UNIVERSITY OF MIAMI
Estrogen receptor-a (ER-a, hereon in, ER represents ER-a) and progesterone receptor (PR) are ligand-induced transcription factors. Accessory proteins, termed coactivators modulate the transcriptional activation of these factors. Coactivators in general are thought to enhance transcription of target genes but recent evidence suggests that they may selectively regulate subsets of receptor target genes. Coactivators form multiprotein complexes that integrate a number of different enzymatic activities that are essential for the process of gene regulation. We have identified WBP-2 as a selective coactivator of ER and PR. WBP-2 is a proline-rich protein that contains three PPXY motifs. The PPXY motif mediates interaction with WW-domain containing proteins and the PPXY motif-WW-domain (PPXY-WW) complex promotes transcriptional stimulation in a number of gene contexts. PPXY-WW modules have been increasingly recognized for their relevance to gene regulation in the last decade, many new roles of the PPXY-WW complex in transcription and other cellular processes are just emerging. WW-domains have been observed on several key regulatory proteins including yes associated protein (YAP1)and WW-domain-containing oxidoreductase (WWOX1). YAP1 has been shown to act as transcriptional coactivator for several transcription factors. We have shown that YAP1, which physically associates with WBP-2, also modulates ER and PR functions. The interaction between PPXY motif and WW-domain is highly specific suggesting that the PPXY-WW complex is a specialized coactivator complex that regulates transactivation functions of a subset of transcription factors. In addition to transcription stimulation, PPXY motif containing proteins have also been shown to suppress transcription when they interact with certain WW-domain containing proteins. We observed that overexpression of the WW-domain containing protein, WWOX1 significantly reduced the coactivation function of WBP-2, moreover, WWOX1 also suppressed the WBP-2-YAP1-mediated transcriptional activities of ER and PR. Based on these observations we hypothesized that the coactivation functions of the oncoprotein YAP1 are dependent on WBP-2 and that the WBP-2:YAP1 complex enhances ER and PR transactivation function to promote breast tumorigenesis. Whereas, WWOX1 acts as a tumor suppressor by physically blocking the formation of the WBP-2:YAP1 complex resulting in the attenuation of its coactivation and oncogenic functions. The overall goal of this award is to comprehensively analyze the coactivation functions of WBP-2:YAP1 complex in an unbiased manner. To achieve this goal, we have purchased a Roche LightCycler 480. Purchase of this equipment have enhanced and expedited our research efforts. Using this equipment, we have shown that WBP-2:YAP1 complex regulates selective estrogen target genes. Our data also suggest that WBP-2:YAP1 complex regulates target gene expression by recruiting p300 to the target gene promoters which leads to chromatin modification. Characterizing the mechanism of action of coactivator proteins and coactivator complexes will provide a prime source for the discovery of new molecular events in the regulation of transcription and their role in physiological and pathological cellular processes. We have identified WBP-2, YAP1 and WWOX1 as key regulators of ER and PR transactivation functions, but the precise mechanisms by which these proteins modulate hormone receptor action remain unknown. WW-domains and PPXY motif containing proteins have been implicated in many diseases, including hormone regulated cancers such as breast cancers but the exact molecular mechanism by which they regulate transcription, cell function and growth are largely unknown. Thus, accomplishing the goal of this proposal will not only provide new insights into the mechanism of action of WBP-2, YAP1 and WWOX1 on ER and PR signaling but also in identifying their novel roles in the pathogenesis of human breast cancer.