Role of BAP1/ASXL3 complex in transcriptional regulation and development - Project Summary Recent advances have shown that dysfunctions prevalent in epigenetic factors play an important role in the developing human diseases, including developmental disorders and cancers. Therefore, understanding the roles of these epigenetic factors may aid in identifying new epigenetic prognostic markers or targetable biomarkers that could contribute towards the establishment of novel therapeutics. Histone H2A lysine 119 ubiquitination (H2AK119ub) is one of the most functional histone marks that plays an essential role in establishing repressive chromatin domains and mediating Polycomb induced transcriptional repression during development. The evolutionarily conserved H2AK119ub histone modification is mainly catalyzed by the Polycomb repressive complex 1 (PRC1) and deubiquitinated by Polycomb repressive-deubiquitinase (PR-DUB) complex (also known as the BAP1 complex in mammals) that antagonizes PRC1’s function. In mammalian cells, the BAP1 complex functions as a multi-protein complex, containing as many as ten different subunits, which are responsible for the chromatin recruitment, protein stability, and enzymatic activity of BAP1. Mutations and dysregulations within subunits in the BAP1 complex are found in patients with developmental diseases, neuronal disorders, and cancer. Therefore, it is critical to understand the molecular basis of how genes are turned on/off by the BAP1/PRC1 epigenetic balance. Our previous studies have characterized a functional epigenetic axis comprised of both BAP1 and Bromodomain and Extraterminal (BET)-containing protein 4 (BRD4), which are physically bridged together by the scaffold protein ASXL3—the largest subunit within the BAP1 complex. Genetic depletion of the linker, ASXL3, dramatically attenuates the establishment of the BAP1/ASXL3/BRD4 epigenetic axis machinery at active enhancers, leading towards a significant reduction in the enhancer-nearby gene expression. In our current studies, we have employed biochemical, molecular, and small-molecule screening approaches to mechanistically understand how the ASXL3/BAP1/BRD4 epigenetic axis regulates transcription and determines cell fate and differentiation ability. Our first goal for this study is to uncover the BAP1/ASXL3 sub-complex’s role at active enhancers and assess the impact of the BAP1/ASXL3/BRD4 epigenetic axis on enhancer activity, chromatin structure, and gene expression. As a major H2AK119 deubiquitinase, the BAP1 complex functions as a general transcriptional activator, antagonizing PRC1’s function and is involved in PRC1-dependent transcriptional regulation. Therefore, our second project is to elucidate the relationship between BAP1 and a distinct PRC1 sub-complex in regulating H2AK119ub levels by utilizing our newly developed BAP1-specific small-molecule inhibitor. Mutations within ASXL3 have been demonstrated to be driver mutations in multiple neuronal diseases. Therefore, we will define the role of ASXL3 in mediating ESC differentiation into neuronal progenitor cells (NPC), and then investigate how ASXL3 and its associated epigenetic factors determine the transcriptome landscape during developmental processes.
