefort H 1996 Biological and clinical significance of cathepsin
D in breast cancer metastasis. Stem Cells 14:642650[Abstract]
-
Confort C, Rochefort H, Vignon F 1995 Insulin-like growth
factors (IGFs) stimulate the release of alpha 1-antichymotrypsin
and soluble IGF-II/mannose 6-phosphate receptor from MCF7 breast cancer
cells. Endocrinology 136:37593766[Abstract]
-
Glikman P, Rogozinski A, Mosto J, Pollina A, Garbovesky C,
Levy C 1997 Relationship between cathepsin-D and other prognostic
factors in human breast cancer. Tumori 83:685688[Medline]
-
Li SA, Liao D-ZJ, Yazlovitskaya EM, Pantazis CG, Li JJ 1997 Induction of cathepsin D protein during estrogen carcinogenesis:
possible role in estrogen-mediated kidney tubular cell damage.
Carcinogenesis 18:13751380[Abstract]
-
Joensuu H, Toikkanen S, Isola J 1995 Stromal cell cathepsin D
expression and long-term survival in breast cancer. Br J Cancer 71:155159[Medline]
-
Touitou I, Vignon F, Cavaillès V, Rochefort H 1991 Hormonal regulation of cathepsin D following transfection of the
estrogen or progesterone receptor into three sex steroid hormone
resistant cancer cell lines. J Steroid Biochem Mol Biol 40:231237[CrossRef][Medline]
-
Augereau P, Miralles F, Cavaillès V, Gaudelet C, Parker
M, Rochefort H 1994 Characterization of the proximal
estrogen-responsive element of human cathepsin D gene. Mol Endocrinol 8:693703[Abstract]
-
Cavaillès V, Augereau P, Rochefort H 1993 Cathepsin D
gene is controlled by a mixed promoter, and estrogens stimulate only
TATA-dependent transcription in breast cancer cells. Proc Natl Acad Sci
USA 90:203207[Abstract]
-
Krishnan V, Wang X, Safe S 1994 Estrogen receptor-Sp1
complexes mediate estrogen-induced cathepsin D gene expression in MCF-7
human breast cancer cells. J Biol Chem 269:1591215917[Abstract/Free Full Text]
-
Wang F, Porter W, Xing W, Archer TK, Safe S 1997 Identification of a functional imperfect estrogen-responsive element in
the 5'-promoter region of the human cathepsin D gene. Biochemistry 36:77937801[CrossRef][Medline]
-
Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schütz
G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995 The nuclear receptor superfamily: the second decade. Cell 83:835839[Medline]
-
Evans RM 1988 The steroid and thyroid hormone receptor
superfamily. Science 240:889895[Medline]
-
Beato M 1989 Gene regulation by steroid hormones. Cell 56:335344[Medline]
-
Horwitz KB, Jackson TA, Bain DL, Richer JK, Takimoto GS, Tung
L 1996 Nuclear receptor coactivators and corepressors. Mol Endocrinol 10:11671177[Abstract]
-
Glass CK, Rose DW, Rosenfeld MG 1997 Nuclear receptor
coactivators. Curr Opin Cell Biol 9:222232[CrossRef][Medline]
-
McEwan IJ, Wright APH, Gustafsson J-Å 1997 Mechanism of
gene expression by the glucocorticoid receptor: Role of protein-protein
interactions. Bioessays 19:153160[Medline]
-
Shikama N, Lyon J, La Thangue NB 1997 The p300/CBP family:
integrating signals with transcription factors and chromatin. Trends
Cell Biol 7:230236[CrossRef]
-
Archer TK Watson CE 1998 Chromatin and receptor-mediated
transcription. In: Freedman LP (ed) Molecular Biology of Steroid and
Nuclear Hormone Receptors. Birkhäuser, Boston, pp 209235
-
Beato M 1991 Transcriptional control by nuclear receptors.
FASEB J 5:20442051[Abstract/Free Full Text]
-
Shibata H, Spencer TE, Onata SA, Jenster G, Tsai SY, Tsao M-J,
OMalley BW 1997 Role of co-activators and co-repressors in the
mechanism of steroid/thyroid receptor action. Recent Prog Horm Res 52:141165[Medline]
-
Archer TK, Fryer CJ, Lee H-L, Zaniewski E, Liang T, Mymryk JS 1995 Steroid hormone receptor status defines the MMTV promoter
chromatin structure in vivo. J Steroid Biochem Mol Biol 53:421429[CrossRef][Medline]
-
Hager GL, Archer TK, Fragoso G, Bresnick EH, Tsukagoshi Y,
John S, Smith CL 1993 Influence of chromatin structure on the binding
of transcription factors to DNA: DNA and chromosomes. Cold Spring
Harbor Symp Quant Biol 58:6371[Medline]
-
Beato M, Herrlich P, Schütz G 1995 Steroid hormone
receptors: Many actors in search of a plot. Cell 83:851857[Medline]
-
Archer TK, Cordingley MG, Marsaud V, Richard-Foy H Hager GL 1989 Steroid transactivation at a promoter organized in a
specifically-positioned array of nucleosomes. In: Gustafsson JA,
Eriksson H, Carlstedt-Duke J (eds) The Steroid/Thyroid Hormone Receptor
Family and Gene Regulation. Birkhäuser Verlag Basel, Berlin, pp
221238
-
Archer TK Mymryk JS 1995 Modulation of transcription factor
access and activity at the MMTV promoter in vivo. In: Wolffe
AP (ed) The Nucleus. JAI Press Inc., Greenwich, CT, pp
123150
-
Archer TK, Lefebvre P, Wolford RG, Hager GL 1992 Transcription
factor loading on the MMTV promoter: a bimodal mechanism for promoter
activation. Science 255:15731576[Medline]
-
Lee H-L, Archer TK 1994 Nucleosome-mediated disruption of
transcription factor-chromatin initiation complexes at the mouse
mammary tumor virus long terminal repeat in vivo. Mol Cell
Biol 14:3241[Abstract]
-
Elnitski L, Miller W, Hardison R 1997 Conserved E boxes
function as part of the enhancer in hypersensitive site 2 of the
ß-globin locus control region. J Biol Chem 272:369378[Abstract/Free Full Text]
-
Gao E, Wang Y, Alcorn JL, Mendelson CR 1997 The basic
helix-loop-helix-zipper transcription factor USF1 regulates expression
of the surfactant protein-A gene. J Biol Chem 272:2339823406[Abstract/Free Full Text]
-
Hoffman PW, Chernak JM 1995 DNA binding and regulatory effects
of transcription factors SP1 and USF at the rat amyloid precursor
protein gene promoter. Nucleic Acids Res 23:22292235[Abstract]
-
Gupta MP, Amin CS, Gupta M, Hay N, Zak R 1997 Transcription
enhancer factor 1 interacts with a basic helix-loop-helix zipper
protein, max, for positive regulation of cardiac
-myosin heavy-chain
gene expression. Mol Cell Biol 17:39243936[Abstract]
-
Maekawa T, Sudo T, Kurimoto M, Ishii S 1991 USF-related
transcription factor, HIV-TF1, stimulates transcription of human
immunodeficiency virus-1. Nucleic Acids Res 19:46894694[Abstract]
-
Scholtz B, Kingsley-Kallesen M, Rizzino A 1996 Transcription
of the transforming growth factor-ß2 gene is dependent on an E-box
located between an essential cAMP response element/activating
transcription factor motif and the TATA box of the gene. J Biol
Chem 271:3237532380[Abstract/Free Full Text]
-
Sirito M, Lin Q, Maity T, Sawadogo M 1994 Ubiquitous
expression of the 43- and 44-kDa forms of transcription factor USF in
mammalian cells. Nucleic Acids Res 22:427433[Abstract]
-
Gregor PD, Sawadogo M, Roeder RG 1990 The adenovirus major
late transcription factor USF is a member of the helix-loop-helix group
of regulatory proteins and binds to DNA as a dimer. Genes Dev 4:17301740[Abstract]
-
di-Fagagna FD, Marzio G, Gutierrez MI, Kang LY, Falaschi A,
Giacca M 1995 Molecular and functional interactions of transcription
factor USF with the long terminal repeat of human immunodeficiency
virus type 1. J Virol 69:27652775[Abstract]
-
Hetman M, Perschl A, Saftig P, von Figura K, Peters C 1994 Mouse cathepsin D gene: molecular organization, characterization of the
promoter, and chromosomal localization. DNA Cell Biol 13:419427[Medline]
-
Porter W, Saville B, Hoivik D, Safe S 1997 Functional synergy
between the transcription factor Sp1 and the estrogen receptor. Mol
Endocrinol 11:15691580[Abstract/Free Full Text]
-
Archer TK, Lee H-L 1997 Visualization of multicomponent
transcription factor complexes on chromatin and nonnucleosomal
templates in vivo. Methods 11:235245[CrossRef][Medline]
-
Heinemeyer T, Wingender E, Reuter I, Hermjakob HKAE, Kel OV,
Ignatieva EV, Ananko EA, Podkolodnaya OA, Kolpakov FA, Podkolodny NL,
Kolchanov NA 1998 Databases on transcriptional regulation: TRANSFAC,
TRRD and COMPEL. Nucleic Acids Res 26:362367[Abstract/Free Full Text]
-
Archer TK, Cordingley MG, Wolford RG, Hager GL 1991 Transcription factor access is mediated by accurately positioned
nucleosomes on the mouse mammary tumor virus promoter. Mol Cell Biol 11:688698[Medline]
-
Archer TK, Lee H-L, Cordingley MG, Mymryk JS, Fragoso G,
Berard DS, Hager GL 1994 Differential steroid hormone induction of
transcription from the mouse mammary tumor virus promoter. Mol
Endocrinol 8:568576[Abstract]
-
Scheinman RI, Gualberto A, Jewell CM, Cidlowski JA, Baldwin Jr
AS 1995 Characterization of mechanisms involved in transrepression of
NF-kB by activated glucocorticoid receptors. Mol Cell Biol 15:943953[Abstract]
-
Archer TK, Hager GL, Omichinski JG 1990 Sequence-specific DNA
binding by glucocorticoid receptor "zinc finger peptides." Proc
Natl Acad Sci USA 87:75607564[Abstract]
-
Singh H, Clerc RG, LeBowitz JH 1989 Molecular cloning of
sequence-specific DNA binding proteins using recognition site probes.
Biotechniques 7:252261[Medline]
-
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith
JA, Struhl K, Albright LM, Coen DM, Varki A 1994 Current
Protocols in Molecular Biology. John Wiley & Sons, Inc., New York,
1994