"""""""" The goal is to understand the molecular basis of the estrogen response. The expression of estrogen responsive genes is controlled in part by a hormone dependent transcription factor, the estrogen receptor (ER). Estradiol ligand induces a dimerization of the receptor, allowing it to bind a DNA sequence called the estrogen responsive element (ERE), and thereby to stimulate the transcription machinery. Previously we defined the DNA sequence and helical structure requirements that allow a DNA segment to act as an ERE. We also showed that with appropriate stereo-alignment, several ER-ERE complexes can synergize to stimulate gene expression to a much greater level than expected from additive interaction. Recently, it was found that many tissues express two subtypes of the ER, called alpha and beta. They display differences in structure that produce differences in their characteristics as transcription factors. The amino-terminal region of ERa has a transcription activation function (AF-1), that we recently showed was essential for the ERa to display synergy. The corresponding region in ERD may not have this capacity, but may be a transcription repressor. We propose use of domain substitution and mutagenesis to define the roles of the AF-1 region in each receptor subtype. Co-synthesis of the two subtypes in native tissue allows them to compete for binding EREs. We will analyze interaction of the ER subtypes with EREs having natural variations from the consensus sequence, to determine whether the ER subtypes display ERE specificity. Induction of gene expression by each receptor subtype will be tested with variant EREs using reporter genes in cell culture. We predict that estrogen versus antiestrogen ligands will produce unique conformations in each receptor subtype, leading to changes in ERE binding capacity and induction ability. Cosynthesis of subtypes also allows the formation of ERa/b heterodimer. Analysis of its biological activity is complicated by the presence of alpha and, b/a homodimers. To allow determination of the functions of a/b we have used a genetic fusion of the two subunit cDNAs that produces an ER as a single polypeptide. The fusion protein displays expected biochemical and biological activity. We are making the fusion heterodimer which will be analyzed for DNA binding and reporter gene activation in the absence of homodimers. Overall, the plan is to determine how the ERa and ERb subtypes interact with natural EREs, various ligands, and with each other to regulate gene expression.""""""""
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