Molecular mechanisms of NKX2.2 function in adult human beta cells

成人β细胞中NKX2.2功能的分子机制

基本信息

批准号：
10603492
负责人：
Yasminye D Pettway
金额：
$ 3.28万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-01-01 至 2027-12-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10603492
关键词：
ATAC-seq Adult Alpha Cell Beta Cell Biology CRISPR/Cas technology Cell Nucleus Cell Separation Cell physiology Cellular biology Chromatin Chronic Collaborations Communication Communities D Cells DNA Binding Development Diabetes Mellitus Environment Event Fellowship Foundations Functional disorder Funding Future Gene Expression Gene Expression Profiling Genes Genetic Genetic Transcription Harvest Homeodomain Proteins Hormones Human Immunodeficient Mouse In Vitro Insulin Islet Cell Islets of Langerhans Knock-out Leadership Maintenance Mediating Microfluidics Molecular Mus Mutation Non-Insulin-Dependent Diabetes Mellitus Paracrine Communication Pathway interactions Patients Phenotype Physicians Play Proteins Research Research Personnel Role Scientist Signal Transduction Structure of beta Cell of islet Testing Tissue-Specific Gene Expression Training Transcript Transcription Coactivator Transcription Repressor Transcriptional Regulation Transplantation United States National Institutes of Health anterior chamber base blood glucose regulation career career development diabetes pathogenesis differential expression endocrine pancreas development endoplasmic reticulum stress eye chamber gene function gene repression homeodomain human tissue improved in vivo innovation insight insulin regulation insulin secretion interest islet knock-down live cell imaging loss of function mouse model multiple omics neonatal diabetes mellitus novel therapeutic intervention single nucleus RNA-sequencing skills transcription factor transcriptome

项目摘要

PROJECT SUMMARY. Loss of pancreatic β cell function and/or mass is central to the development of type 2 diabetes (T2D). Understanding how β cell function is normally regulated in adult human islets will help elucidate mechanisms of dysfunction in T2D, which are not well understood. A large body of work in mouse models suggests that the islet-enriched transcription factor (TF) NKX2.2 is a critical regulator of β cell development and plays a role in the maintenance of adult β cell function. Further, patients with loss-of-function NKX2-2 mutations have neonatal diabetes, highlighting an important role of NKX2.2 in human islet development. However, the role of NKX2.2 in adult human β cells remain undefined. Interestingly, we found increased insulin secretion from primary human pseudoislets following global NKX2-2 knockdown, suggesting different roles of NKX2.2 across species and developmental stages. We hypothesize that, in adult human islets, NKX2.2 regulates insulin secretion via transcriptional repression of β cell-intrinsic pathways. To test this hypothesis, we will first determine the role of NKX2.2 in adult human islet function in a β cell-specific manner. Using florescence-activated cell sorting and CRISPR/Cas9 technology, we will perform targeted knockout of NKX2-2 in adult β cells in primary human pseudoislets. We will assess β cell intracellular Ca2+ signaling events and function in vitro using an integrated live cell imaging and microfluidic platform. To evaluate the impact of chronic loss of NKX2.2, we will examine pseudoislet function in vivo following transplantation into immunodeficient mice. Results of this aim will determine the impact of NKX2.2 on β cell-intrinsic pathways that lead to insulin secretion. Secondly, we will define molecular mechanisms of NKX2.2 function in adult human β cells using a single nucleus (sn)RNA-seq+ ATAC-seq multiome approach on the same nucleus. snRNA-seq will determine if NKX2.2 functions as a transcriptional repressor of insulin secretory machinery in β cells. In combination, snATAC-seq will reveal how NKX2.2 alters chromatin accessibility to regulate the β cell transcriptome. To study the impact of chronic NKX2-2 knockout on β cell phenotype and function, we will analyze harvested pseudoislet transplants for changes in proteins corresponding to top differentially expressed genes of interest. This aim will provide mechanistic insight into how NKX2.2 regulates adult human β cell gene transcription and function at the chromatin and transcript level. Overall, these studies will reveal molecular mechanisms of NKX2.2 function in adult human β cells, with implications for new therapeutic approaches to improve β cell function in T2D. Training under this fellowship will be enhanced by a rich environment, including a large community of islet biology investigators under the NIH-funded Vanderbilt Diabetes Research and Training Center, collaborations with experts in the field, and a variety of opportunities to promote career development, leadership, and scientific communication. Together, the proposed research, training plan, and environment will provide a strong foundation on which to base a career as a physician-scientist.

项目摘要。胰腺β细胞功能和/或质量的丧失对于2型的发展至关重要糖尿病（T2D）。了解通常在成人人类胰岛中通常调节β细胞功能将有助于阐明了T2D功能障碍的机制，这些机制尚不清楚。鼠标中的大量工作模型表明富含胰岛的转录因子（TF）NKX2.2是β细胞的关键调节剂开发并在维持成人β细胞功能中起作用。此外，功能丧失的患者 NKX2-2突变具有新生儿糖尿病，突出了NKX2.2在人类胰岛中的重要作用发展。但是，NKX2.2在成年人类β细胞中的作用仍然不确定。有趣的是，我们发现在全球NKX2-2敲低之后，原代人伪符的胰岛素分泌增加，这表明 NKX2.2在物种和发育阶段的不同作用。我们假设在成年人中 NKX2.2胰岛通过β细胞中心途径的转录表示调节胰岛素分泌。测试这个假设，我们将首先以β细胞特异性方式确定NKX2.2在成年人类胰岛功能中的作用。使用荧光激活的细胞分类和CRISPR/CAS9技术，我们将执行针对性的敲除原代人伪群体中成年β细胞中的NKX2-2。我们将评估β细胞内CA2+信号传导事件并使用集成的活细胞成像和微流体平台在体外功能。评估 NKX2.2的慢性丧失，我们将在移植到体内检查伪符功能免疫缺陷小鼠。该目标的结果将决定NKX2.2对β细胞中性途径的影响导致胰岛素分泌。其次，我们将定义NKX2.2成人人β的分子机制使用单个核（SN）RNA-SEQ+ ATAC-SEQ多组方法在同一核上使用细胞。 snrna-seq 将确定NKX2.2是否起作用β细胞中胰岛素分泌机械的转录表示。组合，SNATAC-SEQ将揭示NKX2.2如何改变染色质的可及性来调节β细胞转录组。为了研究慢性NKX2-2敲除对β细胞表型和功能的影响，我们将分析收获的伪符移植物，以使对应于最高表达的蛋白质的变化感兴趣的基因。这个目标将提供有关NKX2.2如何调节成年人β细胞基因的机械洞察力在染色质和转录水平上的转录和功能。总体而言，这些研究将揭示分子 NKX2.2在成年人类β细胞中功能的机理，对新的治疗方法的影响改善T2D中的β细胞功能。在这个奖学金下的培训将通过丰富的环境增强 NIH资助的范德比尔特糖尿病研究和培训中心，与该领域的专家合作以及促进职业的各种机会发展，领导和科学沟通。共同提议的研究，培训计划和环境将为以身体科学家的身份为基础提供坚实的基础。