ISL1 controls pancreatic alpha cell fate and regulates beta cell differentiation and maturation [CUT&Tag]

Glucose homeostasis is dependent on functional pancreatic α and ß cells. Mechanisms underlying generation and maturation of these endocrine cells remain unclear. Here, we unravel the molecular mode of action of ISL1 in controlling α cell fate and endocrine differentiation in the pancreas. By combining transgenic mouse models, transcriptomic and epigenomic profiling, we uncover that elimination of Isl1 results in a diabetic phenotype with a complete loss of α cells, disrupted pancreatic islet architecture, downregulation of maturation markers of ß cells, and an enrichment in an intermediate endocrine progenitor transcriptomic profile. Mechanistically, apart from the altered transcriptome of pancreatic endocrine cells, Isl1 elimination results in altered silencing H3K27me3 histone modifications in the promoter regions of the essential genes for endocrine cell differentiation. Our results thus show that ISL1 transcriptionally and epigenetically controls α cell fate competence, and ß cell maturation, suggesting that ISL1 is a critical component for generating functional α and ß cells. 10 biological samples pooled from two to three pancreata (biological replicates of 4 control and 6 mutant samples) were analyzed at age P9/10. Approximately 14700 cells/sample FACS-sorted tdTomato+ endocrine cells were used for CUT&Tag sequencing. At age E14.5 were analyzed 12 samples (biological replicates of 6 control and 6 mutant samples). Each sample was pooled together from five to nine pancreata ( ~ 4700 cells/sample) and used for used for CUT&Tag sequencing. Two CUT&Tag validated primary antibodies anti-H3K4me3 and anti-H3K27me3 were used. All samples were analyzed separately. Each developmental stage was enriched of one more sample were IgG antibodies were used for CUT&Tag on input cells mixed from control and mutant.

葡萄糖稳态依赖于功能正常的胰腺α细胞与β细胞。此类内分泌细胞的生成与成熟机制迄今尚未完全阐明。本研究解析了胰岛素增强子结合蛋白1（ISL1）调控胰腺α细胞命运与内分泌细胞分化的分子作用模式。结合转基因小鼠模型、转录组学与表观基因组学谱分析，我们发现敲除Isl1会导致小鼠出现糖尿病表型：α细胞完全缺失、胰腺胰岛结构紊乱、β细胞成熟标志物表达下调，且中间型内分泌祖细胞的转录组特征显著富集。从机制层面来看，除胰腺内分泌细胞的转录组发生改变外，敲除Isl1还会导致内分泌细胞分化必需基因启动子区域的沉默型H3K27me3组蛋白修饰出现异常。综上，本研究结果表明ISL1可通过转录与表观遗传层面调控α细胞的命运潜能以及β细胞的成熟过程，提示ISL1是生成功能正常的α与β细胞的关键调控因子。
在小鼠出生后第9/10天（P9/10），我们分析了10份混合生物学重复样本：对照组4份、突变组6份，每份样本由2~3个胰腺组织混合而成。每份样本通过荧光激活细胞分选术（Fluorescence-Activated Cell Sorting, FACS）分选出约14700个tdTomato阳性内分泌细胞，用于CUT&Tag测序。
在小鼠胚胎第14.5天（E14.5），我们分析了12份生物学重复样本（对照组与突变组各6份），每份样本由5~9个胰腺组织混合而成，每样本约含4700个细胞，同样用于CUT&Tag测序。本研究使用了两种经CUT&Tag验证的一抗：抗H3K4me3抗体与抗H3K27me3抗体。所有样本均单独进行分析。针对每个发育阶段，我们设置了额外的对照样本：将对照组与突变组的输入细胞混合后，使用IgG抗体进行CUT&Tag测序作为阴性对照。