The Pdx1-recruited Swi/Snf chromatin remodeling complex regulates endocrine cell expansion and differentiation in vivo

Pdx1 招募的 Swi/Snf 染色质重塑复合物调节体内内分泌细胞的扩增和分化

• 批准号: 10321296
• 负责人: Jason M Spaeth
• 金额: $ 11.89万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10321296
• 关键词: ATAC-seq; ATP phosphohydrolase; Adult; Affect; Age; Alleles; American; Animal Model; Animals; Apoptosis; Beta Cell; Binding; Blood Glucose; Cell Lineage; Cell Maturation; Cell Proliferation; Cell Therapy; Cell physiology; Cells; Chromatin; Chromatin Remodeling Factor; Complex; Cre-LoxP; DNA Binding; Development; Diabetes Mellitus; Disease; Endocrine; Enhancers; Excision; Failure; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Transcription; Glucose; Glucose Intolerance; Goals; Health; Health Care Costs; Hormone secretion; Hormones; Human; Hyperglycemia; Immunofluorescence Immunologic; Impairment; In Vitro; Insulin; Insulin Resistance; Islet Cell; Islets of Langerhans; Knowledge; Mature B-Lymphocyte; Mediating; Metabolic; Molecular Target; Monitor; Mus; Nature; Non-Insulin-Dependent Diabetes Mellitus; Pancreas; Peripheral; Phenotype; Process; Production; Quality of life; RNA; Reagent; Regulator Genes; Research Personnel; Role; SWI/SNF Family Complex; Serum; Signal Transduction; Stem Cell Development; Structure of beta Cell of islet; Technology; Testing; Tissues; Transcriptional Regulation; Transgenic Animals; Transgenic Model; Type 2 diabetic; Weaning; Work; XCL1 gene; base; beta cell replacement; blood glucose regulation; cell replacement therapy; cell type; chromatin remodeling; combat; data resource; defined contribution; design; diabetic patient; economic cost; embryo tissue; endocrine pancreas development; experience; improved; improved outcome; in vivo; insulin secretion; islet; islet stem cells; mouse Neurog3 protein; mutant; novel; pancreas development; postnatal; progenitor; programs; protein protein interaction; recruit; stem cell proliferation; stem cells; therapy development; transcription factor; transcriptome sequencing

PROJECT SUMMARY/ABSTRACT Pancreatic β-cells within the islets of Langerhans are required for glucose-stimulated insulin secretion and glucose homeostasis. Dysfunctional β-cell activity and identity results in diabetes mellitus (DM), a growing disease affecting millions of Americans, thus creating an enormous fiscal and health burden. Strategies to improve outcomes for the mounting number of diabetic patients and the possibility to replace destroyed and dysfunctional β-cell mass requires a deep understanding of the complex programs that coordinate proper islet formation. Developing upon existing knowledge of how islet enriched transcription factors (TFs) coordinate signals that direct islet cell development will allow us to understand how such programs can be targeted for β- cell replacement therapies. Pdx1, one of the most important TFs in the pancreas and developing islet, has been shown to recruit a diverse set of coregulators which could potentially modulate its activity. This proposal is focused around how the Pdx1 recruited ATP-dependent Swi/Snf chromatin remodeling complex modulates Pdx1 transcriptional activity in the developing islet. Previous work by the PI’s group revealed a critical role for Pdx1:Swi/Snf function in the developing pancreas and the mature β-cell. Preliminary studies thus far have shown that conditional removal of one of the core Swi/Snf ATPase subunits (through Cre-LoxP technology) from developing endocrine progenitor cells results in glucose intolerance, ad libitum fed hyperglycemia and reduced serum insulin levels in mutant animals postnatally. This proposal will test the hypothesis that Pdx1-recruited Swi/Snf chromatin remodeling activities control chromatin accessibility and gene expression programs essential for endocrine progenitor cell expansion and postnatal islet cell function. In Aim 1, the PI will use this novel transgenic animal model to determine islet cell function, expression of islet cell maturation markers, hormone secretion and hormone cell mass in adult pancreata. In Aim 2, embryonic tissues from mutant animals will be used to determine changes in endocrine progenitor cell mass and hormone cell proliferation/apoptosis. The mechanistic actions of Swi/Snf on directing chromatin accessibility and gene expression programs in endocrine progenitors will be determined by ATAC-Seq, RNA-Seq and ChIP-qPCR analyses on flow-sorted Swi/Snf-deficient endocrine precursors. My extensive experience studying transcription factor coregulatory complexes and our available in vivo reagents make my lab uniquely suited to accomplish these Aims. Mechanisms defined by these studies will uncover key processes and unique gene expression signatures dependent on chromatin remodeling during endocrine development, which will provide instrumental knowledge to researchers developing new molecular targets and cell-based therapies to combat diabetes. Importantly, this proposal has also been designed to generate data and resources that the applicant will utilize in an R01 application defining how dysregulated enhancer function from Type 2 diabetic (T2D) islet donors are influenced by altered Pdx1:Swi/Snf actions.

项目概要/摘要 朗格汉斯岛内的胰腺 β 细胞是葡萄糖刺激的胰岛素分泌所必需的 β细胞活性和功能失调会导致糖尿病（DM），这是一种日益严重的疾病。 该疾病影响了数百万美国人，从而造成了巨大的财政和健康负担。 改善越来越多的糖尿病患者的治疗结果以及更换被破坏和损坏的可能性 功能失调的 β 细胞团需要深入了解协调适当胰岛的复杂程序 以胰岛富集转录因子 (TF) 如何协调的现有知识为基础进行开发。 指导胰岛细胞发育的信号将使我们能够了解此类程序如何针对 β- 细胞替代疗法。 Pdx1 是胰腺和发育中的胰岛中最重要的 TF 之一，已被证明可以招募 该提案的重点是如何调节其活动。 Pdx1 招募 ATP 依赖性 Swi/Snf 染色质重塑复合物调节 Pdx1 转录活性 PI 小组之前的工作揭示了 Pdx1:Swi/Snf 功能在发育中的胰岛中的关键作用。 迄今为止的初步研究表明，有条件地去除发育中的胰腺和成熟的β细胞。 来自发育中的内分泌祖细胞的核心 Swi/Snf ATPase 亚基之一（通过 Cre-LoxP 技术） 细胞导致突变体葡萄糖不耐受、随意喂养高血糖和血清胰岛素水平降低 该提案将检验 Pdx1 招募 Swi/Snf 染色质重塑的假设。 控制内分泌祖细胞必需的染色质可及性和基因表达程序的活动 在目标 1 中，PI 将使用这种新型转基因动物模型来进行扩增和出生后胰岛细胞功能。 确定胰岛细胞功能、胰岛细胞成熟标志物的表达、激素分泌和激素细胞 在目标 2 中，突变动物的胚胎组织将用于确定成年胰腺质量的变化。 Swi/Snf 对内分泌祖细胞团和激素细胞增殖/凋亡的机制作用。 内分泌祖细胞中染色质可及性和基因表达程序的指导将由以下因素决定 对流式分选的 Swi/Snf 缺陷内分泌前体进行 ATAC-Seq、RNA-Seq 和 ChIP-qPCR 分析。 我研究转录因子共调节复合物的丰富经验以及我们在体内的应用 试剂使我的实验室非常适合实现这些研究定义的这些目标。 揭示依赖于染色质重塑的关键过程和独特的基因表达特征 内分泌发育，这将为开发新分子的研究人员提供工具知识 重要的是，该提案还旨在对抗糖尿病。 生成申请人将在 R01 申请中使用的数据和资源，定义如何失调 2 型糖尿病 (T2D) 胰岛供体的增强子功能受到 Pdx1:Swi/Snf 作用改变的影响。