Role of Brg1 in Embryo Development and Pluripotency

Brg1 在胚胎发育和多能性中的作用

基本信息

批准号：
8438457
负责人：
Jason Glenn Knott
金额：
$ 27.7万
依托单位：
MICHIGAN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-12 至 2015-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8438457
关键词：
Address Adverse effects Assisted Reproductive Technology Biochemical Biological Assay Biological Models Biological Process Catalytic Domain Cell Count Cell Lineage Cells Chromatin Chromatin Remodeling Factor Chromatin Structure Clinical DNA Sequence Data Defect Development Down-Regulation Embryo Embryo Transfer Embryonic Development Endoderm Enzymes Epiblast Epigenetic Process Etiology Exhibits Failure Fertilization Foundations Future Gene Expression Gene Expression Profile Genes Genetic Transcription Germ Layers Goals Health Human In Vitro Individual Infertility Inner Cell Mass Knockout Mice Knowledge Laboratories Lead Life Malignant Neoplasms Mammals Mediating Messenger RNA Methodology Microinjections Mission Molecular Multiple Pregnancy Mus National Institute of Child Health and Human Development Oocytes Organism Placenta Pregnancy Rate Pregnancy loss Proteins Protocols documentation Regulation Replacement Therapy Role Small Interfering RNA Spontaneous abortion Staging Stem cells Sucrose Technology Testing Time Tissues United States Up-Regulation Work base blastocyst brahma chromatin immunoprecipitation chromatin remodeling cohort embryo culture embryo stage 2 embryonic stem cell gastrulation immunocytochemistry implantation in vivo mortality mouse development mouse model novel offspring oncology pluripotency preimplantation promoter public health relevance self-renewal stem stem cell differentiation transcription factor trophoblast

项目摘要

DESCRIPTION (provided by applicant): The underlying epigenetic mechanisms that govern cell-fate determination and pluripotency during early embryonic development are poorly understood. The long-term goal is to better understand the chromatin-based mechanisms that regulate gene expression changes between days 2.5 (8-cell) to 6.5 (pre-gastrulation) of mouse embryonic development. These stages of development in a mouse model correspond to the stages of development in humans when a large percentage of embryos are lost to either failed implantation or early miscarriage. The overall objective of this application is to investigate the specific role of the Brg1 chromatin-remodeling protein in trophectoderm development and pluripotency. Brg1 is a critical chromatin-remodeling enzyme that is implicated in human cancer and mammalian development. Brg1-deficient embryos arrest around the blastocyst stage, undergo perturbations in gene expression, and exhibit defects in the ICM and trophectoderm. Moreover, disruption of Brg1 function in embryonic stem (ES) cells results in phenotypic changes indicative of differentiation, downregulation of self-renewal and pluripotency genes, and upregulation of differentiation genes. The specific aims of this application are to test the hypotheses that: 1) Brg1 is required for epigenetic silencing of Oct4 and Nanog expression in the trophectoderm. 2) Brg1 and Nanog promote embryonic pluripotency through epigenetic regulation of Oct4 and Klf5. To address these questions a battery of molecular, cellular, and biochemical assays will be performed in vitro and in vivo. These will include microinjection of siRNAs and mRNAs, immunocytochemistry, chromatin immunoprecipitation (ChIP) assays, chromatin- remodeling assays, and real-time qPCR analysis. The proposed studies will enhance our basic knowledge of early mammalian development. Moreover, these studies are relevant to clinical infertility, human oncology, and stem cell differentiation for cell replacement therapies.

描述（由申请人提供）：早期胚胎发育过程中控制细胞命运决定和多能性的潜在表观遗传机制尚不清楚。长期目标是更好地了解基于染色质的机制，该机制调节小鼠胚胎发育第 2.5 天（8 细胞）到 6.5 天（原肠胚形成前）之间的基因表达变化。小鼠模型中的这些发育阶段与人类的发育阶段相对应，此时大部分胚胎因植入失败或早期流产而丢失。本申请的总体目标是研究 Brg1 染色质重塑蛋白在滋养外胚层发育和多能性中的具体作用。 Brg1 是一种关键的染色质重塑酶，与人类癌症和哺乳动物发育有关。 Brg1 缺陷的胚胎在囊胚阶段停滞，基因表达受到干扰，并表现出 ICM 和滋养外胚层缺陷。此外，胚胎干 (ES) 细胞中 Brg1 功能的破坏会导致表明分化的表型变化、自我更新和多能性基因的下调以及分化基因的上调。本申请的具体目的是测试以下假设：1) 滋养外胚层中 Oct4 和 Nanog 表达的表观遗传沉默需要 Brg1。 2) Brg1和Nanog通过Oct4和Klf5的表观遗传调控促进胚胎多能性。为了解决这些问题，将在体外和体内进行一系列分子、细胞和生化测定。这些将包括 siRNA 和 mRNA 的显微注射、免疫细胞化学、染色质免疫沉淀 (ChIP) 测定、染色质重塑测定和实时 qPCR 分析。拟议的研究将增强我们对早期哺乳动物发育的基础知识。此外，这些研究还与临床不孕症、人类肿瘤学和细胞替代疗法的干细胞分化相关。