Defining the role of BRD9-BAF in embryonic stem cell pluripotency

项目总结 自我更新和多能性是胚胎干细胞(ESC)的特征。ESCS必须保持 无差别的，但镇定的状态只有当它收到适当的刺激时，它才开始 分化和承诺，产生组成有机体的不同细胞类型和组织。 干细胞的特性在很大程度上是由主调节器控制的复杂的转录电路决定的。 10月4日，Sox2和Nanog。然而，这些转录因子的结合反过来又高度依赖于 染色质景观的可访问性。为了获得潜在的基因组信息，组蛋白必须 被重新定位或移除，这一功能是由依赖于ATP的染色质重塑复合体执行的。 在ESCS中，存在着一个专门的BAF复合体，esBAF，它对于维持自我更新和 多能性。EsBAF由定义亚基BRG1、BAF155、ARID1A、BAF57、BAF47和6个亚基组成 其他。最近，这位申请人在ESCs中发现了一个新的、非规范的BAF复合体，它包含 含溴结构域蛋白9(BRD9)，但不包括ARID1A和BAF47。到目前为止，对esBAF的研究 已经通过执行BRG1或BAF155的缺失或敲除实验来完成，这两个实验也是 存在于含有BRD9的BAF复合体或BRD9-BAF中。因此，BRD9-BAF在ESC中的具体作用 生物学仍然完全没有被探索过。事实上，我们发现抑制BRD9会导致ESC自身的丧失 更新。该提议假设BRD9-BAF独特的复杂组成使其具有唯一性 功能和靶向对于ESCs中转录程序的特定调控至关重要。这个 本项目的具体目标是：1.确定BRD9-BAF的复杂成分，并 鉴定其体外ATPase和染色质重塑活性；2.阐明其在 BRD9中的BRD9-BAF在ESCs中的基因组定位和功能。组成和生化 BRD9-BAF的活性将通过蛋白质组学、体外ATPase和染色质重塑来确定 化验。BRD9-BAF在胚胎干细胞多能性中的作用将通过评估ES细胞的生长和 BRD9功能丧失时的分化潜力，使用小分子和遗传扰动。 此外，BRD9-BAF的基因组定位和活性将使用以下方法建立 全基因组实验：染色质免疫沉淀(CHIP)-SEQ、RNA-SEQ和转座酶-SEQ 可及染色质(ATAC)-以下这些研究将确立BRD9的S在胚胎干细胞生物学中的作用，并有助于我们的 了解BAF复合体异质性如何有助于ESC转录的精确控制 程序。

PROJECT SUMMARY Self-renewal and pluripotency are defining properties of an embryonic stem cell (ESC). ESCs must maintain an undifferentiated, but poised state when only upon receipt of the proper stimuli does it begin the process of differentiation and commitment, giving rise to the diverse cell types and tissues that make up an organism. Stem cell identity is largely dictated by an intricate transcriptional circuit controlled by the master regulators Oct4, Sox2 and Nanog. However, the binding of these transcriptional factors is in turn highly dependent on the accessibility of the chromatin landscape. To access the underlying genomic information, histone proteins must be repositioned or removed, a function that is performed by ATP-dependent chromatin remodeling complexes. In ESCs, a specialized BAF complex, esBAF exists, which is essential for maintaining self-renewal and pluripotency. esBAF consists of the defining subunits - BRG1, BAF155, ARID1A, BAF57, BAF47 and six others. Recently, this applicant discovered a novel, non-canonical BAF complex in ESCs that contains the Bromodomain-containing protein 9 (BRD9), but excludes ARID1A and BAF47. Thus far, studies on esBAF have been done by performing deletion or knockdown experiments of BRG1 or BAF155, both of which are also present in the BRD9-containing BAF complex, or BRD9-BAF. Thus, the specific function of BRD9-BAF in ESC biology remains completely unexplored. Indeed, we find that inhibition of BRD9 results in loss of ESC self renewal. This proposal hypothesizes that BRD9-BAF’s distinct complex composition gives it unique function and targeting critical for the specific regulation of transcriptional programs in ESCs. The specific aims of this project are 1. to identify the complex composition of BRD9-BAF and to characterize its in vitro ATPase and chromatin remodeling activities and 2. to elucidate the role of BRD9 in BRD9-BAF’s genomic localization and function in ESCs. The composition and biochemical activities of BRD9-BAF will be defined using proteomics and in vitro ATPase and chromatin remodeling assays. The role of BRD9-BAF in ESC pluripotency will be elucidated by assessing ES cell growth and differentiation potential upon loss of BRD9 function using both small molecule and genetic perturbations. Furthermore, the genomic localization and activity of BRD9-BAF will be established using the following genome-wide experiments: chromatin immunoprecipitation (ChIP)-seq, RNA-seq, and Assay for Transposase- Accessible Chromatin (ATAC)-seq. These studies will establish BRD9’s role in ESC biology and aid in our understanding of how BAF complex heterogeneity contributes to the precise control of the ESC transcription program.