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

项目摘要 自我更新和多能性是胚胎干细胞（ESC）的定义属性。ESC必须保持 一种未分化的，但平衡的状态，只有在收到适当的刺激时，它才开始 分化和定型，产生构成生物体的不同细胞类型和组织。 干细胞的身份在很大程度上取决于一个复杂的转录电路控制的主监管机构 Oct 4、Sox 2和Nanog。然而，这些转录因子的结合反过来又高度依赖于转录因子。 染色质景观的可及性。为了获得潜在的基因组信息，组蛋白必须 重新定位或移除，这是由ATP依赖性染色质重塑复合物执行的功能。 在ESC中，存在一种专门的BAF复合物esBAF，它对维持自我更新和 多能性。esBAF由定义亚基-BRG 1、BAF 155、ARID 1A、BAF 57、BAF 47和六个亚基组成。 他人最近，本申请人在ESC中发现了一种新的非典型BAF复合物，其含有 含溴结构域蛋白9（BRD 9），但不包括ARID 1A和BAF 47。到目前为止，关于esBAF的研究 已经通过进行BRG 1或BAF 155的缺失或敲低实验来完成，这两者也是 存在于含有BRD 9的BAF复合物或BRD 9-BAF中。因此，BRD 9-BAF在ESC中的具体功能 生物学仍然完全未被探索。事实上，我们发现BRD 9的抑制导致ESC自身的丧失， 退款该提案假设BRD 9-BAF独特的复杂组成使其具有独特的 功能和靶向对于ESC中转录程序的特异性调节至关重要。的 该项目的具体目标是1.鉴定BRD 9-BAF的复杂组成， 表征其体外ATP酶和染色质重塑活性和2.阐明 BRD 9在BRD 9-BAF的基因组定位和在ESCs中的功能。其组成和生化 将使用蛋白质组学和体外ATP酶和染色质重塑来定义BRD 9-BAF的活性 测定。BRD 9-BAF在ESC多能性中的作用将通过评估ES细胞生长和分化来阐明。 在BRD 9功能丧失后，使用小分子和遗传扰动两者，可以提高BRD 9的分化潜力。 此外，将使用以下方法确定BRD 9-BAF的基因组定位和活性 全基因组实验：染色质免疫沉淀（ChIP）-seq、RNA-seq和转座酶测定- 可溶性染色质（ATAC）-序列这些研究将确定BRD 9在ESC生物学中的作用，并有助于我们的研究。 了解BAF复合物的异质性如何有助于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.