The expression order determines the pioneer functions of Ngn3 and NeuroD1 in pancreatic endocrine differentiation -2

This study investigates the roles of Ngn3 and NeuroD1 in pancreatic endocrine differentiation. Using a new mouse model, we mapped NGN3 binding sites by CUT&RUN and demonstrated its pioneering role in making chromatin accessible by scATAC-seq. According to conditional knock-out and over-expression experiments and scRNA-seq, we domonstrated that NGN3's pioneering function was dose tolerance, with low doses sufficing, while NeuroD1, as a conventional TF, has no significant pioneer effects under normal phenomena. But when NGN3 was absent, NeuroD1 can alternatively acted as a pioneer factor, and sequential expression of NeuroD1 ahead of Ngn3 predominantly drives a-cell generation. Overall design: Six types of datasets were generated in this project. 1. CUT&RUN of Ngn3-flag, NeuroD1 or P300 in Ngn3low or Ngn3high cells isolated from E13.5 or E14.5 fetal pancreas. CUT&RUN of NeuroD1 in GFP+ cells isolated from E15.5 Pdx1CreER;Neurod1OE or Ngn3KI;Ngn3Cre;Neurod1OE mice. 2. 10x genomics snATAC-seq of endocrine or pancreatic cells collected from Ngn3CreER;RossaRFP or Pdx1Cre;RossaRFP mice. 10x genomics snATAC-seq of Epcam+ cells collected from Neurod1KO or Ngn3Cre;Ngn3fl mice and their corresponding WT groups (CKO/OE and WT were distinguished by gender). 3. ATAC-seq of Ngn3low or Ngn3high cells in E13.5 fetal pancreas. ATAC-seq of the E13.5 GFP+ cells isolated from Ngn3KI mice, E15.5 or E17.5 Venus+ cells isolated from Neurod1KO mice, E15.5 GFP+ cells isolated from Pdx1CreER;Ngn3fl;Neurod1OE, Pdx1CreER;Neurod1OE or Ngn3KI;Ngn3Cre;Neurod1OE mice. 4. 10x genomics scRNA-seq of the Epcam+ cells isolated from E14.5 Ngn3Cre;Ngn3fl, E14.5 or E17.5 Ngn3Cre;Ngn3OE and E15.5 Neurod1KO mice and their corresponding control WT groups (CKO/OE and WT were distinguished by gender). 5. Smartseq3 scRNA-seq of the E12.5 or E15.5 GFP+ cells isolated from Pdx1CreER;Ngn3fl;Neurod1OE, Pdx1CreER;Neurod1OE or Ngn3KI;Ngn3Cre;Neurod1OE mice, and Epcam+ cells isolated from WT mice. 6. Smartseq2 scRNAseq of Ngn3flagGFPlow, Ngn3flagGFPhigh and GFP+ cells isolated from Ngn3KI mice cells.

本研究探究了转录因子Ngn3与NeuroD1在胰腺内分泌细胞分化中的功能。本研究构建了新型小鼠模型，通过CUT&RUN测序技术绘制了NGN3的全基因组结合位点图谱，并借助单细胞转座酶可及性测序（scATAC-seq）验证了其介导染色质开放的先锋功能。通过条件性敲除、过表达实验以及单细胞RNA测序（scRNA-seq）分析，我们证实NGN3的先锋功能具有剂量耐受性，低剂量即可发挥核心作用；而作为经典转录因子的NeuroD1，在正常生理条件下未表现出显著的先锋因子活性。但当NGN3缺失时，NeuroD1可替代其行使先锋因子功能，且若NeuroD1的表达时序早于Ngn3，则主要驱动α细胞的生成。

本项目共生成六类测序数据集：
1. 针对从胚胎发育第13.5天（E13.5）或第14.5天（E14.5）胎鼠胰腺中分离的Ngn3低表达（Ngn3low）、Ngn3高表达（Ngn3high）细胞，开展Ngn3-flag、NeuroD1或P300的CUT&RUN测序；针对从E15.5 Pdx1CreER;Neurod1OE或Ngn3KI;Ngn3Cre;Neurod1OE小鼠的GFP+细胞中分离的样本，开展NeuroD1的CUT&RUN测序。
2. 针对从Ngn3CreER;RossaRFP或Pdx1Cre;RossaRFP小鼠中收集的内分泌细胞或胰腺细胞，开展10x Genomics单细胞核转座酶可及性测序（snATAC-seq）；针对从Neurod1敲除（Neurod1KO）或Ngn3条件性敲除（Ngn3Cre;Ngn3fl）小鼠及其对应野生型（WT）对照组的Epcam+细胞，开展10x Genomics snATAC-seq（实验组与对照组通过性别进行区分）。
3. 针对E13.5胎鼠胰腺中的Ngn3low、Ngn3high细胞开展转座酶可及性测序（ATAC-seq）；针对从Ngn3敲入（Ngn3KI）小鼠中分离的E13.5 GFP+细胞、Neurod1KO小鼠中分离的E15.5或E17.5 Venus+细胞、Pdx1CreER;Ngn3fl;Neurod1OE、Pdx1CreER;Neurod1OE或Ngn3KI;Ngn3Cre;Neurod1OE小鼠中分离的E15.5 GFP+细胞开展ATAC-seq。
4. 针对从E14.5 Ngn3Cre;Ngn3fl小鼠、E14.5或E17.5 Ngn3Cre;Ngn3OE小鼠以及E15.5 Neurod1KO小鼠及其对应野生型对照组分离的Epcam+细胞，开展10x Genomics scRNA-seq（实验组与对照组通过性别进行区分）。
5. 针对从Pdx1CreER;Ngn3fl;Neurod1OE、Pdx1CreER;Neurod1OE或Ngn3KI;Ngn3Cre;Neurod1OE小鼠中分离的E12.5或E15.5 GFP+细胞，以及野生型小鼠分离的Epcam+细胞，开展Smart-seq3 scRNA-seq。
6. 针对从Ngn3KI小鼠中分离的Ngn3flagGFPlow、Ngn3flagGFPhigh及GFP+细胞，开展Smart-seq2 scRNA-seq。