Estrogens play crucial roles in regulating gene expression in physiological and disease states. Estrogens acts through estrogen receptors (ERs) and their binding sites in genomic DNA to modulate transcription by RNA polymerase II. Although recent gene-specific and genomic analyses have provided considerable information about of estrogen-dependent transcription, many aspects of the estrogen signaling network have not yet been elucidated. The goal of my studies was to uncover new information about the immediate and direct effects of estrogen signaling at the cell membrane, in the cytoplasm, and in the nucleus to elucidate the underlying regulatory networks. First, I investigated an ER transcriptional coregulators, SWI/SNF, an ATPdependent chromatin remodeling complex. I explored the molecular functions of the BAF57 and BAF180 subunits of SWI/SNF using a quantitative proteomic approach called SILAC (Stable Isotope Labeling by Amino Acids in Cell Culture). I found that depletion of BAF57 results in a significant depletion of BAF180 from the SWI/SNF complex without decreasing the total cellular BAF180 levels, resulting in an accumulation of cells in the G2/M phase. Knockdown of BAF57 also causes transcriptional misregulation of cell cycle-related genes involved in the late G2 checkpoint. Collectively, these studies have elucidated the role of BAF57 and BAF180 in the transcriptional control of cell proliferation. Second, I have used GRO-Seq (Global Nuclear Run-On and Massively Parallel Sequencing) to explore the immediate effects of estrogen signaling on the transcriptome of breast cancer cells. I found that estrogen directly regulates a strikingly large fraction of the transcriptome in a rapid, robust, and unexpectedly transient manner. In addition to protein coding genes, estrogen regulates the distribution and activity of all three RNA polymerases, and virtually every class of non-coding RNA that has been described to date. I also identified a large number of previously undetected estrogen-regulated intergenic transcripts, many of which are found proximal to ER[alpha] binding sites. These results provide the most comprehensive measurement of the primary and immediate estrogen effects to date. I expect that genome-wide inferences based on the direct estrogen-regulated transcriptome in combination with estrogen-regulated signaling pathway will be useful for understanding estrogen biology. Advisors/Committee Members: Kraus, William Lee (chair), Collins, Ruth N. (committee member), Lis, John T (committee member).

Prostate cancer relies on androgens for growth and survival. Androgens elicit their function primarily through the androgen receptor (AR), a transcription factor that is required for the development and progression of prostate cancer. Therefore, current treatment options for disseminated disease are aimed at either inhibiting androgen synthesis or preventing activation of the receptor. These treatment options are initially effective; however, recurrent disease ultimately arises within two to three years that typically present as therapy resistant. From this, it is obvious that more effective means of targeting this pathway are necessary to treat prostate cancer. The present study exploited a specific aspect of the AR activation pathway and uncovered a novel means for potentially targeting AR action and subsequent prostate cancer proliferation. The SWI/SNF chromatin remodeling complex is one such necessary component involved in mediating AR activation. Specifically, the BRM core ATPase was demonstrated to exhibit a preferential response to activating AR target genes which was dependent on promoter context. Subsequent studies identified the BAF57 subunit of the SWI/SNF complex as the primary interacting AR subunit, thus indicating a possible role in complex recruitment to AR target DNA. BAF57 proved to be critical for AR mediated transcriptional activation, AR enhanced coactivator activation, and prostate cancer proliferation. Based on these findings, the potential for targeting BAF57 as a prostate cancer therapeutic option was examined. First, expression of BAF57 was verified in human prostate tissue specimens, with possible enhanced expression observed in metastatic samples. Subsequent molecular analyses determined that the primary region of interaction occurred between the DBD/hinge region of AR and the proline-rich/HMG domain of BAF57. The N-terminal binding region of BAF57 was then shown to reduce AR recruitment to AR target genes, inhibit AR target gene activity, and decrease prostate cancer cell proliferation. Together, the data herein establish the SWI/SNF chromatin remodeling complex, specifically BAF57, as a valid target for the treatment of prostate cancer. Advisors/Committee Members: Knudsen, Dr. Karen (Advisor).