ESTROGEN RECEPTOR INTERACTION WITH DNA

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The estrogen receptor is an important component in development, differentiation and growth. It is thought to play a role in the development of some breast cancers. Elucidation of the mechanism of estrogen regulation of transcription is important in understanding the cause of these cancers and in the design of effective treatments. An initial step in estrogen regulation of transcription is the binding of the receptor to a specific DNA sequence called an estrogen responsive element (ERE). A consensus ERE has been derived which is a perfect palindromic sequence. This consensus ERE has been shown to confer specific estrogen responsiveness. The interaction of the hormone-activated receptor with the ERE influences the activity of RNA polymerase II by an unknown mechanism. The objectives of this project are to determine the mechanism by which hormone activated-estrogen receptor recognizes EREs which contain deviations from the consensus ERE and the mechanism by which the receptor activates RNA polymerase II. There is evidence which suggests that components, that is, flanking sequences and proteins in addition to the estrogen receptor are required for imperfect EREs to be functional in vivo. The hypothesis will be tested that these components allow imperfect EREs to function in vivo by increasing the binding affinity of the estrogen receptor for the ERE. The imperfect ERE and flanking sequences from the upstream region of the rat luteinizing hormone B gene will be analyzed in either transient transfection experiments into HeLa cells or gel mobility shift assays. The binding of the receptor to the ERE does not appear to be sufficient for signal transduction. There is evidence which suggests that steroid receptors appear to compete for common limiting transcription factors which are not components of the basal transcription initiation machinery. The hypothesis will be tested that proteins which specifically interact with the hormone activated-estrogen receptor aid the receptor in influencing RNA polymerase 11 activity. These estrogen receptor-binding proteins will be identified by either gel mobility shift assays or immunoprecipitation with an isolated epitope tagged N-terminal domain of the estrogen receptor or by use of an estrogen receptor affinity column. The function of these proteins will be analyzed in either "transcriptional interference" experiments or steroid receptor-dependent in vitro transcription.