NKX2.2 and KLF4 cooperate to regulate α cell identity [sorted_alpha_nkx2.2_KO_RNAseq]

Transcription factors (TF) are indispensable for maintaining cell identity through regulating cell specific gene expression. Distinct cell identities derived from a common progenitor are frequently perpetuated by shared TFs; yet the mechanisms that facilitate their cell specific regulatory targets are poorly characterized. We report that the TF NKX2.2 is critical for the identity of pancreatic islet α cells by directly activating α cell genes and repressing alternate islet cell fate genes. When compared to the known role of NKX2.2 in islet β cells, we demonstrate that NKX2.2 regulates novel α cell target genes, facilitated in part by α cell specific DNA binding at gene promoters. Furthermore, we have identified the reprogramming factor KLF4 as having enriched expression in α cells, where it co-occupies NKX2.2-bound α cell promoters and is necessary for NKX2.2 binding in α cells to co-regulate many NKX2.2 α cell transcriptional targets. Misexpression of Klf4 in β cells is sufficient to manipulate chromatin accessibility, increase binding of NKX2.2 at α cell specific promoters sites, and alter expression of NKX2.2-regulated cell specific targets. This study identifies KLF4 is a novel α cell identity factor that cooperates with NKX2.2 to regulate α cell identity. To investigate the gene expression regulation by NKX2.2 in in vivo pancreatic alpha cells. Pancreatic alpha cells were FACS purified from C57B1/6J mouse pancreatic iselts using the genotypes Nkx2.2fl/fl;Gcg-iCre;Rosa25:tdTomato or Nkx2.2+/+;Gcg-iCre;Rosa25:tdTomato Using 3 mutant samples and 4 control samples differential gene expression analysis on this RNAseq were conducted using DESEQ2 with no batch correction.

转录因子（Transcription Factor, TF）是通过调控细胞特异性基因表达以维持细胞身份不可或缺的核心分子。由共同祖细胞分化产生的不同细胞身份，常由共享的转录因子加以维持，但此类转录因子实现细胞特异性调控靶标的具体机制仍有待深入解析。
本研究发现，转录因子NKX2.2可通过直接激活α细胞特征基因并抑制其他胰岛细胞命运相关基因，对胰腺胰岛α细胞的身份维持至关重要。相较于NKX2.2在胰岛β细胞中的已知功能，我们证实NKX2.2可调控全新的α细胞靶基因，这一调控过程部分依赖于α细胞特异性的基因启动子区域DNA结合活性。此外，我们鉴定出重编程因子KLF4在α细胞中富集表达，其可与NKX2.2共同结合NKX2.2靶向的α细胞启动子区域，且对于NKX2.2在α细胞中结合并共同调控众多NKX2.2介导的α细胞转录靶标是必需的。
在β细胞中异位表达Klf4足以改变染色质可及性，增强NKX2.2在α细胞特异性启动子位点的结合能力，并改变NKX2.2调控的细胞特异性靶基因的表达水平。本研究鉴定KLF4为一种全新的α细胞身份维持因子，可与NKX2.2协同调控α细胞身份。
为探究NKX2.2在体内胰腺α细胞中的基因表达调控机制，我们从携带Nkx2.2fl/fl;Gcg-iCre;Rosa25:tdTomato（突变组）及Nkx2.2+/+;Gcg-iCre;Rosa25:tdTomato（对照组）基因型的C57B1/6J小鼠胰岛中，通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）纯化得到胰腺α细胞，其中突变组样本3例，对照组样本4例。采用DESEQ2对该RNA测序（RNAseq）数据进行差异基因表达分析，未进行批次校正。