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

基本信息

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
项目类别：
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

项目摘要

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（ESC）的特性。 ESC必须保持一个只有在收到适当的刺激后，未分化但中毒状态才开始差异化和承诺，引起了组成组织的潜水细胞类型和组织。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。但是，这些转录因子的结合又高度依赖于染色质景观的可及性。要访问基本信息，Hisstone蛋白必须重新定位或去除，这是由ATP依赖性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多能。 ESBAF由定义亚基-BRG1，BAF155，ARID1A，BAF57，BAF47和六其他的。最近，这种适用含溴群的蛋白9（BRD9），但不包括ARID1A和BAF47。那远，对ESBAF的研究通过执行BRG1或BAF155的删除或敲低实验来完成，这两个都是存在于含BRD9的BAF复合物或BRD9-BAF中。那，brd9-baf在ESC中的特定功能生物学仍然完全出乎意料。确实，我们发现BRD9的抑制会导致ESC自我丧失更新。该提议假设BRD9-BAF独特的复杂构图使其具有独特功能和定位对于ESC中转录程序的特定调节至关重要。这该项目的具体目的是1。确定BRD9-BAF的复杂组成和表征其体外ATPase和染色质重塑活性和2。 BRD9-BAF在ESC中的基因组定位和功能中的BRD9。构图和生化 BRD9-BAF的活性将使用蛋白质组学和体外ATPase和染色质重塑定义测定。通过评估ES细胞生长和使用小分子和遗传扰动丧失BRD9功能的分化潜力。此外，将使用以下内容建立BRD9-BAF的基因组定位和活性全基因组实验：染色质免疫沉淀（芯片） - 塞克，RNA-Seq和转座酶 - 测定法可访问的染色质（ATAC）-Seq。这些研究将确立BRD9在ESC生物学中的作用，并帮助我们了解BAF复合物异质性如何有助于ESC转录的精确控制程序。