Role of BAP1/ASXL3 complex in transcriptional regulation and development

项目摘要 最近的进展表明，表观遗传因素中普遍存在的功能障碍在遗传过程中起着重要作用。 发展人类疾病，包括发育障碍和癌症。因此，了解角色 这些表观遗传因素可能有助于确定新的表观遗传预后标志物或靶向生物标志物 这可能有助于建立新的治疗方法。组蛋白H2 A赖氨酸119泛素化 （H2 AK 119 ub）是最具功能的组蛋白标志物之一，在建立抑制性免疫应答中起着重要作用。 染色质结构域和介导Polycomb诱导的转录抑制在发展过程中。的 进化上保守的H2 AK 119 ub组蛋白修饰主要由Polycomb抑制蛋白催化， 复合物1（PRC 1）并被Polycomb阻遏-去泛素化酶（PR-DUB）复合物（也称为 作为哺乳动物中的BAP 1复合物），其拮抗PRC 1的功能。在哺乳动物细胞中，BAP 1复合物 作为一种多蛋白复合物，含有多达10个不同的亚基，负责 BAP 1的染色质募集、蛋白质稳定性和酶活性。基因突变和失调 BAP 1复合物中的亚基存在于发育性疾病、神经元疾病和 癌因此，了解基因如何被基因启动/关闭的分子基础是至关重要的。 BAP 1/PRC 1表观遗传平衡。我们以前的研究已经描述了一个功能性表观遗传轴， BAP 1和Bromodomain和含外末端（BET）的蛋白4（BRD 4），它们在物理上是 由支架蛋白ASXL 3桥接在一起，ASXL 3是BAP 1复合物中最大的亚基。基因耗竭 接头ASXL 3的表达显著减弱了BAP 1/ASXL 3/BRD 4表观遗传轴的建立 在活性增强子处的机械，导致增强子附近基因表达的显著减少。 在我们目前的研究中，我们采用了生物化学、分子和小分子筛选方法， 机械地理解ASXL 3/BAP 1/BRD 4表观遗传轴如何调节转录， 细胞命运和分化能力。我们这项研究的第一个目标是揭示BAP 1/ASXL 3亚复合物的作用， 并评估BAP 1/ASXL 3/BRD 4表观遗传轴对增强子活性的影响， 染色质结构和基因表达。作为一种主要的H2 AK 119去泛素化酶，BAP 1复合物的功能是 一种通用的转录激活因子，拮抗PRC 1的功能，并参与PRC 1依赖的 转录调控因此，我们的第二个项目是阐明BAP 1与一个 通过利用我们新开发的BAP 1特异性的， 小分子抑制剂。已证明ASXL 3内的突变是多个细胞中的驱动突变。 神经元疾病因此，我们将确定ASXL 3在介导ESC分化为神经元细胞中的作用。 祖细胞（NPC），然后研究ASXL 3及其相关的表观遗传因子如何决定 在发育过程中的转录组景观。

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.