Role of Brg1 in Embryo Development and Pluripotency

Brg1 在胚胎发育和多能性中的作用

• 批准号: 8055952
• 负责人: Jason Glenn Knott
• 金额: $ 28.84万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-12 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8055952
• 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; 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; Pattern; 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

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. PUBLIC HEALTH RELEVANCE: In the long-term the proposed studies will lead to better methodologies for selecting high quality embryos and/or protocols for augmenting embryo viability (eg, optimization of embryo culture protocols). Enhanced embryo viability in assisted eproduction technologies (ART) will lead to reduced numbers of embryos transferred per cycle and correspondingly reduced multiple pregnancy rates; and furthermore, proposed studies on the role of Brg1 in early embryos and ES cells have direct implications in human oncology and ES cell differentiation for cell replacement therapies. Collectively, these studies are highly relevant to the mission of the NICHD.

描述（由申请人提供）：对早期胚胎发育期间细胞剂量确定和多能性的基本表观遗传机制的理解很少。长期目标是更好地理解基于染色质的机制，这些机制调节基因表达在第2.5天（8细胞）至6.5（淋巴结前）之间的变化小鼠胚胎发育。当大部分胚胎因失败的植入或早期流产而丢失了很大一部分胚胎时，小鼠模型中的这些发育阶段对应于人类的发展阶段。该应用的总体目的是研究BRG1染色质复制蛋白在滋养外胚层发育和多能性中的特定作用。 BRG1是一种关键的染色质复制酶，与人类癌症和哺乳动物发育有关。 BRG1缺陷型胚胎在胚泡阶段停滞，在基因表达中受到扰动，并在ICM和Trophectododerm中表现出缺陷。此外，胚胎干细胞（ES）细胞中BRG1功能的破坏会导致表型变化，指示分化，自我更新和多能基因的下调以及分化基因的上调。本应用的具体目的是检验以下假设：1）BRG1是滋养剂中OCT4和Nanog表达表观遗传沉默所必需的。 2）BRG1和Nanog通过OCT4和KLF5的表观遗传调节促进胚胎多能性。为了解决这些问题，将在体外和体内进行分子，细胞和生化测定的电池。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展的基本知识。此外，这些研究与细胞替代疗法的临床不育症，人肿瘤学和干细胞分化有关。 公共卫生相关性：从长远来看，拟议的研究将为选择高质量的胚胎和/或协议来增强胚胎生存能力（例如，优化胚胎培养方案）。增强的辅助电子生产技术（ART）中的胚胎生存力将导致每个周期转移的胚胎数量减少，并相应降低多重妊娠率；此外，对BRG1在早期胚胎和ES细胞中的作用的提出的研究在人类肿瘤学和ES细胞分化中对细胞替代疗法具有直接影响。总的来说，这些研究与NICHD的使命高度相关。