Project Description Enteroendocrine cells (EECs) are one of five epithelial cell lineages in the intestine that arise from intestinal stem cells. EECs are notable for their secretion of peptide hormones and biogenic amines. Secreted products regulate food intake, energy homeostasis, insulin secretion and the function of most digestive organs. Relatively little is known about the transcriptional programs that drive enteroendocrine cell differentiation. Until recently, it has difficult to isolate enough EECs for gene expression analysis since EECs represent less than 2% of the intestinal epithelium. The ability to collect fluorescently labeled EECs from transgenic mice combined with technological improvements in high throughput sequencing, make it possible to consider gene expression studies in EECs that previously could not be done. Two basic helix loop helix transcription factors are critical for EEC differentiation. Neurogenin3 (Neurog3) is required for the earliest stages of EEC specification but can give rise to nonendocrine cell types. Expression of the bHLH protein NeuroD1 is expressed in all EECs, restricting cells to an endocrine cell fate. As a relatively weak transcriptional activator, it is not known how NeuroD1 drives cells to become EECs. Our understanding is further limited by the paucity of identified NeuroD1 targets in enteroendocrine cells. An increasing body of information has revealed that tissue specific expression depends on both the local chromatin environment and enhancer occupancy by multiple tissue specific transcription factors. The overall goals of this proposal are to identify NeuroD1 transcriptional targets, to identify other proteins that occupy sites close to NeuroD1, and determine how NeuroD1 activity is influenced by the local chromatin environment. In addition, the contribution of ubiquitously expressed transcription factors bound to nearby sites, to NeuroD1 transcriptional activity will be examined. The goal of Aim 1 is to identify genes that are activated by NeuroD1 in EECs and to identify other transcription factors that bind to DNA in close proximity with NeuroD1 to enhance target gene expression. Studies in Aim 2 will examine the broad role of RREB1 and LSD1, two members of the CtBP co-repressor complex that associate with NeuroD1 to potentiate transcription. The goal of Aim 3 is to determine the importance open chromatin subtypes and enhancer occupancy by multiple transcription factors in NeuroD1 driven tissue specific gene expression. The final goal of Aim3 will be to determine if any identified NeuroD1 enhancer clusters are linked to disease associated variants (SNPs) in the GWAS catalogue. Completion of the proposed studies will expand our knowledge about enteroendocrine cell differentiation and their potential impact on common diseases like diabetes and obesity.