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

与许多其他生物系统类似，胰岛细胞类型的维持受转录控制 因子函数NKX2.2是一种这样的转录因子，其对胰岛细胞发育的所有阶段都至关重要， 包括α和β细胞8 -12。携带Nkx2.2无效突变的小鼠具有受损的α和β细胞分化， 出生后不久就死了9.在成年β细胞中，NKX2.2通过调节β细胞的分化，促进并积极维持β细胞的特性。 几个细胞特异性基因，包括抑制α细胞主调节因子Arx 13。NKX2.2也表达于 α细胞群体，但其在维持α细胞身份和功能中的作用尚不清楚9。以确定 无论NKX2.2是否对α细胞的身份和功能也很重要，我建立了一个组成性α细胞特异性 敲除（KO）Nkx2.2。初步的体内数据显示，α细胞表达面积减少， 似乎伴随着α细胞身份基因的丢失，伴随着非α胰岛细胞基因的获得。 这表明，在α细胞中，NKX2.2促进α细胞转录程序，而抑制交替转录程序。 内分泌细胞基因在鉴定NKX2.2 α细胞特异性DNA占有率的初步研究中， 用α细胞系（αTC）14进行NKX2.2染色质免疫沉淀， 测序（ChIP-seq）和qPCR。有趣的是，这项分析确定了一个新的α细胞特异性占据位点， Arx的启动子区-β细胞中受NKX2.2抑制的α基因。当与来自 pilot αTC RNA-seq在αTC细胞中的Nkx2.2敲低显示出显著的Arx下调，这些数据 表明Arx是NKX2.2在α细胞中直接转录靶点。此外，表达式之间的比较 α细胞与β或δ细胞的图谱显示，在α细胞内，NKX2.2与显著更多的β或δ细胞结合， 基因比α细胞基因。这表明NKX2.2在α细胞中的主要作用是抑制交替的细胞凋亡。 胰岛转录程序。最后，NKX2.2在α细胞中更频繁地与启动子区域结合; 而它主要占据β细胞中的基因间增强子区域，这表明在 α与β细胞中的NKX2.2占有率13。总之，以前的研究和初步证据支持 总体假设，在α细胞中，NKX2.2通过促进α细胞分化， 细胞基因转录和抑制β和δ细胞基因。本提案的目标1评估了NKX2.2的作用 在使用形态测量学、基因表达分析和功能分析维持α细胞身份和功能方面， 在α细胞特异性Nkx2.2 KO小鼠中的测定。目的2确定NKX2.2同一性的分子机制 使用Nkx2.2 ChIP相关的RNA-seq寻找直接靶点，ChIP-seq探索α细胞中的 NKX2.2占有率的功能表观遗传学，以及与质谱联用的免疫共沉淀（Co-Immunoprecipitation coupled with mass spectrometry， IP-MS）来识别相互作用的因素。这些实验将进一步明确NKX2.2在α细胞中的作用。 NKX2.2在α细胞中的维持和功能以及NKX2.2在α细胞中的分子机制。

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.