Role of Chromatin Remodeling Complex BAF in Immunity and Lymphoma

项目摘要/摘要 为了保持染色质的紧凑结构并在需要时确保访问和功能， 真核基因组利用多单位染色质重塑复合体，如BAF，实现动态结合 转录因子对DNA的影响。在基因组调控中发挥如此重要的作用，BAF-Complex就不足为奇了 在癌症中，基因最常受到体细胞突变的影响，在所有患者中有20%，在23%的患者中 弥漫性大B细胞淋巴瘤(DLBCL)。然而，BAF促进恶性的机制 转化和淋巴增生症尚不清楚。根据我们的初步分析，BAF综合体似乎是一个 生发中心B细胞的重要调节因子，DLBCL细胞的起源。我们假设BAF能使染色质 生发中心B细胞分化相关因子的可及性及阻止活化B细胞 保持快速骑行的致瘤状态。为了研究潜在的机制，我们将(目标1)定义 BAF在正常体液免疫反应中的生物学作用和作用机制。为此，我们将 利用基因组学中的计算和实验方法确定BAF-复合体组成 原代生发中心B细胞染色质可及性的基因组结合和BAF依赖性变化 在淋巴瘤患者中发现的BAF突变失活。此外，我们还将确定曝气生物滤池在 一种新的计算方法在生发中心细胞中的核小体移动和定位。梅尔尼克家族 实验室发现，核结构的调节在生发中心B细胞生物学中起着关键作用 而核拓扑学中涉及的因素的扰动会导致淋巴瘤。但是，这些因素，比如 作为粘附素复合体，在淋巴瘤中很少发生突变。通过研究原子核拓扑结构的变化 与BAF-复合体的结合相关，我们将测试这种差异是否可以通过BAF突变来解释 可能会执行这些建筑功能。此外，我们将(目标2)确定BAF在 通过对染色质可塑性的影响，淋巴瘤和其他肿瘤的起始和克隆性进化。我们 BAF复合体通过整体诱导核小体移动和暴露发挥作用的假说 转录因子基序。一旦BAF亚基发生突变，核小体的一般流动性 如果核小体丢失，核小体可能优先锁定在不利于染色质的位置。这可能会导致 恶性程序的随机激活。为了解决这个问题，我们计划扩展我们的计算 用公开的方法模拟受BAF突变影响的癌症中染色质硬度的变化 可用的数据。此外，我们将建立一个简单易用的平行单细胞转录组和染色质 可及性分析，并将其应用于我们的BAF突变小鼠模型的淋巴瘤肿瘤。总而言之， 拟议的项目将为BAF介导的淋巴瘤形成机制提供深入的见解。以防我们的 研究结果支持BAF是B细胞肿瘤抑制的主要调节因子的假说，我们将 能够为BAF突变患者确定新的治疗靶点，并进一步对这些肿瘤进行分类。

PROJECT SUMMARY/ABSTRACT In order to maintain the compact structure of chromatin yet ensure access and functionality when required, eukaryotic genomes utilize multiunit chromatin remodeling complexes such as BAF, to enable dynamic binding of transcription factors to DNA. Being so instrumental in genome regulation, it is not surprising that BAF-complex genes are the most frequently affected by somatic mutations in cancer, in 20% of all patients and in 23% of diffuse large B cell lymphoma (DLBCL). However, the mechanism by which BAF promotes malignant transformation and lymphomagenesis is unclear. Based on our initial analysis, the BAF complex seems to be an important regulator of germinal center B cells, DLBCL cell-of-origin. We hypothesize that BAF enables chromatin accessibility for factors involved in germinal center B cell differentiation and prevents activated B cells from staying in the tumorigenic state of rapid cycling. To investigate the underlying mechanism, we will (Aim 1) define the biological role and mechanism of action of BAF in the normal humoral immune response. To this aim, we will use computational and experimental methods in genomics to determine BAF-complex composition, BAF genomic binding and BAF-dependent changes in chromatin accessibility in primary germinal center B cells with inactivating BAF mutations found in lymphoma patients. Furthermore, we will determine the role of BAF in nucleosome mobility and positioning in germinal center cells using a novel computational approach. The Melnick lab has discovered that regulation of nuclear architecture plays a critical role in germinal center B cell biology and that perturbation of factors involved in nuclear topology leads to lymphoma. However, these factors, such as the cohesin complex, are rarely mutated in lymphoma. By investigating changes in nuclear topology associated with binding of the BAF-complex, we will test if this discrepancy is explained by BAF mutations that might carry out these architectural functions. Furthermore, we will (Aim 2) determine the role of BAF in the initiation and clonal evolution of lymphoma and other tumors through effects on chromatin plasticity. We hypothesize that BAF complex exerts its function by globally inducing nucleosome mobility and exposing transcription factor motifs. Once a mutation in a BAF subunit occurs and the general fluidity of nucleosomes is lost, nucleosomes might be preferentially locked in an unfavorable chromatin position. This might lead to stochastic activation of malignant programs. To address this question, we plan to expand our computational approach to model changes in chromatin stiffness within cancers affected by BAF mutations using publicly available data. Furthermore, we will establish a simple-to-use parallel single-cell transcriptome and chromatin accessibility assay and apply it to lymphoma tumors from our BAF mutant mouse models. Taken together, the proposed project will provide insights into the mechanism of BAF-mediated formation of lymphoma. In case our findings support the hypothesis of BAF being the master regulator of tumor suppression in B cells, we will be able to identify novel therapeutic targets for patients with BAF mutations and further classify those tumors.