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

项目摘要/摘要 郎格罕胰岛内的胰腺β细胞是葡萄糖刺激的胰岛素分泌所必需的 和葡萄糖动态平衡。功能失调的β细胞活动和身份识别导致糖尿病(DM)，一种不断增长的 这种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。战略以实现 改善越来越多的糖尿病患者的预后，并有可能取代被摧毁的和 功能障碍的β细胞团需要对协调适当胰岛的复杂程序有深刻的理解 队形。基于丰富的胰岛转录因子(TF)如何协调的现有知识 引导胰岛细胞发育的信号将使我们了解这些程序如何针对β- 细胞替代疗法。 PDX1是胰腺和发育中的胰岛中最重要的转录因子之一，已被证明招募了一种 不同的联合监管机构，这可能会调节其活动。这项提案的重点是如何 PDX1招募的依赖ATP的SwI/SNF染色质重塑复合体调节Pdx1的转录活性 在发育中的小岛上。PI小组之前的工作揭示了Pdx1的关键作用：Swi/Snf功能在 发育中的胰腺和成熟的β细胞。到目前为止的初步研究表明，有条件地移除 发育内分泌前体的核心SWI/SNF ATPase亚基之一(通过Cre-loxP技术) 细胞导致葡萄糖耐受，随意喂食高血糖和降低突变的血清胰岛素水平 动物出生后。这一提议将检验Pdx1招募的Swi/Snf染色质重塑的假设 控制染色质可及性和内分泌祖细胞必需的基因表达程序的活动 胰岛细胞的扩增和出生后的功能。在目标1中，PI将使用这种新的转基因动物模型来 测定胰岛细胞功能、胰岛细胞成熟标志物表达、激素分泌及激素细胞 成人胰腺肿块。在目标2中，突变动物的胚胎组织将被用来确定 内分泌祖细胞质量和激素细胞增殖/凋亡。SWI/SNF的机械作用 指导内分泌前体细胞的染色质可及性和基因表达程序将由以下因素决定 流动分选SWI/SNF缺陷内分泌前体的ATAC-Seq、RNA-Seq和ChIP-qPCR分析。 我研究转录因子共调节复合体的丰富经验和体内可用的 试剂使我的实验室非常适合实现这些目标。这些研究定义的机制将 发现依赖于染色质重塑的关键过程和独特的基因表达特征 内分泌开发，这将为研究人员开发新分子提供工具性知识 对抗糖尿病的靶点和基于细胞的疗法。重要的是，这项提议还旨在 生成数据和资源，申请者将在R01申请中使用这些数据和资源，以定义如何失控 2型糖尿病(T2D)胰岛供体的增强子功能受Pdx1：Swi/SNF变化的影响。

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.