Swi/Snf chromatin remodeling activity is required for islet cell development in vivo

Swi/Snf 染色质重塑活性是胰岛细胞体内发育所必需的

• 批准号: 10314133
• 负责人: Rebecca K Davidson
• 金额: $ 3.08万
• 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: 2021-09-01 至 2022-08-23
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10314133
• 关键词: ATAC-seq; ATP phosphohydrolase; Age; Alleles; Alpha Cell; Anatomy; Apoptosis; Area; Automobile Driving; B-Cell Development; Beta Cell; Binding; Biology; Cell Differentiation process; Cell physiology; Cells; Chromatin; Chromatin Remodeling Factor; Core Facility; Data; Development; Diabetes Mellitus; Educational workshop; Embryo; Endocrine; Enhancers; Evaluation; Excision; Faculty; Fasting; Fellowship; Functional disorder; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Glucose; Hormone secretion; Hormones; Human; Hyperglycemia; Immunofluorescence Immunologic; Impairment; In Vitro; Indiana; Insulin; Insulin Resistance; Islet Cell; Islets of Langerhans; Mature B-Lymphocyte; Metabolic Diseases; Methods; Modeling; Mus; Mutant Strains Mice; Nature; Non-Insulin-Dependent Diabetes Mellitus; Pancreas; Patients; Peripheral; Phenotype; Physiological; Plasma; Play; Production; Protocols documentation; Regulation; Research; Research Proposals; Role; Series; Stains; Stem Cell Development; Testing; Time; Training; Universities; Weaning; XCL1 gene; blood glucose regulation; career development; cell type; chromatin remodeling; effective therapy; endocrine pancreas development; experience; functional loss; genetic corepressor; glucose tolerance; impaired glucose tolerance; in vivo; islet; islet stem cells; medical schools; mutant; novel; pancreas development; pancreatic islet function; postnatal; pre-doctoral; precursor cell; progenitor; programs; promoter; protein protein interaction; recruit; stem cell differentiation; stem cell proliferation; stem cells; transcription factor; transcriptome sequencing; transdifferentiation

PROJECT SUMMARY: Type 2 diabetes is associated with loss of activity and expression of pancreas-enriched transcription factors (TFs) that control gene expression programs necessary for mature β-cell function. Among these is Pdx1, which plays key roles during early pancreas development, islet endocrine cell differentiation, and β-cell development and function. Pdx1, like most TFs, requires additional regulation from recruited coregulators to modulate specific regulatory programs. Numerous coregulators have been identified as Pdx1-interacting partners including the ATP-dependent Swi/Snf chromatin remodeling complex. In the early developing pancreas, Swi/Snf plays a critical role in progenitor cell expansion, where early pancreatic progenitor cell deletion of one essential Swi/Snf ATPase subunit, Brg1, results in a 50% reduction in final pancreas mass. In the mature β-cell, deletion of both Swi/Snf ATPase subunits, Brg1 and Brm, impairs whole-body glucose tolerance through severe loss of insulin production, which is largely driven by a loss of Pdx1 occupancy on the insulin gene promoter. While Swi/Snf plays an essential role in the early developing pancreas and the mature β-cell, the contribution of Swi/Snf chromatin remodeling activity to islet endocrine progenitor development has not yet been explored. Herein, I will test the hypothesis that the Pdx1-recruited Swi/Snf chromatin remodeling complex dynamically controls the chromatin landscape and expression of genes essential for endocrine progenitor cell development and postnatal islet function. Mutant mice with endocrine-specific deletions of either Brg1, Brm, or both subunits were generated to determine the mechanistic actions of Swi/Snf at this stage of islet development and evaluate the postnatal consequences of losing Swi/Snf during islet endocrine cell development. Preliminary results demonstrate that loss of the Brg1 subunit, but not Brm, from endocrine progenitors leads to severe glucose dyshomeostasis beginning at 4 weeks of age with a reduction in plasma insulin levels, suggesting that Brg1 is essential for proper islet development and function. Remarkably, no mice deficient for both subunits have been recovered at weaning, indicating that total loss of Swi/Snf activity results in postnatal lethality. Aim 1 will explore the anatomical and physiological attributes driving the postnatal phenotype observed in the Swi/Snf mutants through quantitation of islet cell mass and evaluation of islet function through perifusion analysis on isolated islets. Aim 2 will investigate the mechanistic actions of Swi/Snf in controlling chromatin accessibility, TF recruitment, and gene expression programs in endocrine progenitor cells with RNA-sequencing, ATAC-sequencing, and ChIP-qPCR approaches. With this F31 Predoctoral Fellowship, I will be able to commit my time to completing the research described in the Aims outlined in this application, while also focusing on career development and enhancing my scientific skillset through attending seminars and workshops. Indiana University School of Medicine is equipped with experienced faculty and state-of-the-art core facilities to assist in carrying out this research proposal and provide guidance in my training.

项目摘要：2 型糖尿病与富含胰腺的活性和表达丧失相关 转录因子 (TF) 控制成熟 β 细胞功能所需的基因表达程序。 这些是 Pdx1，它在早期胰腺发育、胰岛内分泌细胞分化和 与大多数 TF 一样，Pdx1 的发育和功能需要来自招募的核心调节因子的额外调节。 调节特定的调控程序已被确定为与 Pdx1 相互作用。 合作伙伴包括 ATP 依赖性 Swi/Snf 染色质重塑复合物 在早期发育的胰腺中， Swi/Snf 在祖细胞扩增中发挥着关键作用，其中早期胰腺祖细胞缺失一个 必需的 Swi/Snf ATP 酶亚基 Brg1 导致成熟 β 细胞中最终胰腺质量减少 50%。 Swi/Snf ATPase 亚基 Brg1 和 Brm 的缺失会通过严重损害全身葡萄糖耐量 胰岛素产生的损失，这主要是由于胰岛素基因启动子上 Pdx1 占据的损失造成的。 虽然 Swi/Snf 在早期发育的胰腺和成熟的 β 细胞中发挥着重要作用，但 Swi/Snf 染色质重塑活性对胰岛内分泌祖细胞发育的影响尚未被探索。 在这里，我将检验以下假设：Pdx1 招募的 Swi/Snf 染色质重塑复合物 动态控制染色质景观和内分泌必需基因的表达 祖细胞发育和出生后胰岛功能的突变小鼠。 生成 Brg1、Brm 或两个亚基以确定此阶段 Swi/Snf 的机械作用 胰岛发育并评估胰岛内分泌细胞期间失去 Swi/Snf 的产后后果 初步结果表明，内分泌导致了 Brg1 亚基的损失，但 Brm 没有损失。 祖细胞从 4 周龄开始导致严重的葡萄糖稳态失调，并伴有血浆减少 胰岛素水平，表明 Brg1 对于胰岛的正常发育和功能至关重要。值得注意的是，没有小鼠。 断奶时两个亚基的缺陷都已恢复，表明 Swi/Snf 活性完全丧失 目标 1 将探索驱动产后致死率的解剖学和生理学特征。 通过胰岛细胞质量定量和胰岛功能评估在 Swi/Snf 突变体中观察到的表型 通过对孤立胰岛的灌注分析，目标 2 将研究 Swi/Snf 在中的机制作用。 控制内分泌祖细胞中的染色质可及性、TF 募集和基因表达程序 通过 RNA 测序、ATAC 测序和 ChIP-qPCR 方法，我获得了 F31 博士前奖学金。 将能够投入时间完成本申请中概述的目标中描述的研究，同时 还注重职业发展并通过参加研讨会和提高我的科学技能 印第安纳大学医学院配备了经验丰富的教师和最先进的核心人员。 协助实施这项研究计划并为我的培训提供指导的设施。