Functional Characterization of m-Bop, a Transcriptional Repressor Essential for Heart Development

Dedication To my parents, family, and Ashley for their undying support and encouragement. v Acknowledgements There are many friends and co-workers who contributed to the completion of this work. I would like to thank the Gottlieb laboratory members and neighboring labs for their advice and reagents. I am very grateful to Dr. Bose (and laboratory), who was gracious enough to treat me as one of his own. Sean O'Malley isolated three additional skNAC clones while screening the yeast two-hybrid skeletal muscle library. Li Zhu performed excellent work expressing and purifying recombinant m-Bop as well as performing m-Bop in vitro interaction assays. Elizabeth Weihe was our resident staining expert and was truly the driving force behind the immunofluorescence staining presented in this study. I would like to especially thank June Harriss for her countless aid and instruction throughout my stay. In addition to becoming a close friend, Andrew Liss' contribution to my progress cannot be measured. My parents' strong support paved my way to success. Ashley Brown helped me in countless ways and continuously makes me a better person. Finally, I would like to thank Paul Gottlieb for his advice and support during my tenure in his lab. He convinced me that this project had the potential to be something special; once again, he was right. The Bop gene encodes MYND and SET domain-containing proteins that are expressed in cytotoxic T lymphocytes (t-Bop) and in skeletal and cardiac muscle (m-Bop). MYND and SET domains are found in numerous transcriptional regulators and chromatin remodeling proteins. The MYND domain is believed to function as a protein-protein interaction motif, while the SET domain was recently demonstrated to participate in core histone covalent modification. Previous studies in mice demonstrated that Bop is required for proper cardiac development, specifically the right ventricle. Additionally, targeted inactivation of the Bop gene altered the expression of chamber-specific transcription factors. While much has been learned from prior experimentation, the molecular function of Bop has not been previously described. In these studies, Bop is shown to function as a histone deacetylase-dependent transcriptional repressor. When tethered to the GAL4-DNA binding domain, m-Bop and t-Bop repressed the expression of a luciferase reporter in a dose-dependent fashion. The addition of trichostatin A, a potent inhibitor of histone deacetylase (HDAC) activity, significantly reduced the vii repression potential of m-Bop. Coimmunoprecipitation experiments identified that m-Bop physically associates with class I and class II HDACs in cell …