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