Transcription factor dynamics, oscillation, and functions in human enteroendocrine cell differentiation

Enteroendocrine cells (EECs) secrete serotonin (enterochromaffin [EC] cells) or specific peptide hormones (non-EC cells) that serve vital metabolic functions. The basis for terminal EEC diversity remains obscure. By forcing activity of the transcription factor (TF) NEUROG3 in 2D cultures of human intestinal stem cells, we replicated physiologic EEC differentiation and examined transcriptional and cis-regulatory dynamics that culminate in discrete cell types. Abundant EEC precursors expressed stage-specific genes and TFs. Before expressing pre-terminal NEUROD1, post-mitotic precursors oscillated between transcriptionally distinct ASCL1+ and HES6hi cell states. Loss of either factor accelerated EEC differentiation substantially and disrupted EEC individuality; ASCL1 or NEUROD1 deficiency had opposing consequences on EC and non-EC cell features. These TFs mainly bind cis-elements that are accessible in undifferentiated stem cells, and they tailor subsequent expression of TF combinations that underlie discrete EEC identities. Thus, early TF oscillations retard EEC maturation to enable accurate diversity within a medically important cell lineage. Enteroendocrine differentiation was induced by transient (48h) NEUROG3 activation in 2D human ISCs. This triggered full and efficient EEC differentiation over a period of 6-8 days during which we examined step-wise dynamic profile of enhancers and genes at bulk and single-cell resolution. We identify the binding pattern of crucial intermediate factors ASCL1 and NEUROD1 using CUT&RUN and CRISPR-edited specific transcription factors to determine their role in differentiation program.