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

项目总结：2型糖尿病与胰腺富集的活性和表达的丧失有关。 这些转录因子（TF）控制成熟β细胞功能所必需的基因表达程序。之间 这就是Pdx 1，它在早期胰腺发育、胰岛内分泌细胞分化和 β细胞发育和功能。像大多数TF一样，Pdx 1需要来自募集的协同调节因子的额外调节 以调节特定的调节程序。许多协同调节因子已被确定为Pdx 1相互作用 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在祖细胞扩增中起关键作用，其中早期胰腺祖细胞缺失一个 必需的Swi/Snf ATP酶亚基Brg1导致最终胰腺质量减少50%。在成熟的β细胞中， 缺失Swi/Snf ATP酶亚基Brg 1和Brm，通过严重的葡萄糖耐量降低全身葡萄糖耐量。 胰岛素产生的损失，这主要是由胰岛素基因启动子上Pdx 1占据的损失驱动的。 虽然Swi/Snf在早期发育的胰腺和成熟的β细胞中起着重要作用，但是， 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研究。 在此，我将检验Pdx 1募集的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。内分泌特异性缺失的突变小鼠 产生Brg 1、Brm或两种亚基以确定Swi/Snf在该阶段的机制作用 的胰岛发育和评估出生后的后果失去Swi/Snf在胰岛内分泌细胞 发展初步结果表明，从内分泌系统中丢失Brg 1亚基，而不是Brm， 祖细胞导致严重的葡萄糖稳态失调，从4周龄开始， 胰岛素水平，表明Brg1是必要的适当胰岛发育和功能。值得注意的是， 两种亚基都缺乏的小鼠在断奶时恢复，表明Swi/Snf活性的完全丧失导致 产后致死率目的1将探讨驱动出生后发育的解剖学和生理学属性。 通过定量胰岛细胞质量和评价胰岛功能在Swi/Snf突变体中观察到的表型 通过对分离的胰岛进行灌流分析。目的2将研究Swi/Snf在 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 RNA测序、ATAC测序和ChIP-qPCR方法。通过F31博士前奖学金，我 我将能够承诺我的时间来完成本申请中概述的目标所描述的研究，而 我还专注于职业发展，并通过参加研讨会来提高我的科学技能， 工作坊.印第安纳州大学医学院配备了经验丰富的教师和最先进的核心 协助实施本研究计划并在培训中提供指导的设施。

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.