Dissecting the Mechanism of Polycomb Eviction by the BAF Complex

项目摘要 在过去的十年中，数千个癌症样本的测序显示， mSWI/SNF（BAF）复合物在20%的癌症中发生突变。BAF中的这些突变如何有助于 肿瘤的发生机制还不清楚，部分原因是BAF亚单位的作用和功能还不清楚。 很好的描述。BAF复合物的组合多样性使研究进一步复杂化， 在细胞类型内和细胞类型之间。总之，这些因素使得很难定义机械的 尽管有大量的癌症基因组数据，但BAF突变对癌症的贡献仍然很大。我的研究 克拉布特里实验室将利用一种快速、可逆的分析方法，这种方法将化学诱导的邻近性和染色质结合起来， 免疫沉淀（ChIP）以诱导性地将BAF募集到活细胞中的特定基因组位点，并测量 直接下游效应。开发这种检测方法是研究BAF的重要一步 复杂的，因为以前的体外研究观察并不总是在活细胞中。使用 通过这项试验，我们的实验室发现BAF重塑染色质的一种机制是通过驱逐Polycomb， 抑制复合物（PRCs）。事实上，突变的BAF复合物， 或在恶性横纹肌样瘤（MRT）中发现的蛋白质不能从染色质中驱逐PRC。重要的是，MRT 在临床试验中，患者对PRC抑制剂有积极反应。因此，至少在MRT的情况下， 破坏BAF的PRC驱逐功能有助于可通过PRC抑制剂治疗的癌症状态。MRT为 一种罕见的癌症，其中100%的疾病病例在单个BAF亚基中包含无效突变。对于绝 在所有涉及BAF突变的癌症中，20%的大多数突变对特定的 癌症类型。在这个研究计划中，我将利用我们实验室的体内试验来研究染色质重塑， 生物化学和基因组学方法以及癌症突变数据，以（1）确定PRC所需的所有BAF亚基 对抗，特别是通过PRC驱逐;和（2）定义在癌症中发现的BAF亚基突变的范围 其产生不能从染色质中驱逐PRC的突变BAF复合物。这是一个重要的问题， 因为携带这种突变的患者可能会损害BAF介导的PRC驱逐， 对PRC抑制剂有积极反应。因此，这项研究的结果将立即确定潜在的新的 可以用PRC抑制剂治疗的癌症亚群。更广泛地说，理解BAF和 Polycomb将为治疗开发确定新的靶点。

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.