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

定义 BRD9-BAF 在胚胎干细胞多能性中的作用

• 批准号: 9542127
• 负责人: Jovylyn Gatchalian
• 金额: $ 5.87万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9542127
• 关键词: ATP phosphohydrolase; Address; Antibodies; Binding; Biochemical; Biological Assay; Bromodomain; Chromatin; Chromatin Remodeling Factor; Complex; Coupled; DNA; Dependence; Development; Differentiation and Growth; Digestion; Disease model; Electrophoretic Mobility Shift Assay; Embryo; Genes; Genetic; Genetic Transcription; Genomics; Heterogeneity; Histones; Immunofluorescence Immunologic; Immunoprecipitation; In Vitro; Inner Cell Mass; Investigation; Knowledge; Light; Maintenance; Malignant Neoplasms; Names; Nucleosomes; Organism; Pharmacology; Process; Property; Proteins; Proteomics; Regenerative Medicine; Research; Role; SMARCA4 gene; SMARCB1 gene; SMARCC1 gene; SMARCE1 gene; Sedimentation process; Stains; Stimulus; Testing; Thinness; Tissues; Transcriptional Regulation; Transposase; Undifferentiated; cell growth; cell type; chromatin immunoprecipitation; chromatin remodeling; density; embryonic stem cell; experimental study; genome-wide; in vitro activity; in vivo; knock-down; mutant; novel; pluripotency; programs; restriction enzyme; self-renewal; small molecule; stem cell biology; stem cells; transcription factor; transcriptome sequencing

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.

项目摘要 自我更新和多能性是定义胚胎干细胞的特性（ESC）。 当最佳开始procin过程的过程中 区分和承诺，引起构成有机体的各种细胞类型和组织。 干细胞身份在很大程度上由主调节器控制的复杂转录电路决定 oct4，sox2和nanog。 染色质景观的能力。 被报告或删除，通过ATP化力染色质重塑复合物执行的函数。 在ESC中，ESBAF是ESBAF的专业综合体，这对于维持自我更新至关重要 ESBAF由BRG1，BAF155，ARID1A，BAF57，BAF47和六 其他人。 含溴群的蛋白质9（BRD9），但排除了ARID1A和BAF47。 通过执行BRG1或BAF155的划定或敲低实验来完成，这两个都是 存在于BRD9的BAF复合物或BRD9-BAF中。 生物学仍然没有探索。 续约。 对ESC中转录程序的特定调节的功能和靶向 该项目的具体目的是1。确定BRD9-BAF的组成和 特征是体外ATPase和染色质重塑活性，2。 BRD9中的BRD9在ESC中的基因组定位和功能。 BRD9-BAF的活性将使用蛋白质组学和体外ATPase和染色质重塑定义 通过评估ES细胞生长和 使用小分子和遗传扰动丧失BRD9功能的差异潜力。 此外，将使用tollowing建立BRD9-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和转置的测定法 可访问的染色质（ATAC） - 这些研究将确立BRD9在ESC生物学中的作用 了解BAF复合物异质性如何有助于对ESC转录的精确控制 程序。