Functional characterization of m-Bop, a transcriptional repressor essential for heart development

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Sims, Robert Joseph

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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 repression potential of m-Bop. Coimmunoprecipitation experiments identified that m-Bop physically associates with class I and class II HDACs in cell culture. The nuclear receptor co-repressor N-CoR interacted with m-Bop in similar assays. Furthermore, m-Bop displayed the ability to associate with multiple components of the SIN3 repression complex in vivo. While SET domain-dependent histone methyltransferases (HMTases) have been recently identified, m-Bop did not demonstrate HMTase activity in vitro, suggesting that Bop may function as a ‘mute’ HMTase. Physical interaction between m-Bop and skNAC, a reported muscle-specific transcriptional activator, was detected in the yeast two-hybrid system and binding assays in vitro and in vivo. m-Bop and skNAC were induced and localized in a similar manner during skeletal myogenesis in vitro. The muscle-specific and MYND domains of m-Bop were required for skNAC association in mammalian cells. skNAC residues important for m-Bop interaction were located near a carboxy terminal proline-rich region. Computational analysis of Bop identified it as the charter member of a family of MYND and SET domain proteins found in a broad range of species including yeast, plants, worms, flies, mice, and humans.