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

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 α-cell generation. 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.

本研究探讨了神经元素3（Ngn3）与神经分化因子1（NeuroD1）在胰腺内分泌分化中的调控作用。我们依托全新构建的小鼠模型，通过CUT&RUN技术绘制了NGN3的全基因组结合位点，并借助单细胞转座酶可及性测序（scATAC-seq）证实了其介导染色质开放的先驱因子功能。基于条件性敲除、过表达实验以及单细胞RNA测序（scRNA-seq）结果，我们证明NGN3的先驱因子活性具有剂量耐受性，低剂量即可发挥功能；而作为经典转录因子（TF）的NeuroD1，在正常生理状态下无显著的先驱因子活性。但当NGN3缺失时，NeuroD1可替代性地充当先驱因子；若NeuroD1的表达时序早于Ngn3，则主要驱动胰岛α细胞的生成。本项目共生成六类数据集：1. 从E13.5或E14.5胎鼠胰腺中分离的Ngn3low、Ngn3high细胞中，针对Ngn3-flag、NeuroD1或P300的CUT&RUN测序；从E15.5 Pdx1CreER;Neurod1OE或Ngn3KI;Ngn3Cre;Neurod1OE小鼠中分离的GFP+细胞中，针对NeuroD1的CUT&RUN测序。2. 从Ngn3CreER;RossaRFP或Pdx1Cre;RossaRFP小鼠中收集的内分泌细胞或胰腺细胞的10x Genomics单细胞核转座酶可及性测序（snATAC-seq）；从Neurod1KO或Ngn3Cre;Ngn3fl小鼠及其对应野生型（WT）组中分离的Epcam+细胞的10x Genomics单细胞核转座酶可及性测序（CKO/OE与野生型通过性别区分）。3. E13.5胎鼠胰腺中Ngn3low、Ngn3high细胞的ATAC测序；从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+细胞的ATAC测序。4. 从E14.5 Ngn3Cre;Ngn3fl、E14.5或E17.5 Ngn3Cre;Ngn3OE以及E15.5 Neurod1KO小鼠中分离的Epcam+细胞及其对应野生型对照组的10x Genomics单细胞RNA测序（scRNA-seq）（CKO/OE与野生型通过性别区分）。5. 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+细胞的Smart-seq3单细胞RNA测序。6. 从Ngn3KI小鼠中分离的Ngn3flagGFPlow、Ngn3flagGFPhigh以及GFP+细胞的Smart-seq2单细胞RNA测序。