Analysis of the estrogen responsive region of the inositol 1,4,5-trisphosphate receptor gene promoter
Chrzan, Brian Gary
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The molecular basis of estrogen action in tissues is still not completely understood. The regulation of estrogen-responsive genes is now known to be highly complex. The relative expression of estrogen receptor (ER) subtypes, transcription factors and the context of the gene promoter all contribute to the effect of various ligands on estrogen-responsive genes. Recent studies have highlighted this complexity, describing the development of selective estrogen receptor modulators (SERMs) and novel non-classical actions of estrogen receptors at promoters. For the development of therapies to treat estrogen-dependent tumors, osteoporosis, or to eliminate potential side effects of hormone replacement therapy, a complete understanding of all the mechanisms controlling estrogen-dependent gene regulation is required. This thesis investigated the effects of estrogen and phytoestrogens on classical estrogen response element (ERE) -dependent and non-classical ERE-independent transcriptional regulation. The results of these investigations indicate that gene expression regulated by phytoestrogens and estrogen are similar. Specifically, genistein and daidzein were shown to be significantly more potent in stimulating ERß-mediated transcription compared to ERα-mediated transcription, although both are less potent than 17ß-estradiol (E2). Comparison with E2-regulated gene expression in MCF7 mammary epithelial cells and E2-inhibition of stimulated-IL-6 secretion from G-292 osteosarcoma cells showed that both genistein and daidzein mimic these estrogenic activities. These results are consistent with the hypothesis that these dietary phytoestrogens may provide potential therapeutic effects for menopausal symptoms and osteoporosis. As part of this thesis, the ITPR1 gene promoter was analyzed to characterize the estrogen responsive region of the promoter. Previous studies had shown transcription of the type 1 ITPR gene is repressed in osteoblasts and osteosarcoma cells by 17ß-estradiol, despite the lack of consensus EREs in the promoter. Experiments were conducted to determine whether estrogen receptors mediate the estrogen-dependent regulation of ITPR1 expression and whether estrogen receptors and other regulatory factors assemble in transcriptional complexes on the ITPR1 promoter. The studies on G-292 cells and primary human osteoblasts indicated that activator protein 2α (AP-2α) and ERß1 are bound to the ITPR1 promoter in osteoblastic cells in situ and that ERß1 mediates E2-dependent transcriptional repression. In G-292 cells, overexpression of ERß1 potentiated the E2-mediated repression of ITPR1 promoter activity, whereas overexpression of ERα inhibited promoter activity. These actions appeared to be mediated by an AP-2 binding element in the ITPR1 proximal promoter. Following these observations, the MCF7 cell line was chosen for further analysis of the relationship between AP-2α and estrogen receptors on binding to and regulation of the ITPR1 promoter. In contrast to the E2-mediated repression observed in osteoblasts, E2 treatment of MCF7 cells activated transcription from the ITPR1 promoter. As in osteoblasts, the estrogen responsive region of the promoter was found to be the -141/-110 region. This promoter region lacks a consensus ERE but does contain an AP-2 site. Promoter affinity resin experiments and chromatin immunoprecipitation experiments demonstrated binding in situ of both AP-2α and ERα to the promoter in MCF7 cells. These results indicate that AP-2α and ERs bind to the ITPR1 promoter and that AP-2α and ER mediate E2-dependent transcriptional regulation, supporting the proposed hypothesis that AP-2α acts as a tether to link the ITPR1 promoter to ERs to regulate transcription. Furthermore, these studies demonstrate that cell-specific expression of estrogen receptor subtypes can result in significantly different expression of estrogen-responsive genes. This thesis represents the first reporting and analysis of the non-classical transcriptional regulation by AP-2α and estrogen receptors. It also highlights the significance of the differential expression of estrogen receptor subtypes and the influence of such differential expression on estrogen-responsive genes. Information gained through these studies will advance knowledge of the actions of estrogen in various target tissues.