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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo). In contrast, within the same cells ß2-microglobulin mRNA levels were unaffected by E2 treatment (Fig. 1AGo). 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. 1BGo). The E2 induction was specific for the CD promoter as the CAT reporter plasmid without the promoter was unresponsive to E2 (Fig. 1BGo).



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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. 2BGo). This 5'-boundary is consistent with a protein or proteins bound at or adjacent to the E box sequence within the CD promoter (Fig. 2AGo), 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.



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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, {phi}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. 3Go, compare lanes 1–3). Interestingly, and in contrast to what was seen with the in vivo footprinting assay (Fig. 2BGo), there was no significant increase in binding with extracts prepared from E2-treated cells compared with untreated cells (Fig. 3Go, 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. 3Go, 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. 3Go, 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. 3Go).



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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. 4Go, compare lanes 1–3). 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. 4Go, 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. 5Go, compare lanes 1–7). 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. 5Go., 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. 5Go, 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. 5Go, lane 11). Further, proteinase K digestion completely eliminated all DNA protein binding (Fig. 5Go, 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. 3–5GoGoGo).



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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.

 


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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. 6Go). The EMSA with purified USF-1 protein displayed a similar migration of the DNA-protein complex as in MCF-7 nuclear extracts (Fig. 6AGo, 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. 6BGo, 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. 6BGo, compare lanes 3–6). 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. 6BGo, 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. 6BGo, 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. 2BGo).



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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. 3Go. 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. 3Go, 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. 7AGo, compare lanes 9–11). In addition, the mutated probe failed to inhibit binding to the wild-type probe, even at 250 molar excess (Fig. 7AGo, 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.



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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. 3Go. 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. 2Go, using a site-directed mutated construct (pMCD/355) as well as a wild-type construct (pCD/355). Lane 1, {phi}x 175 DNA size marker; lanes 2–5, 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 10–13, 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. 1BGo, 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. 7BGo, 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. 7BGo, 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. 7CGo). 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. 7CGo). 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. 7CGo). 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. 7CGo). 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go and 2Go). 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. 2Go). Subsequent biochemical experiments demonstrated that these proteins correspond to the ubiquitous transcription factors USF-1 and USF-2 (39, 40) ( Figs. 3–6GoGoGoGo). 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. 4Go and 5Go, 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. 7Go, 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. 2BGo and 7BGo). 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. 7Go). 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. 7BGo). 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. 2AGo) (15). Our biochemical assays find no evidence for interaction of the ER with USF-1/2 on the E box ( Figs. 3–6GoGoGoGo). 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha} 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 manufacturer’s 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 [{gamma}-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 [{alpha}-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.


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