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Molecular Endocrinology 20 (12): 3105-3119
Copyright © 2006 by The Endocrine Society

Stimulation of Steroid Receptor Coactivator-3 (SRC-3) Gene Overexpression by a Positive Regulatory Loop of E2F1 and SRC-3

Paola Mussi, Chundong Yu, Bert W. O’Malley and Jianming Xu

Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Jianming Xu, Department of Molecular and Cellular Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas 77030. E-mail: jxu{at}bcm.tmc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroid receptor coactivator 3 (SRC-3, amplified in breast cancer 1, or ACTR) is a transcriptional coactivator for nuclear receptors and certain other transcription factors such as E2F1. SRC-3 is overexpressed in breast cancers, and its overexpression is sufficient to cause mammary carcinomas in vivo. However, the mechanisms controlling endogenous SRC-3 overexpression are unknown. In this study, we identified the first exon and analyzed the 5' regulatory sequence of the SRC-3 gene. We found three evolutionarily conserved regions (ECRs) in the 5' SRC-3 regulatory sequence, and ECR2 makes a major contribution to the SRC-3 promoter activity. The ECR2 region (bp –250/+350) contains several specificity protein 1 (Sp1) binding sites and two E2F1 binding sites. We show that E2F1 can significantly activate the ECR2 promoter activity in a dose-dependent manner. Furthermore, overexpression of E2F1 significantly increases the promoter activity of the endogenous SRC-3 gene and boosts SRC-3 expression in vivo. Conversely, knockdown of E2F1 reduces SRC-3 expression. We demonstrate that the mechanism of E2F1 activity on SRC-3 promoter is independent of the E2F binding sites but relies on the Sp1 element located at bp +150/+160. Sp1, E2F1, and SRC-3 are specifically recruited to this Sp1 site and the interaction between E2F1 and Sp1 is essential to modulate SRC-3 expression. Moreover, SRC-3 coactivates E2F1 activity and thereby additively stimulates a further increase in SRC-3 expression in vivo. These results suggest that in cells with hyperactive E2F1, such as the case encountered in breast cancer cells, there is a positive feedback regulatory loop consisting of E2F1 and SRC-3 to maintain high levels of SRC-3 and E2F1 activity, which may partially interpret the oncogenic role of SRC-3 overexpression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE STEROID RECEPTOR coactivator 3 (SRC-3) is a member of the p160 SRC coactivator family (1, 2, 3, 4, 5). SRC-3, similar to the other two family members, can interact with nuclear receptors in a ligand-dependent manner and enhance their transcriptional activity (6). In addition, members of the p160 SRC family can coactivate many other transcription factors, such as signal transducer and activator of transcription (7), nuclear factor-{kappa}B (8, 9), activating protein 1 (10), p53 (11), and E2F1 (12). Genetic disruption of the SRC-3 gene in mice results in growth retardation, impaired mammary gland development, reduced female reproductive functions and reduced inhibition of neointima formation by estrogen after vascular injury (13, 14), suggesting that SRC-3 plays an important function in many physiological and pathological processes.

Several lines of evidence demonstrated that SRC-3 is a new oncogene. First, the SRC-3 gene is amplified and overexpressed in a proportion of human breast cancers either positive or negative to the estrogen receptor {alpha} (ER{alpha}) (1, 15, 16, 17, 18). One study reported that SRC-3 is amplified and overexpressed in 9.5% and 64% breast tumors, respectively (1); another study reported that SRC-3 protein is overproduced in about 10% breast tumors (17). SRC-3 is also overexpressed in certain ovarian cancers (15), endometrial carcinomas (19), prostate cancers (20), liver cancers (21), and gastric cancers (22). Second, overexpression of SRC-3 from a mammary epithelium-specific transgene in mice is sufficient to cause a high frequency of mammary carcinoma (23). Third, disruption of the SRC-3 gene in mice significantly suppresses oncogene and carcinogen-induced mammary tumor initiation and progression (24, 25). These findings indicate that overexpression or overactivation of SRC-3 is an important risk factor for breast cancer.

Despite the critical role of SRC-3 in tumorigenesis and its frequent overexpression in human cancers, only a few studies assessed certain factors that may affect SRC-3 levels. The proteasome-mediated posttranslational protein turnover plays an important role in regulation of the SRC-3 protein levels (26). In terms of SRC-3 gene transcription, all-trans retinoic acid can increase SRC-3 mRNA in HL60 and NB4 promyelocytic leukemia cell lines (27). Antiestrogens (ICI and tamoxifen) and TGF-ß can increase SRC-3 mRNA levels, whereas estrogen can suppress SRC-3 gene expression in human MCF-7 breast cancer cells (28). Although these observations suggest that steroid hormones and peptide growth factors both can regulate SRC-3 expression, the molecular mechanisms that regulate SRC-3 promoter activity and mRNA overexpression in cancer cells are unclear. The goal of this study is to address this problem by analyzing the 5' regulatory sequence and identifying the essential transcription factors that regulate SRC-3 promoter activity.

We demonstrate that the transcription factor E2F1 is required for stimulating SRC-3 promoter activity for SRC-3 expression. We provide evidence to show that E2F1 activates the SRC-3 promoter not through binding to its consensus DNA elements, but by cooperating with the transcription factor specificity protein 1 (Sp1). SRC-3 also is a transcriptional coactivator for E2F1 (12) and enhances the expression of its own gene in an E2F1-dependent manner. Thus, a positive feedback regulatory loop consisting of E2F and SRC-3 can maintain high levels of SRC-3 and E2F-dependent target gene transcription in cells with hyperactive E2F, such as the case encountered in breast cancer cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of the First Exon of the SRC-3 Gene
The SRC-3 gene is located in the 20q12 chromosomal region in human cells. To determine the genomic organization of the human SRC-3 gene, we aligned its mRNA sequence (GenBank accession no. NM_181659) to two genomic DNA sequences (GenBank accession no. AL353777 and AL034418). This alignment identified 23 exons, spanning over a 155-kb region (Fig. 1AGo). In comparison with the results published previously for the mouse SRC-3 gene (29), we discovered a new 118-bp exon, termed exon 1a, which is located more than 80 kb upstream from the first exon detected by Liao et al. (29). Two evidences indicate that exon 1a is the first exon of the SRC-3 gene. First, no splice acceptor sequence can be found at the 5' region of exon 1a, whereas exon 1 is flanked by a consensus splice donor sequence (AG) and a splice acceptor site (GT). Second, the matched clones in the human expressed sequence tag database at National Center for Biotechnology Information aligned with exon 1a and exon 1 sequence, but no additional 5' sequence was present. To investigate whether exon 1a is expressed, we performed RT-PCR assay with RNA prepared from MCF-7 and T47D human breast cancer cells. PCR using the primer P1 in exon 1a and the primer P2 in exon 2 amplified a 300-bp fragment spanning exon 1a, exon 1, and exon 2 from both MCF-7 and T47D cells cDNA templates; and no product was amplified from the control samples that were not reversely transcribed (Fig. 1BGo), indicating that exon 1a is expressed and appropriately linked with exon 1 and exon 2 after RNA splicing. In contrast, PCR using the primer P3 located upstream exon 1a and P2 did not generate any product (Fig. 1BGo), suggesting that the region upstream exon 1a is not expressed. These results suggest that the transcriptional initiation of SRC-3 mRNA begins at exon 1a.


Figure 1
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  		Fig. 1. Genomic Structure of SRC-3 Gene

A, Exon and intron organization of the human SRC-3 gene. Black bars indicate exon regions coding for SRC-3 protein. Open boxes indicate transcribed but not translated sequences. Exon 1a, exon 1, and exon 2 are indicated. The lengths of the first and second intron are marked and so is the length of the entire exon and intron region of the SRC-3 gene. P1, P2, and P3 indicate the locations of forward and reverse primers used for RT-PCR analysis shown in panel B. B, RT-PCR analysis of Exon 1a. Total RNA was extracted from T47D and MCF-7 cells and subjected to RT reaction. The cDNA was amplified using primer pairs P1/P2 and P3/P2. Input materials without addition of reverse transcriptase (–RT) were used as a reagent control. The primer P3 upstream the transcriptional initiation site was used as a negative control.

 
Identification of the SRC-3 Promoter
We next addressed whether the genomic DNA sequence upstream and downstream of exon 1a is important for the control of SRC-3 gene expression. It is known that regulatory sequences can lie far away from the transcriptional starting site. Therefore, we decided to analyze a large region surrounding exon 1a. Reasoning that critical DNA sequences for the control of gene expression are evolutionarily conserved, we compared human SRC-3 genomic sequence to orthologous intervals in mouse and rat. The sequence alignment identified three evolutionary conserved regions (ECR), designated as ECR1, ECR2, and ECR3 (Fig. 2AGo). Each human ECR contains two highly conserved regions longer than 100 bp and with more than 70% identity compared with mouse and rat SRC-3 DNA. ECR1 lays 8 kb upstream exon 1a, whereas ECR3 is located in intron 1 and 1.4 kb downstream exon 1a. Interestingly, ECR2 corresponds to the region straddling exon 1a and is the highest conserved interval among human, rat, and mouse DNA sequences, suggesting that it may play an important function.


Figure 2
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  		Fig. 2. ECRs and Activities of the 5' SRC-3 Regulatory Sequences

A, Identification of ECR1, ECR2, and ECR3 in the human 5' SRC-3 sequence. Exon 1a (ex. 1a) is indicated. The first bp of exon 1a is designated as bp +1. The sequence from –8300 to +1400 was compared. The peaks-graphical plot represents the alignment of human sequence with mouse and rat DNA sequences. The vertical peaks indicate the homologous percentage of conserved regions, in a predefined interval, among human, rat, and mouse sequences. The bars above the peaks indicate the conserved regions with more than 70% identity among human, mouse, and rat sequences. B, SRC-3 promoter-luciferase reporter constructs and transfection assays. Crossed bars in the diagrams indicate ECR1, ECR2, and ECR3. The 5' SRC-3 sequences included in the constructs are indicated in reporter nomenclatures. Open boxes indicate Sp1 binding sites (GC boxes). The black box represents exon 1a. The arrow indicates the beginning of exon 1a. The reporter constructs or the empty parent basic vector pGL3 were transfected into MCF-7 cells and the luciferase activity was measured 48 h later. Luciferase counts were normalized to ß-galactosidase activity expressed from the cotransfected pRSV-ß-galactosidase plasmids. Values are presented as relative luciferase activities, with that of the empty vector as 1.

 
To analyze the transcriptional activity of these three conserved regions, we constructed several reporters containing one, two or three ECRs (Fig. 2BGo). When these promoter-reporter constructs were transfected into MCF-7 cells, they resulted in different luciferase activities. The SRC-3(–8300/+1400) construct, carrying all three ECRs exhibited 9-fold higher activity compared with the pGL3 basic empty vector (Fig. 2BGo). Interestingly, the SRC-3(–750/+1400) reporter holding ECR2 and ECR3 and the SRC-3(–250/+350) containing only ECR2 showed a similar activity compared with SRC-3(–8300/+1400) reporter, but the SRC-3(+800/+1400) reporter carrying ECR3 alone presented a very low activity (Fig. 2BGo). Similar transcriptional profiles were also obtained by using Hela cells (data not shown). These results demonstrate that ECR2 is the most active region and that ECR1 and ECR3 do not contain additional important regulatory sequences. Thus, the critical positive regulatory sequence for the SRC-3 promoter activity is located in the –250- to +350-bp region containing ECR2.

Knowing that vertebrate gene promoters are frequently associated with GC-rich regions (30, 31), we calculated the GC content of ECR2 and we compared this value with the average percentage of GC in the 10-kb region bracketed by ECR1 and ECR3. Interestingly, the GC content in ECR2 reaches 80%, whereas the GC content in the 10-kb region is about 40–60%. This provides additional evidence that the SRC-3 promoter is located in this region. We looked further for promoter elements, such as TATA box, initiator, CAAT box, or GC box in this region. Sequence analysis identified several GC boxes but did not find initiator sequence, TATA or CAAT boxes in this region (Fig. 2BGo). Multiple GC boxes are commonly found in many gene promoters. These GC boxes serve as binding sites for the transcription factor Sp1 (32). Taken together, these results demonstrate that the sequence associated with ECR2 works as an SRC-3 minimal promoter. Moreover, because this region does not contain any TATA box, CAAT box or initiator sequence, the four Sp1 sites straddling exon 1a most likely serve as essential active elements of the SRC-3 proximal promoter.

E2F1 Enhances SRC-3 Promoter Activity
Because our results demonstrate that the SRC-3 promoter region containing ECR2 plays a pivotal role in SRC-3 promoter activity, we analyzed its sequence and searched for potential regulatory elements involved in SRC-3 gene transcription. Recognition of transcription factor binding sites within evolutionary conserved sequences has been particularly useful in dissection of gene activation mechanisms (33, 34). Those regulatory elements conserved in human, mouse, and rat SRC-3 promoter regions are very likely to be functional elements important for SRC-3 expression. Interestingly, our analysis identified two conserved E2F binding sites (EBS), EBS1 and EBS2, in the ECR2 region.

Because E2F is overactivated in most of tumors due to deregulation of E2F/Rb pathway (35, 36, 37) and SRC-3 is overexpressed in cancer cells (1, 36, 37), we were particularly interested in determining whether E2F transcription factors are involved in the regulation of SRC-3 promoter activity. To investigate the role of E2F in SRC-3 promoter activation, we cotransfected MCF-7 cells with E2F1 and an SRC-3(–250/+350) luciferase reporter carrying EBS1 and EBS2 in the ECR2 region. In MCF-7 cells, E2F1 significantly enhanced the reporter activity in a dose-dependent manner; at the highest E2F1 dosage examined, E2F1 stimulated an 18-fold increase in the SRC-3(–250/+350) reporter activity (Fig. 3AGo). We also obtained similar results from transfected T47D and Hela cells (data not shown). These findings indicate that E2F1 can play a decisive role in activation of the SRC-3 promoter in multiple cell types.


Figure 3
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  		Fig. 3. Activation of SRC-3 Promoter Activity by E2F1

A, E2F1 stimulates transcriptional activity of the SRC-3(–250/+350) reporter. This reporter contains two E2F binding sites, EBS1 and EBS2 (open circles), and four Sp1 binding sites (open boxes). The black box represents exon 1a. MCF-7 cells were transfected with 0.5-µg reporter DNA and without (–) or with increasing amounts of E2F1 plasmids (0.01, 0.2, and 0.5 µg). Luciferase counts were normalized to cotransfected ß-galactosidase activity. Values are presented as relative luciferase activity with that of the reporter alone as 1. **, P < 0.01. B, E2F1 is recruited to the SRC-3 endogenous promoter. ChIP assay was performed in MCF-7 cells, by precipitating endogenous E2F1-DNA complex with an antibody specific to E2F1 or normal IgG as a negative control. Precipitated DNA was analyzed by quantitative real-time PCR using SRC-3 promoter-specific primers or primers that amplify albumin promoter as a negative control. Normal IgG was used to substitute specific antibody to serve as a negative control. Input material represents 2% of the total chromatin used for immunoprecipitation. **, P < 0.01.

 
We next performed chromatin immunoprecipitation (ChIP) assays to address whether E2F1 is directly recruited to the promoter region of the endogenous SRC-3 gene in MCF-7 cells. Indeed, our quantitative analysis showed that antibody specific to E2F1, but not the nonimmune IgG control, successfully coimmunoprecipitated E2F1 and the SRC-3 promoter region (+60/+180) (Fig. 3BGo), indicating that E2F1 is recruited to the endogenous SRC-3 promoter under physiological conditions. To prove that the E2F1 recruitment is specific for the SRC-3 promoter, we also analyzed E2F1 binding to the non-E2F1 target gene albumin, as a negative control. As shown in Fig. 3BGo, E2F1 is not recruited to the albumin gene promoter, supporting that our ChIP assay for E2F1 recruitment to the SRC-3 promoter is valid. Taken together, the dose-dependent activation of the SRC-3 promoter by E2F1 reflects a direct regulatory role of E2F1 in SRC-3 transcription.

E2F-Binding Motifs Are Not Required for Activation of SRC-3 Promoter by E2F1
To test whether E2F1 activates the SRC-3 promoter by directly binding to the EBS1 and EBS2 sites, we mutated the two E2F recognition motifs separately or in combination by using the SRC-3(–250/+350) reporter as a parent construct (Fig. 4Go) and examined the E2F1-dependent activation of these mutated SRC-3 promoters by measuring the luciferase reporter activity. In the absence of E2F1 transfection, both the single EBS mutation and the double EBS1 and EBS2 mutations did not obviously alter the basal activities of these reporters. After E2F1 transfection, the transcriptional activities of all of the reporters including SRC-3(–250/+350) and its EBS1, EBS2, and EBS1–2 mutants were significantly induced at a similar extent (Fig. 4Go, lower left panel). However, the fold induction over the basal activity in all the reporters was not significantly different from the parent SRC-3 (–250/+350) construct (Fig. 4Go, lower right panel). Because E2F1 did not stimulate the luciferase activity of the pGL3 basic vector that has no SRC-3 promoter sequence (Fig. 4Go), the E2F1-enhanced activity of these wild-type or mutant reporters was mediated by the SRC-3 promoter region. Taken together, these results suggest that direct binding to DNA motifs is not likely to be the major mechanism by which E2F1 boosts SRC-3 promoter activity. Instead, E2F may regulate the SRC-3 promoter through an indirect protein recruitment mechanism.


Figure 4
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  		Fig. 4. Mutational Analysis of E2F Binding Sites

Point mutations for EBS1, EBS2, or both of them were introduced into the SRC-3(–250/+350) promoter-reporter construct as shown and their activities were assayed by transfection. Hela cells were transfected with 0.5 µg of reporter DNA alone (open bars) or together with 0.25 µg of E2F1 (black bars). Luciferase counts were normalized to coexpressed ß-galactosidase activity. In the right panel, values from the same experiment are expressed as fold induction over the reporter activity in the absence of E2F1, with that of the empty vector taken as 1. Open circles represent E2F binding elements EBS1 or EBS2; crossed circles represent mutated EBS1 or EBS2; open boxes represent Sp1 binding sites.

 
E2F1 Activates SRC-3 Promoter through an Interaction with Sp1
Because E2F factors can physically interact with different transcription factors such as Sp1 (38, 39), YY1 (40), TFE3 (41), and different transcription factors may be required for the E2F activity (42), we hypothesized that E2F1 activates SRC-3 promoter by cooperating with Sp1, which may directly bind to its DNA elements located in the regulatory region of the SRC-3 promoter. To dissect more precisely the region containing functional regulatory cis elements for E2F1 action on the SRC-3 promoter, we further mapped the E2F1-responsive sequence in the SRC-3(–250/+350) promoter by using serial deletion mutants in transfection assays (Fig. 5AGo). A 100-bp deletion from the 3' terminus of ECR2 [SRC-3(–250/+250)] caused an increase in both the basal reporter activity and its activation in presence of E2F1, possibly due to the presence of some unknown repressor sequences in the 100-bp removed fragment (Fig. 5AGo, lower left panel). However this deletion did not affect the reporter activity stimulated by E2F1 (Fig. 5AGo, lower right panel). Additional removal of the entire exon 1a [SRC-3(–250/+1)], containing a Sp1 binding site and EBS2, significantly reduced the E2F1-stimulated reporter activity more than 8-fold (Fig. 5AGo). Further deletion of the most promoter region [SRC-3(–250/–100)] maintained the insensitivity of the reporter to E2F1 transfection (Fig. 5AGo). These results suggest that the region from +1 to +250 bp, which contains EBS2 and a Sp1 binding site, is essential for mediating E2F1 activity.


Figure 5
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  		Fig. 5. Identification of the Sequence Responsible for E2F1-Mediated Activation of the SRC-3 Promoter

A, Serial deletion mutants and a Sp1 deletion mutant were made by using the SRC-3(–250/+350) luciferase promoter-reporter construct as shown. The crossed box indicates the deleted Sp1 binding site (upper panel). The transcriptional activities of these constructs were measured by transfection using 0.5 µg of reporter DNA and 0.25 µg of E2F1 plasmids. Luciferase activity was assessed 48 h after transfection and was normalized to the coexpressed ß-galactosidase activity. Values are expressed as bars indicating the reporter activity in absence (open bars) or presence (black bars) of E2F1 transfection (left panel), and as fold induction over the reporter activity in the absence of E2F1 transfection (right panel). B, Detection of E2F1 and SRC-3 associated with the Sp1 binding site. 293 Cells were transfected with wild-type or mutant constructs as indicated. ChIP assays were performed with antibodies specific to Sp1, E2F1, or SRC-3 and a pair of PCR primers specific to all three promoter-reporter constructs. Bands intensities were quantified, and averages over different experiments are represented in the bar graph. Normal IgG was used as a negative control for immunoprecipitation. Input material represents 2.5% of the total chromatin used for immunoprecipitation. *, P < 0.05; **, P < 0.01. C, ChIP-ReChIP assay was performed in MCF-7 cells. Chromatin was immunoprecipitated sequentially with E2F1 and Sp1 antibodies. The SRC-3 promoter DNA bound to E2F1 and Sp1 was analyzed by PCR. One of the three similar experiments is shown as gel image. Relative band intensity is presented in the bar graph. Input material represents 2% of the total chromatin used.

 
Because we have demonstrated that the EBS2 mutation (Fig. 4Go) or its deletion (data not shown) does not affect SRC-3 promoter responsiveness to E2F1, we investigated whether the mapped Sp1 binding site is required for E2F1 activity on the SRC-3 promoter. We made the SRC-3(–250/+350)-Sp1-del reporter carrying an exact deletion of the Sp1 site in the SRC-3 promoter and performed a transfection assay. Although deletion of this Sp1 binding site did not significantly modify the reporter activity in the absence of E2F1 transfection, this deletion completely diminished the promoter activity stimulated by E2F1 transfection (Fig. 5AGo), indicating that this specific Sp1 site is essential for SRC-3 promoter activation by E2F1. Therefore, E2F1 may enhance SRC-3 promoter activity through interacting with Sp1 at the GC box (Sp1 binding site) located downstream exon 1a rather than directly binding to the E2F sites located in the promoter region.

To understand how E2F is recruited to the SRC-3 promoter, we performed ChIP assays using transfected 293 cells containing SRC-3(–250/+350) reporter or the same reporter with mutated EBS1 and EBS2 sites or deleted Sp1 site (Fig. 5BGo). The 293 cell line was used because the conditions for ChIP assay was previously described in this cell line (43). To achieve specific detection of the transfected DNA, PCR was executed with a forward primer upstream EBS2 and a reverse primer in the luciferase sequence. We performed three independent experiments with repeats in each experiment and presented the results of ChIP assays as bar graphs, where the bars indicate the mean values of gel bands intensities and error bars show SD (Fig. 5BGo). These analyses showed that Sp1 is associated with the wild-type SRC-3(–250/+350) promoter and with the SRC-3(–250/+350)-EBSs-mut promoter carrying EBS1 and EBS2 mutations (Fig. 5BGo). In contrast, Sp1 is not associated with the SRC-3(–250/+350)-Sp1-del promoter lacking the Sp1 site (Fig. 5BGo). Because we hypothesized that E2F1 activates SRC-3 promoter by interacting with Sp1, we investigated whether E2F1 was also recruited to the Sp1 site. Indeed, E2F1 was associated with the wild-type SRC-3(–250/+350) promoter as well as the mutant SRC-3(–250/+350)-EBSs-mut promoters but did not interact with the SRC-3(–250/+350)-Sp1-del promoter. These results demonstrate that the Sp1 binding site is required for E2F1 binding to SRC-3 promoter.

Recently, it has been shown that SRC-3 physically interacts with E2F1 and works as a strong coactivator to enhance the expression of E2F1 target genes (12). For this reason, we were interested in understanding whether SRC-3 was also recruited to the SRC-3 promoter with E2F1. Our analysis revealed that SRC-3 was bound to the wild-type SRC-3(+250/–350) promoter and the SRC-3(–250/+350)-EBS1–2-mut construct with mutated E2F binding sites. However, SRC-3 was not recruited to SRC-3(–250/+350)-Sp1-del construct with the deletion of the Sp1 binding site (Fig. 5BGo). These results suggest that both E2F1 and SRC-3 are recruited to the SRC-3 promoter, and their recruitments are dependent on the Sp1 binding site required for the SRC-3 promoter activity induced by E2F1.

To demonstrate that E2F1 is recruited to the SRC-3 promoter through an in vivo interaction with Sp1, we performed ChIP-ReChIP assays. Cross-linked and fragmented chromatin was prepared from MCF-7 cells and sequentially subjected to the first step ChIP with E2F1 antibody and the second step IP with Sp1 antibody. As shown in Fig. 5CGo, the two-step ChIP-ReChIP successfully precipitated the SRC-3 promoter, indicating that E2F1 and Sp1 form a protein complex at the SRC-3 promoter. Taken together, these results demonstrate that E2F1 controls SRC-3 promoter activity by interacting with Sp1, which provides a molecular mechanism to understand why the Sp1 site downstream exon 1a is essential but the EBS1 and EBS2 sites are not required for the E2F1-stimulated SRC-3(–250/+350) promoter activity.

E2F1 Enhances the Endogenous SRC-3 Gene Expression
Considering a potential role of E2F1 in the regulation of an endogenous SRC-3 promoter in cells, we determined whether E2F1 could stimulate the SRC-3 promoter and increase SRC-3 gene expression in vivo. To address this question, we took advantage of mouse embryonic fibroblasts (MEFs) derived from SRC-3 knockout (KO) mice. In these animals, the SRC-3 gene has been genetically modified by removing the sequence from exon 2 to exon 13 and substituting it with the lacZ sequence, coding for the ß-galactosidase. The LacZ sequence is fused with the SRC-3 gene sequence coding the 10th amino acid residue within exon 2 (13). Expression of the ß-galactosidase in SRC-3 mutant mice is controlled by the intact endogenous SRC-3 promoter, which has made this mouse line extremely useful for measuring SRC-3 promoter activity and SRC-3 expression patterns in different types of tissues and cells (13, 14, 25). Because the ß-galactosidase expression in SRC-3 KO MEFs is regulated by the endogenous SRC-3 promoter, SRC-3 expression can be monitored by measuring ß-galactosidase activity in SRC-3 KO MEFs. Because we were particularly interested in understanding whether E2F1 overactivation can boost SRC-3 expression, we transfected SRC-3 KO MEFs with increasing dosages of E2F1 and measured the E2F1-induced ß-galactosidase activity. As shown in Fig. 6AGo, the activity of the endogenous SRC-3 promoter grows in an E2F1 dose-dependent fashion and increases almost 4-fold with the highest E2F1 concentration. These results indicate that E2F1 stimulates SRC-3 expression in vivo.


Figure 6
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  		Fig. 6. E2F1 and SRC-3 Enhance SRC-3 Expression

A, E2F1 stimulates SRC-3 transcription in vivo. SRC-3 KO MEFs were transfected with empty vector (–) or with 0.02, 0.2, or 0.5 µg of E2F1 expression plasmids. The endogenous SRC-3 promoter-directed expression of the knock-in ß-galactosidase activity was measured 48 h after transfection and normalized to luciferase counts of the cotransfected pGL3 control vector. The results are expressed as relative ß-galactosidase activity, with that of the empty vector transfection taken as 1. **, P < 0.01. B, SRC-3 protein levels in Hela cells transfected with empty vector (–) or with 2 or 3 µg of E2F1 expression vector were analyzed by immunoblotting. ß-Actin was assayed as loading controls. Bar graph indicates the intensity of SRC-3 bands normalized on ß-actin. Where bars represent the mean value and error bars indicate the SD for three independent experiments. *, P < 0.05. C, E2F1 down-regulation results in lower SRC-3 protein levels. Hela cells were transfected with 20 or 40 nmol of E2F1-specific siRNA or 40 nmol of control siRNA. The bar graph represents E2F1 (black bars) or SRC-3 (open bars) band intensity, normalized to ß-actin of three independent experiments. *, P < 0.05; **, P < 0.01. D, SRC-3 coactivates E2F1 to stimulate its own expression. SRC-3–/– MEFs were transfected with parent empty vectors (–), with 0.25 µg of SRC-3 or 0.25 µg of E2F1 expression plasmids alone, or with 0.25 µg of SRC-3 and 0.25 µg of E2F1 plasmids. ß-galactosidase activity was measured 48 h after transfection and normalized to luciferase counts of the cotransfected pGL3 control plasmids. The results are presented as relative ß-galactosidase activity, with that of the empty vector control taken as 1. *, P < 0.05 calculated for cells transfected with E2F1 vs. cells transfected with both E2F1 and SRC-3.

 
Next, we further examined whether SRC-3 expression stimulated by E2F1 causes an increase in cellular SRC-3 protein in Hela cells. As shown in Fig. 6BGo, overexpression of E2F1 significantly elevated SRC-3 protein levels in a dose-dependent manner, whereas it has no effect on the level of ß-actin in Hela cells. These results indicate that E2F1 enhances not only the SRC-3 promoter activity but also the SRC-3 protein levels.

To confirm that SRC-3 expression is specifically controlled by E2F1, we knocked down endogenous E2F1 by small interfering RNA (siRNA) and determined the down-regulation effect on SRC-3 protein levels. Figure 6CGo shows that the transfection of two different dosage of siRNA was effective and caused a dose-dependent decrease of E2F1 levels, with the highest dosage lowering E2F1 more than 80% of the siRNA control. In parallel to E2F1, SRC-3 protein amount significantly decreased in a dose-dependent fashion in the presence of E2F1 siRNA but not in the presence of control siRNA, indicating that SRC-3 protein drop is specifically linked to E2F1 reduction. In conclusion, these experiments demonstrate that SRC-3 expression is regulated by E2F1.

Because SRC-3 works as an E2F1 coactivator (12) and SRC-3 is recruited with E2F1 to its own promoter (Fig. 5BGo), we reasoned that, if E2F1 enhances SRC-3 expression, the higher levels of SRC-3 coactivator will further potentiate E2F1 activity, and therefore this positive feedback regulatory loop will continuously boost SRC-3 transcription. For this reason, we decided to verify whether SRC-3 overexpression sustains E2F1 activity in vivo. We transfected SRC-3 KO MEFs with E2F1 expression plasmid, alone or together with SRC-3 expression plasmid and we analyzed SRC-3 expression by measuring the ß-galactosidase activity. Our experiments revealed that coexpression of both E2F1 and SRC-3 can induce a higher ß-galactosidase activity compared with the expression of E2F1 alone, whereas expression of SRC-3 alone was unable to significantly enhance SRC-3 promoter activity, despite the fact that lower levels of E2F1 are present in MEFs (Fig. 6DGo). These observations suggest that the effect of E2F1 and SRC-3 is additive only when both are present at high cellular concentrations or activities. In summary, our results suggest that activation of E2F1 enhances SRC-3 expression and protein levels; the resultant higher amounts of SRC-3 coactivator then can further coactivate E2F1 to sustain SRC-3 expression, forming a positive feedback regulatory loop that maintains a high level of cellular SRC-3 concentration when E2F1 is hyperactive.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Despite numerous evidence indicating that SRC-3 is a cancer-overexpressed oncogene (1, 12, 17, 18, 23, 24, 25, 29, 44), the molecular mechanisms of its transcriptional regulation remained unknown. The present study demonstrates that there are three ECRs located in the 5' regulatory sequence of the SRC-3 gene with ECR2 responsible for the major activity of the proximal SRC-3 promoter. The ECR2 transcriptional activity is mediated by a Sp1 site that specifically recruits Sp1, E2F1, and SRC-3. E2F1, by working through Sp1, not only stimulates the ECR2 promoter-reporter activity but also significantly increases the activity of the endogenous SRC-3 promoter and the levels of SRC-3 protein in vivo. In turn, elevated SRC-3 can further promote its own expression by serving as a coactivator for active E2F1. Our results suggest that E2F1 regulates SRC-3 expression through interacting with Sp1 and SRC-3. E2F1 enhances its own transcriptional capability by increasing SRC-3 expression. Therefore, in those cancer cells with hyperactive E2F and with impaired apoptosis pathways, there can be a dangerous positive regulatory loop consisting of active E2F and high levels of SRC-3 to drive cell proliferation and tumorigenesis. These findings provide molecular mechanisms, at least in part, for understanding the oncogenic role of SRC-3 mRNA overproduction.

It has been suggested that most sequences regulating gene expression are evolutionarily conserved among species, in particular among species like human, mouse and rat at moderate phylogenetic distances with roughly 70 to 100 million years of divergence (45). In the 5' regulatory sequence of the SRC-3 gene, all three ECRs are longer than 100 bp, consistent with a previous study showing that regulatory elements are typically composed of conserved long sequences (46). Our data suggest that ECR2 functions as an SRC-3 promoter. This promoter is TATA less, but it contains several Sp1 binding sites and two E2F consensus sequences, a feature reminiscent of many known E2F-responsive genes (47).

Several lines of evidence indicate that E2F1 regulates the expression of the SRC-3 gene. First, the SRC-3 promoter-reporter is activated by E2F1 in a dose-dependent manner; second, endogenous E2F1 is associated with the SRC-3 promoter; third, overexpression of E2F1 enhances the endogenous SRC-3 promoter activity and increases SRC-3 protein in vivo; and fourth, specific E2F1 down-regulation lowers SRC-3 endogenous protein levels. However, the mechanism by which E2F1 controls SRC-3 expression appears not to be direct. In fact, E2F1 does not work through its consensus binding sequences located in the proximal promoter region because mutation of all E2F1 binding elements in this region does not affect the E2F1-induced promoter activity of this region. Instead, the Sp1 binding site at bp +150/+160 is absolutely required for SRC-3 promoter activation by E2F1. Our data show that this Sp1 binding site is necessary for the recruitment of Sp1, E2F1, and SRC-3 to the SRC-3 reporter. In vivo, E2F1 binds to Sp1 on the endogenous SRC-3 promoter. Therefore, E2F1 stimulates SRC-3 expression by interacting with Sp1 and modulating its activity. This result is consistent with previous findings showing that E2F1 can directly interact with Sp1 and that this interaction is necessary for activation of certain gene promoters (38, 39). Interestingly, the histone deacetylase 1 (HDAC1), a transcriptional repressor, can associate with Sp1 in vivo. E2F1 and HDAC1 compete each other for binding to the C-terminal region of Sp1 (48). Therefore, it is possible that activated E2F1 binds to Sp1 and displaces HDAC1, leading to an activation of the SRC-3 promoter. Other studies also have revealed that the interaction of E2F1 with other transcription factors represents one of the mechanisms to control transcriptional specificity of target genes (49, 50). These studies showed that the binding of both E2F1 and its interactive transcription factor to their respective DNA elements can synergistically activate their target gene transcription, and failure of recruitment of either one of the factors impairs gene activation. However, our experiments demonstrated that the E2F1 binding site is not required for recruitment of E2F1 to the SRC-3 promoter and for the activation of the SRC-3 promoter by E2F1 in this study; a Sp1 binding site is essential and sufficient for mediating the SRC-3 promoter response to E2F1. These observations enable us to propose that the Sp1 element alone is sufficient to determine virtual full sensitivity of the SRC-3 promoter to E2F1.

The finding that SRC-3 expression is regulated by E2F has an important implication under pathological conditions such as cancer. It is generally accepted that most transcriptional coactivators have limiting concentrations under normal physiological conditions (51). In response to extracellular stimuli, normal cells can regulate their gene expression levels through changing their coactivator concentrations and activities by posttranslational modifications including phosphorylation, ubiquitinization, and inhibition of non-ubiquitin-dependent or ubiquitin-dependent proteasome turnover, rather than by an increase in gene transcription (26, 52, 53, 54). In tumor cells, many oncoproteins or factors involved in the carcinogenic process including E2F1, HER2, and the SRC-3 coactivator are either overproduced or hyperactive. The E2F transcription factors are key regulators of cell cycle, differentiation, apoptosis, and DNA damage response. Because normal apoptosis and differentiation pathways are likely impaired during oncogenesis, E2F activities for cell proliferation are positively correlated with oncogenesis in a large number of distinct human tumors (36, 37). Our findings suggest that SRC-3 overexpression in a subset of cancers may be caused by E2F overactivation in these cancers. This notion is further supported by other studies showing that SRC-3 overexpression in breast cancers correlates with the expression of HER2 that activates E2F transcriptional activity (16, 36, 55). Moreover, it has been reported that tamoxifen-treated human breast tumors with high levels of HER2 and SRC-3 develop earlier tamoxifen resistance, and the patients with these types of tumors have lower rates of disease-free survival compared with patients with tumors containing lower levels of either HER2 or SRC-3 or both of these two proteins (18). This clinical observation has been partially explained by the finding that HER2 activates SRC-3 by phosphorylation, resulting in tumor growth and tamoxifen resistance (56). Here, our results may add an additional interpretation to this clinical observation by reasoning that HER2-activated E2F1 can further promote cell proliferation by increasing SRC-3 expression.

Several studies have recently used microarray technologies to reveal the pattern of E2F regulated genes in normal and cancer cells. Muller et al. (57) generated the U2OS osteosarcoma cell lines that express an E2F1 fusion protein linked to the estrogen receptor ligand-binding domain (E2F1-ERLBD). Using tamoxifen to activate transcriptional activity of the E2F1 fusion protein, the authors assessed tamoxifen-inducible gene expression changes and validated the system with a number of known direct E2F1 target genes such as cyclin E and c-Myb. Intriguingly, in this system SRC-3 expression was found to be down-regulated by the tamoxifen-bound E2F1-ERLBD (57), which is opposite to our results. Although it is unclear what exactly causes this discrepancy, it is possible that the E2F1-ER could not be recruited to the Sp1-binding site on the SRC-3 promoter due to an impaired interaction with Sp1. If the overexpressed E2F1-ER could still interact with SRC-3, it would compete with the endogenous wild-type E2F1 for the SRC-3 coactivator and decrease the transcriptional activity of the endogenous SRC-3 promoter. In addition, other groups have analyzed E2F1 target genes in mouse embryonic fibroblasts (58, 59, 60) or cells derived from NIH3T3 fibroblasts (61), and they found that even after E2F1 expression SRC-3 expression was too low to be reliably measured (61). This is expected because, as mentioned before, SRC-3 expression levels are very low and are largely regulated by posttranslational modifications rather than novel synthesis in untransformed cells.

SRC-3 was initially characterized as a coactivator for steroid receptors, and its amplification and overexpression were first found in tumors derived from steroid hormone-responsive tissues, such as breast, ovary, uterus, and prostate. However, SRC-3 overexpression also was recently observed in steroid hormone-independent tumors, such as gastric and liver cancers (21, 22). Our results showing E2F1 enhances SRC-3 expression and SRC-3 potentiates E2F1 transcriptional activity suggest that SRC-3 has a general role in cell growth, proliferation, and tumorigenesis in addition to its function for steroid receptors, supporting the notion that SRC-3 promotes tumorigenesis not only for steroid hormone-promoted cancers but also for steroid hormone-independent cancers.

Although the mechanisms for SRC-3 as an oncogenic protein are still not fully understood, recent studies have shown that over produced SRC-3 can enhance the transcriptional activity of ER{alpha} in the absence of estrogen or presence of tamoxifen (56), increase estrogen-induced cyclin D1 expression (62), stimulate AKT activity (63), promote E2F1 activity (12), transform fibroblasts in culture (53), and potentiate IGF-I signaling pathway and mammary epithelial proliferation (23). On the other hand, inactivation of SRC-3 decreases the levels of insulin receptor substrates and AKT activity, and suppresses cell proliferation and oncogene and carcinogen-induced mammary tumorigenesis (24, 25, 44). These findings indicate that multiple pathways and gene networks are involved in the process of SRC-3 overexpression-mediated oncogenesis. Therefore, the positive feedback regulatory loop formed by E2F and its coactivator, SRC-3, may play an important role in maintenance of both high concentrations of SRC-3 and high activities of E2F; the overexpressed SRC-3 can subsequently activate other pathways described above to stimulate oncogenesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Bioinformatics
Sequence alignment and searching of expressed sequence tag databases were performed using the National Center for Biotechnology Information services (www.ncbi.nih.gov). Sequence GC content was analyzed using EMBOSS CpG Plot Program (http://www.ebi.ac.uk/emboss/cpgplot/). Potential transcription factor binding elements in the promoter region were searched using Genomatix MatInspector Program (www.genomatix.de). Alignment of human, mouse, and rat SRC-3 sequences and identification of the evolutionary conserved regions (ECRs) were performed using ECR Browser (64) (http://ecrbrowser.dcode.org/). Searching for transcription factor binding elements in the species-conserved sequences was executed using rVista 2.0 Program (http://rvista.dcode.org/).

RT-PCR
Total RNA was extracted from T47D and MCF-7 breast cancer cells using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations. RNA was subjected to deoxyribonuclease I treatment and reverse transcription using SuperScript II enzyme (Invitrogen). SRC-3 cDNA fragments were amplified by PCR using the following primers: 5'-TTTAAAGCTGAGCTGCGAG (P1), 5'-TGTCCTGGAGTATCACATGC (P2) and 5'-CATACCAGAAACGTCCAC (P3).

Plasmid Construction
The 5' regulatory sequence of the SRC-3 gene was amplified by PCR using LA PCR kit 2.1 (Takara Mirus Bio, Madison, WI) from the RP11-456N23 human BAC clone containing the 5' portion of the SRC-3 gene (GenBank accession no. AL353777) using the primers 5'-ATTGGGGTACCTCTCTCTTCTGCTCTCTCTTTC and 5'-TAACCGCTCGAGTCCTCCAATCACCTTCTCAACAAC. The PCR product was digested with KpnI and XhoI restriction enzymes and cloned into the pGL3 basic vector (Promega, Madison, WI) to make the SRC-3(–8300/+1400) luciferase reporter. The 5' serial deletions were obtained by PCR using a reverse primer 5'-TAACCGCTCGAGTCCTCCAATCACCTTCTCAACAAC and the following forward primers: 5'-AAGACTCCAGGCTAGATGTTGTG for SRC-3(–750/+1400); and 5'-ATGGGGGTACCAAACTGCTTGTCTTGTACCTGCC for SRC-3(+800/+1400). These PCR products were digested with KpnI and XhoI or EcoRI and XhoI, in the case of SRC-3(–750/+1400), and cloned into the pGL3 basic vector.

The SRC-3(–250/+350) promoter sequence corresponding to evolutionarily conserved region 2 (ECR2) of the 5' SRC-3 regulatory sequence was obtained by BamH1 digestion of the SRC-3(–750/+1400) PCR product and also cloned into the pGL3 basic vector. Mutations of the E2F binding sites were introduced using QuikChange II XL Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). Point mutations of the E2F binding sites were designed according to previous studies (65, 66). More specifically, EBS1 and EBS2 were mutated, respectively, into GATCGAAA and GATAGGAA. Progressive 3' deletion mutants in the SRC-3(–250/+350) promoter region were generated by PCR using the forward primer 5'-CTAGCAAAATAGGCTGTCCC and the following reverse primers: 5'-CCCCCGCTCGAGTTGGCAGATCTGAAGCCGCT for SRC-3(–250/+250); 5'-TGTCCGCTCGAGGAGACAGCAGCAGCAGCTAAAGT for SRC-3(–250/+1); and 5'-CCTCCGCTCGAGGGCCTAGGGTTCCAGCGAC for SRC-3(–250/–100). The PCR products were subcloned into KpnI/XhoI sites of the pGL3 basic vector. The mutant with deletion of the Sp1 binding site (SRC-3(–250/+350)-Sp1-del) was generated by two rounds of PCR. Initially two partial and overlapping products were produced using the 5'-CTAGCAAAATAGGCTGTCCC primer with the 5'-GCCCCTCTTCCCTCCGCCTCGCCCGCCACC primer and the 5'-AGGAGGAGGCGGTGGCGGGCGAGGCGGAGG primer with the 5'-CTTTATGTTTTTGGCGTCTTCCA primer. In the second round, the two PCR products with partial overlapping sequence obtained from the first round of PCR were alleled to serve as templates for PCR using the two terminal primers. The final product also was subcloned into KpnI/XhoI sites of the pGL3 basic vector.

Cell Culture and Transient Transfections
MCF-7 cells and Hela cells were cultured in DMEM containing 10% fetal calf serum. Transient transfections were performed with Fugene 6 (Roche, Basel, Switzerland) according to manufacturer’s protocol. The 2 x 105 cells were transfected with 0.5 µg of reporter plasmids together with 0.1 µg of pRSV-ß-galactosidase as an internal control, in the presence or absence of 0.25 µg (or different dosage wherever indicated) of PCR3.1-E2F1 plasmid. Total amount of DNA was matched among experimental groups by using parent plasmid DNA. Cells were harvested 48 h after transfection and luciferase activity, ß-galactosidase activity and protein concentration were measured. Luciferase counts were always normalized to ß-galactosidase activity.

Immortalized SRC-3 KO MEFs were developed from SRC-3 null embryos according to protocols described previously and cultured in DMEM containing 10% fetal calf serum (13, 24). MEFs were transfected with PCR3.1-E2F1 plasmids by using Fugene 6. The ß-galactosidase activity was analyzed 48 h after transfection. To set a control, cells were cotransfected with 0.03 µg of pGL3 basic vector. Values shown in figures represent ß-galactosidase activity normalized to luciferase counts. The values presented in all figures are the averages of three different experiments and SDs are shown as error bars.

ChIP Assay and ChIP-ReChIP
MCF-7 cells were grown until 70–80% confluence. The proteins bound to DNA in cells were cross-linked using 1% formaldehyde for 10 min. Cells were incubated 5 min with 0.125 M glycine to stop the reaction and washed in PBS twice before been harvested. Cells were lysed in 5 mM PIPES (pH 8.0), 85 mM KCl, 0.5% Nonidet P40, and nuclei were recovered by centrifugation. Nuclear membranes were destroyed with 1% sodium dodecyl sulfate (SDS) in 50 mM Tris-HCl (pH 8.0). Chromatin was sonicated to obtain fragments with lengths between 200 and 1000 bp. After immunoclearing with salmon sperm DNA-protein A agarose (Upstate, Lake Placid, NY) and IgG, the protein-DNA complexes were immunoprecipitated with 5 µg of E2F1 antibody (sc-193x; Santa Cruz Biotechnology, Santa Cruz, CA) or IgG as a negative control. After reversal of cross-links by heating at 65 C for 4 h, the samples were treated with proteinase K and DNA was recovered by phenol/chloroform extraction followed by ethanol precipitation. The association of E2F1 with SRC-3 promoter was analyzed by quantitative real-time PCR using the SYBR mix (Applied Biosystems, Foster City, CA) and the following primers: 5'-GCGAGTTTCCGATTTAAAGC and 5'-GCCTTGGCAGATCTGAAG. To prove the specificity of E2F1 binding, its recruitment to albumin, a non E2F regulated gene was analyzed as a negative control, using the following primers: 5'-TGGGGTTGACAGAAGAGAAAAGC and 5'-TACATTGACAAGGTCTTGTGGAG, as described previously (50).

For ChIP assays using transfected 293 cells, cells were transfected with 10 µg of SRC-3(–250/+350), SRC-3(–250/+350)-EBS1–2-mut or SRC-3(–250/+350)-Sp1-del luciferase constructs using Lipofectamin 2000 reagent (Invitrogen). Six hours after the transfection, cells were cross-linked and lysed for ChIP assays as described above. Chromatin-protein complexes were immunoprecipitated using Sp1 (sc-59x; Santa Cruz Biotechnology), E2F1 or SRC-3 antibody (sc-7216x; Santa Cruz Biotechnology) or IgG as negative control. Purified DNA was analyzed by a pair of specific primers, 5'-GCGAGTTTCCGATTTAAAGC (complementary to the 5' SRC-3 promoter sequence) and 5'-CTTTATGTTTTTGGCGTCTTCCA (complimentary to the 5' luciferase sequence). Band intensity was measured with Scion (Frederick, MD) Image software, and data were expressed as a bar graph, where bars indicate the mean value of the band intensity of at least three independent experiments and error bars show the SD.

ChIP-ReChIP was performed with MCF-7 cells. After cells were fixed as described above, cells were extracted with solution A containing 10 mM HEPES (pH 7.9), 0.25% Triton X-100, 10 mM EDTA, and 0.5 mM EGTA, and then with solution B containing 10 mM HEPES (pH 7.9), 200 mM NaCl, 1 mM EDTA, and 0.5 mM EGTA. Cell pellets were resuspended in solution C containing 1 mM EDTA, 150 mM NaCl, 50 mM HEPES (pH 7.5), 0.1% SDS, 1% Triton X-100, and 0.1% sodium deoxycholate. After sonication, chromatin was precleared using salmon sperm DNA-protein A/G agarose and IgG and incubated overnight with 5 µg of E2F1 antibody or IgG as negative control. After several washings, the beads were incubated with 50 µl of 0.5% SDS and 0.1 M NaHCO3 for 10 min at 65 C. The supernatant was collected after spinning, diluted with 1 mM EDTA, 150 mM NaCl, 50 mM HEPES (pH 7.5), 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate, and incubated with 3 µg of the Sp1 antibody overnight. After washing, Protein-DNA complexes were eluted from beads and treated with proteinase K overnight. DNA was purified with QIAquik PCR purification kit (QIAGEN) and the binding to the SRC-3 endogenous promoter was quantified by PCR using the primers described above. The experiment was repeated three independent times with similar results and a significant gel image is shown. Gel bands intensity was quantified with Scion Image software, and values of this representative experiment were represented by bar graph.

RNA Interference and Western Blot Analysis
To analyze SRC-3 content, Hela cells were transfected using lipofectamine 2000 reagent (Invitrogen) with 2 and 3 µg of pCR3.1 E2F1 expression plasmids or 3 µg of pCR3.1 empty vector as control. Twenty-four hours after the transfection, cells were lysed in 50 mM Tris-HCl (pH 7.4), 0.2% SDS, and proteinase inhibitors.

E2F1 siRNA and control siRNA were purchased from Dharmacon, Lafayette, CO (SMARTpool reagent). Twenty or 40 nmol of E2F1 siRNA and 40 nmol of control siRNA were transfected into Hela cells using TransIT-TKO reagent (Mirus), following the manufacturer’s protocol. Cells were harvested 3 d after transfection and proteins were prepared as before. Fifty-microgram proteins in cell extracts were loaded on a 7.5% acrylamide gel. E2F1 and SRC-3 protein were detected using the E2F1 antibody or a specific SRC-3 antibody described previously (53). As loading control, ß-actin content was analyzed using a specific antibody (AC-74; Sigma, St. Louis, MO). The bands intensity was measured with Scion Image software and the values of three independent experiments, normalized on each ß-actin control were presented by a bar graph.

Statistical Analysis
All experiments were performed at least three independent times. Data were analyzed using unpaired Student’s t test for comparison of two groups or one-way ANOVA for comparison of more than two groups. Statistical results are indicated in each figure by asterisks, where * corresponds to P value < 0.05 (significant) and ** P < 0.01 (very significant).


   ACKNOWLEDGMENTS
 
We thank the Sanger Institute (Hinxton, Cambridge, UK) for providing the BAC clone used in this study, Dr. Ray-Chang Wu for SRC-3 antibody, Ms. Lan Liao for preparing SRC-3 KO MEFs, and Drs. Jiemin Wong, Dennis Dowhan, Qingtian Li, Xiaotao Li, and Sang Jun Han (Baylor College of Medicine) for discussion and technical assistance.


   FOOTNOTES
 
This work is partially supported by grants from the National Institutes of Health (to J.X. and to B.W.O., respectively) and by an American Cancer Society Scholar Award (to J.X.).

Author Disclosure Summary: P.M., C.Y., B.W.O., and J.X. have nothing to declare.

First Published Online August 17, 2006

Abbreviations: ChIP, Chromatin immunoprecipitation; EBS, E2F binding sites; ECR, evolutionarily conserved region; ER, estrogen receptor; HDAC1, histone deacetylase 1; KO, knockout; MEF, mouse embryonic fibroblast; SDS, sodium dodecyl sulfate; siRNA, small interfering RNA; Sp1, specificity protein 1; SRC-3, steroid receptor coactivator-3.

Received for publication December 19, 2005. Accepted for publication August 8, 2006.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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