Genomic Structure of the Promoters of the Human Estrogen Receptor-α Gene Demonstrate Changes in Chromatin Structure Induced by AP2γ

Abstract

Expression of human estrogen receptor-␣ (ER␣) involves the activity from several promoters that give rise to alternate untranslated 5 exons. However, the genomic locations of the alternate 5 exons have not been reported previously. We have developed a contig map of the human ER␣ gene that includes all of the known alternate 5 exons. By using S1 nuclease and 5-rapid amplification of cDNA ends, the cap sites for the alternate ER␣ transcripts E and H were identified. DNase I-hypersensitive sites specific to ER␣-positive cells were associated with each of the cap sites. A DNase I-hypersensitive site, HS1, was localized to binding sites for AP2 in the untranslated region of exon 1 and was invariably present in the chromatin structure of ER␣-positive cells. Overexpression of AP2␥ in human mammary epithelial cells generated the HS1-hypersensitive site. The ER␣ promoter was induced by AP2␥ in mammary epithelial cells, and trans-activation was dependent upon the region of the promoter containing the HS1 site. These results demonstrate that AP2␥ trans-activates the ER␣ gene in hormone-responsive tumors by inducing changes in the chromatin structure of the ER␣ promoter. These data are further evidence for a critical role for AP2 in the oncogenesis of hormone-responsive breast cancers.There are at least two nuclear receptors for estrogen receptor, ER␣ 1 (1, 2) and ER␤ (3). Most breast cancers that occur in post-menopausal women overexpress ER␣ (4). Patients with breast cancers that express ER␣ are more likely to respond to hormonal therapy (4, 5) and have an improved prognosis compared with patients with ER␣-negative tumors (4, 6, 7). Studies of breast cancer cell lines (8) and primary tumors (9, 10) have indicated that transcription of the ER␣ gene plays an important role in regulating the expression of ER␣. Thus, understanding transcriptional regulation of the ER␣ gene will likely provide critical insights into the pathogenesis of hormone-responsive breast cancers.Transcription of the ER␣ gene is complex and involves activity of several distinct promoters (11-14). Functional promoter studies have concluded that ER␣ expression in breast cancer cell lines and various tissues is likely to involve transacting factors that have a specific cell or tissue distribution pattern (15-19). There appear to be a variety of factors that interact with the ER␣ promoter with trans-activating (15-17) or trans-repressing (20) functions. There is also evidence that ER␣ can autoregulate its own transcription (21,22). Other studies suggest that the lack of ER␣ expression in ER␣-negative breast cancer cell lines and tumors may be controlled by methylation of CpG islands in the 5Ј end of the ER␣ gene (23,24).The main ER␣ promoter, P1, initiates transcription at a cap site previously mapped at the start of exon 1 (1). Exon 1 has a 233-base 5Ј-untranslated region preceding the AUG codon that initiates translation of the ER␣ protein. Studies in ER␣-positive breast cancer cell lines have shown that transcription initiated at exon 1 accounts for 50 -9...

TFAP2C Controls Hormone Response in Breast Cancer Cells through Multiple Pathways of Estrogen Signaling

“…2A, ChIP analysis was done with anti-TFAP2C antibody, which pulled down the regulatory region of the human ERa promoter containing the AP2-binding site in the ERa-positive cell lines MCF7, ZR75-1, and T47-D cells but not in ERa-negative cell lines HCT116, HBL-100, and MDA-MB-231. These results are consistent with a model in which TFAP2C directly targets the AP2 sites in the ERa promoter and is consistent with earlier studies that identified an interaction between TFAP2C and the chromatin of the ERa promoter in the AP2 regulatory region (8).…”

“…We propose that TFAP2C regulates expression of ERa by directly targeting the ERa promoter at the AP2-regulatory region (8). ChIP was used to investigate the interaction between TFAP2C and the ERa promoter.…”

“…The TFAP2A and TFAP2C proteins are commonly expressed in breast cancer and overexpression of these factors can upregulate the cloned ERa promoter (7). Furthermore, overexpression of TFAP2A and TFAP2C can induce a hypersensitive site at the AP2 regulatory region of the ERa promoter in human mammary epithelial cells (8). However, several critical questions remain to be addressed.…”

Molecular and Cellular Determinants of Estrogen Receptor α Expression

“…ERF-1 has been shown to transactivate promoter A, whereas ERBF-1 is necessary for transcription from promoter B (defined in reference 71). Overexpression of ERF-1 in human mammary epithelial cells resulted in the generation of a DNase I-hypersensitive site in the untranslated region of exon 1 (54). Such sites are associated with regions of open chromatin conformations, thereby indicating that ERF-1 can not only transcriptionally regulate the ER gene but can also alter its chromatin structure (54).…”

“…Two transcription factors, ER factor 1 (ERF-1) and ER-B factor 1 (ERBF-1), which regulate ER expression, potentially in conjunction with or as an alternative to methylation in breast cancer cell lines and tissues, have been described previously (13,54,71). ERF-1 has been shown to transactivate promoter A, whereas ERBF-1 is necessary for transcription from promoter B (defined in reference 71).…”