Aim 1 addresses the role of estrogen response elements in determining gene activation. The hypothesis addresses whether DNA binding acts as an allosteric effector to direct ER conformation which in turn leads to recruitment of coactivators or corepressors. The role of ER affinity for particular EREs and the induction of different conformations by the ERE that alters ER interaction with coregulators will be investigated. The effect of increased ER binding to the ERE will be determined in relation to coregulator interaction and transactivation when bound to estradiol and 4-hydroxytamoxifen. ER domains involved in communicating specific ERE binding to coregulator interactions will be established.
Aim 2 makes use of ER bound to different imperfect EREs to determine the effect of exchanging EREs in native promoters to test coregulator recruitment, activity in cell-free transcription assays and in transiently and stably transfected cell lines.
In aim 3, a cell free system of deproteinized templates will be used to identify rate limiting ER coregulators. A chromatin assembly system is proposed to test the effects of DNA replication, nucleosome assembly and position, cell specific factors, and histone deacetylation.
In aim 4, stably transfected ER+ HepG2 cells will be used to test whether elongation rather than initiation is limiting in ER induction of vitellogenin gene expression. Chromatin immunoprecipitation assays will be performed to identify differences in the patterns of ER and HNF3a binding and histone acetylation when transcription of the estrogen inducible VIT and PI-9 promoters is weakly or strongly activated.
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