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

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）。了解成人胰岛中 β 细胞功能的正常调节方式将有助于 阐明 T2D 功能障碍的机制，目前尚不清楚。鼠标方面的大量工作 模型表明富含胰岛的转录因子 (TF) NKX2.2 是 β 细胞的关键调节因子 发育并在维持成体 β 细胞功能中发挥作用。此外，功能丧失的患者 NKX2-2突变导致新生儿糖尿病，凸显NKX2.2在人类胰岛中的重要作用 发展。然而，NKX2.2 在成人 β 细胞中的作用仍不清楚。有趣的是，我们发现 在全局 NKX2-2 敲除后，原代人伪胰岛的胰岛素分泌增加，表明 NKX2.2 在不同物种和发育阶段的不同作用。我们假设，在成年人中 在胰岛中，NKX2.2 通过 β 细胞内在途径的转录抑制来调节胰岛素分泌。为了测试这个 假设，我们将首先以 β 细胞特异性方式确定 NKX2.2 在成人胰岛功能中的作用。 利用荧光激活细胞分选和CRISPR/Cas9技术，我们将进行靶向敲除 原代人伪胰岛成人 β 细胞中的 NKX2-2。我们将评估 β 细胞胞内 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发挥作用。评估影响 NKX2.2 的慢性丧失，我们将在移植到体内后检查体内的假胰岛功能 免疫缺陷小鼠。该目标的结果将确定 NKX2.2 对 β 细胞内在途径的影响， 导致胰岛素分泌。其次，我们将定义 NKX2.2 在成人β中发挥作用的分子机制 在同一核上使用单核（sn）RNA-seq ATAC-seq 多组方法的细胞。 snRNA测序 将确定 NKX2.2 是否充当 β 细胞中胰岛素分泌机制的转录抑制因子。在 结合起来，snATAC-seq 将揭示 NKX2.2 如何改变染色质可及性来调节 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 分析收获的伪胰岛移植物中与顶部差异表达相对应的蛋白质的变化 感兴趣的基因。这一目标将为 NKX2.2 如何调节成人 β 细胞基因提供机制见解 染色质和转录本水平的转录和功能。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，对新的治疗方法具有影响 改善 T2D 中的 β 细胞功能。丰富的环境将加强该奖学金下的培训，包括 NIH 资助的范德比尔特糖尿病研究中心下的一个由胰岛生物学研究人员组成的大型社区 培训中心、与该领域专家的合作以及各种促进职业发展的机会 发展、领导力和科学交流。拟议的研究、培训计划和 环境将为医生科学家的职业生涯奠定坚实的基础。

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.