Determining the role of Nkx2.2 in the maintenance of pancreatic a cell identity

与许多其他生物系统相似，胰岛细胞类型的维持由转录控制。 因子函数。NKX2.2是一种对胰岛细胞发育的所有阶段都至关重要的这样的转录因子， 包括α和β细胞8-12。携带Nkx2.2零突变的小鼠α和β细胞分化受损， 出生后不久就去世了。在成年β细胞中，NKX2.2通过调控促进和主动维持β细胞的特性 几个细胞特异性基因，包括抑制α细胞主调控子Arx13。NKX2.2也表示为 α细胞群，但其在维持α细胞特性和功能中的作用尚不清楚。要确定 NKX2.2是否对α细胞的特性和功能也很重要，我产生了一个结构性的α细胞特异性 Nkx2.2的击倒(KO)。初步体内数据显示，α细胞表达面积减少 似乎伴随着α细胞识别基因的丢失，伴随着非α胰岛细胞基因的获得。 这表明，在α细胞中，NKX2.2正在促进α细胞的转录程序，同时抑制交替 内分泌细胞基因。在初步研究中确定NKX2.2DNA细胞特异的α占有率，永生化 用α细胞系(αTC)14进行NKX2.2染色质免疫沉淀 测序(芯片序列)和定量聚合酶链式反应。有趣的是，这项分析发现了一个新的α小区特有的占用位置 Arx-α基因的启动子区域，在β细胞中被NKX2.2抑制。当与来自 这些数据显示，αTC细胞中Nkx2.2基因敲除的试点αTC RNA序列显示ARX显著下调 提示Arx是NKX2.2在α细胞中的直接转录靶点。此外，表达式之间的比较 α细胞与β或δ细胞的比较表明，在α细胞中，NKX2.2与显著更多的β或δ细胞结合 特异性基因多于α细胞基因。这表明在α细胞中，NKX2.2的S作用的主要部分是抑制交替 胰岛转录程序。最后，NKX2.2更频繁地与α细胞中的启动子区域结合； 而它主要占据β细胞中的基因间增强子区，这表明在 NKX2.2在α与β小区的入住率13。总而言之，先前的研究和初步证据支持 最重要的假设是，在α细胞中，NKX2.2对于维持α细胞特性是重要的，它通过促进α 细胞基因转录和抑制β和δ细胞基因。本提案的目标1评估NKX2.2的作用 使用形态计量学、基因表达分析和功能分析来维持α细胞的特性和功能 在α细胞特异性Nkx2.2KO小鼠体内进行检测。目的2确定NKX2.2同一性的分子机制 在α细胞调控中利用Nkx2.2芯片相关RNA-SEQ寻找直接靶点，CHIP-SEQ探索 NKX2.2占位的功能性表观遗传学，以及免疫共沉淀-质谱仪(Co-MS) IP-MS)以确定相互作用的因素。这些实验将进一步确定NKX2.2在α细胞中的作用 α细胞中NKX2.2‘S的分子机制。

Similar to many other biological systems, maintenance of pancreatic islet cell types is governed by transcription factor function. NKX2.2 is one such transcription factor that is critical for all stages of islet cell development, including α and β cells8-12. Mice carrying Nkx2.2 null mutations have impaired differentiation of α and β cells and die shortly after birth9. In adult β cells, NKX2.2 promotes and actively maintains β cell identity by regulating several cell specific genes, including repressing the α cell master regulator Arx13. NKX2.2 is also expressed in the α cell population, but its role in maintaining α cell identity and function is not yet known9. To determine whether NKX2.2 is also important for α cell identity and function, I generated a constitutive α cell specific knockout (KO) of Nkx2.2. The preliminary in vivo data shows there is a reduction in α cell expression area that appears to be accompanied by a loss of α cell identity genes, with a concomitant gain of non-α islet cell genes. This suggests that in α cells, NKX2.2 is promoting the α cell transcriptional program, while repressing alternate endocrine cell genes. In preliminary studies to identify NKX2.2 α cell-specific DNA occupancy, an immortalized α cell line (αTC)14 was used to perform NKX2.2 chromatin immunoprecipitation coupled with next generation sequencing (ChIP-seq) and qPCR. Interestingly, this analysis identified a novel α cell-specific occupancy site in the promoter region of Arx – an α gene that is repressed by NKX2.2 in β cells. When combined with data from a pilot αTC RNA-seq of a Nkx2.2 knockdown in αTC cells that shows significant Arx downregulation, these data suggest Arx is a direct transcriptional target of NKX2.2 in α cells. Furthermore, comparisons between expression profiles of α cells versus β or δ cells shows that within α cells, NKX2.2 is binding to significantly more β or δ cell specific genes than α cell genes. This suggests that a major part of NKX2.2’s role in α cells is repressing alternate islet transcriptional programs. Lastly, NKX2.2 binds more frequently overall to promoter regions in α cells; whereas it predominantly occupies intergenic enhancer regions in β cells, suggesting global differences in NKX2.2 occupancy in α versus β cells13. Together, previous research and preliminary evidence supports the overarching hypothesis that in α cells, NKX2.2 is important for maintaining α cell identity by promoting α cell gene transcription and repressing β and δ cell genes. Aim 1 of this proposal assesses the role of NKX2.2 in the maintenance of α cell identity and function using morphometrics, gene expression analysis, and functional assays in α cell specific Nkx2.2 KO mice. Aim 2 determines the molecular mechanism of NKX2.2 identity regulation in α cells using Nkx2.2 ChIP-correlated RNA-seq to find direct targets, ChIP-seq to explore the functional epigenetics of NKX2.2 occupancy, and co-immunoprecipitation coupled with mass spectrometry (Co- IP-MS) to identify interacting factors. These experiments will further define the role of NKX2.2 in α cell maintenance and function, and NKX2.2’s molecular mechanism in α cells.