Dissecting the Mechanism of Polycomb Eviction by the BAF Complex

项目总结 在过去的十年里，数以千计的癌症样本被测序，结果表明染色质重构体 MSWI/SNF(BAF)复合体在20%的癌症中发生突变。BAF中的这些突变是如何导致 对肿瘤发生的了解很少，部分原因是单个BAF亚单位的角色和功能不是 很好的特点。BAF复合体的组合多样性使研究进一步复杂化 在单元格类型内和跨单元格类型。这些因素加在一起，使得很难定义机械的 尽管癌症基因组数据丰富，但BAF突变对癌症的贡献。我在中国的研究 克拉布特里实验室将利用一种快速、可逆的检测方法，将化学诱导的邻近性和染色质结合起来 免疫沉淀法(CHIP)在活细胞中诱导将BAF募集到特定的基因组位点并测量 直接的下游影响。发展这种检测方法是研究生物滤池的重要一步。 体内的复杂性，因为以前的体外研究观察到的情况并不总是在活细胞中站得住脚。vbl.使用 在这项测试中，我们的实验室发现，BAF重塑染色质的一个机制是通过驱逐Polycomb 来自染色质的抑制复合体(PRC)。事实上，突变的生物滤池复合体是催化不活跃的 或在恶性横纹肌样肿瘤(MRT)中发现，它们不能将原癌细胞从染色质中驱逐出去。重要的是，捷运 在临床试验中，患者对PRC抑制剂的反应积极。因此，至少在MRT的情况下， 破坏BAF的PRC驱逐功能有助于形成一种可通过PRC抑制剂治疗的癌症状态。捷运是 一种罕见的癌症，其中100%的疾病病例在单个BAF亚单位中包含零突变。为广大民众 在涉及BAF突变的所有癌症的20%中，大多数突变都不是特定的 癌症类型。在这个研究计划中，我将利用我们实验室的体内实验来研究染色质重构体， 生化和基因组学方法，以及癌症突变数据，以(1)定义PRC所需的所有BAF亚基 反对，特别是通过PRC驱逐；和(2)定义在癌症中发现的BAF亚单位突变的范围 这会产生一个突变的BAF复合体，不能将PRC从染色质中驱逐出去。这是一个重要的问题 答案是因为携带这种破坏BAF介导的PRC驱逐的突变的患者可能会 对PRC抑制剂做出积极反应。因此，这项研究的结果将立即确定潜在的小说 可用PRC抑制剂治疗的癌症亚群。更广泛地说，理解BAF和BAF之间的关键对立 Polycomb将为治疗开发确定新的目标。

PROJECT SUMMARY Thousands of cancer samples sequenced over the past decade have revealed that the chromatin remodeler complex, mSWI/SNF (BAF), is mutated in 20% of all cancers. How these mutations in BAF contribute to tumorigenesis is poorly understood, in part because the roles and functions of individual BAF subunits are not well characterized. Studies are further complicated by the combinatorial diversity of the BAF complex both within and across cell types. Together, these factors have made it difficult to define the mechanistic contribution of BAF mutations to the cancer, despite the abundance of cancer genome data. My research in the Crabtree lab will utilize a rapid, reversible assay that combines chemical-induced proximity and chromatin immunoprecipitation (ChIP) to inducibly recruit BAF to a specific genomic locus in living cells and measure immediate downstream effects. Developing this assay has been a major step forward in studying the BAF complex in vivo, as observations from previous studies in vitro have not always held up in living cells. Using this assay, our lab discovered that one mechanism by which BAF remodels chromatin is by evicting Polycomb repressive complexes (PRCs) from chromatin. Indeed, mutated BAF complexes that are catalytically inactive or found in malignant rhabdoid tumors (MRTs) are unable to evict PRCs from chromatin. Importantly, MRT patients have responded positively to PRC inhibitors in clinical trials. Thus, at least in the case of MRTs, disrupting BAF's PRC eviction function contributes to a cancer state that is treatable by PRC inhibitors. MRT is a rare cancer in which 100% of disease cases contain a null mutation in a single BAF subunit. For the vast majority of the 20% of all cancers involving BAF mutations, mutations are rather nonspecific to a particular cancer type. In this research plan I will utilize our lab's in vivo assay for studying chromatin remodelers, biochemical and genomics methods, and cancer mutation data to (1) define all BAF subunits required for PRC opposition, especially via PRC eviction; and (2) define the range of BAF subunit mutations found in cancers which produce a mutant BAF complex that cannot evict PRC from chromatin. This is an important question to answer because patients carrying such mutations that impair BAF-mediated PRC eviction would potentially respond positively to PRC inhibitors. Thus, results from this study will immediately identify potential novel cancer subsets treatable with PRC inhibitors. More broadly, understanding the critical opposition between BAF and Polycomb will identify new targets for therapeutic development.