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

项目总结。胰腺β细胞功能和/或肿块的丧失是2型糖尿病发展的核心 糖尿病(T2D)。了解成人胰岛β细胞功能是如何正常调节的将有所帮助 阐明T2D功能障碍的机制，目前还不是很清楚。鼠标中的一大堆工作 模型表明胰岛富集型转录因子(Tf)NKX2.2是β细胞的关键调控因子 并在成人β细胞功能的维持中发挥作用。此外，功能丧失的患者 NKX2-2突变患有新生儿糖尿病，突显NKX2.2在人类胰岛中的重要作用 发展。然而，NKX2.2在成人β细胞中的作用仍不明确。有趣的是，我们发现 全球NKX2-2基因敲除后，原发人类假性胰岛胰岛素分泌增加，提示 NKX2.2在不同物种和不同发育阶段的作用不同。我们假设，在成年人类中 NKX2.2通过转录抑制β细胞内在途径来调节胰岛素的分泌。为了测试这一点 假设，我们将首先以β细胞特异性的方式确定NKX2.2在成人胰岛功能中的作用。 利用荧光激活的细胞分选和CRISPR/Cas9技术，我们将进行有针对性的基因敲除 Nkx2-2在原代人胰岛中的成体β细胞中的表达。我们将评估β细胞内钙信号事件 并使用集成的活细胞成像和微流控平台在体外发挥作用。评估……的影响 NKX2.2的慢性缺失，我们将在体内检测移植到 免疫缺陷小鼠。这一目标的结果将确定NKX2.2对β细胞内在通路的影响 导致胰岛素分泌。其次，我们将确定NKX2.2在成人β中发挥作用的分子机制 使用单核(SN)RNA-SEQ ATAC-SEQ多组体方法的细胞在同一细胞核上。单链RNA序列 将确定NKX2.2是否在β细胞中作为胰岛素分泌机制的转录抑制因子发挥作用。在……里面 结合起来，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基因敲除对β细胞表型和功能的影响，我们将 分析收获的伪胰岛移植细胞中与TOP差异表达对应的蛋白质的变化 感兴趣的基因。这一目标将为NKX2.2如何调控成人β细胞基因提供机械性的见解 在染色质和转录本水平上的转录和功能。总的来说，这些研究将揭示分子 NKX2.2在成人β细胞中的作用机制及其对新的治疗方法的启示 改善T2D的β细胞功能。这项研究金下的培训将因丰富的环境而得到加强，包括 美国国立卫生研究院资助的范德比尔特糖尿病研究和研究中心下的一个大型胰岛生物学研究社区 培训中心，与该领域的专家合作，以及各种促进职业发展的机会 发展、领导和科学交流。总之，建议的研究、培训计划和 环境将为作为内科科学家的职业生涯提供坚实的基础。

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.