ABSTRACT HNF1A haploinsufficiency underlies the most common form of human monogenic diabetes (HNF1A-MODY) and hypomorphic HNF1A variants confer type 2 diabetes risk, but a lack of experimental systems has limited our understanding of how the transcription factor HNF1α regulates adult human islet function. Here, we combined human islet genetics, RNA sequencing, Cleavage Under Targets &amp; Release Using Nuclease (CUT&amp;RUN) chromatin mapping, patch-clamp electrophysiology and transplantation-based assays to elucidate HNF1α-regulated mechanisms in mature pancreatic α and β cells. shRNA-mediated suppression of HNF1A in primary human pseudoislets led to blunted insulin output and dysregulated glucagon secretion both in vitro and after transplantation into immunocompromised mice, recapitulating phenotypes observed in HNF1A-MODY patients. These deficits corresponded with altered expression of genes encoding factors critical for hormone secretion, including calcium channel subunits, ATP-transporters and extracellular matrix constituents. Additionally, HNF1A loss led to upregulation of transcriptional repressors, providing evidence for a mechanism of transcriptional de-repression through HNF1α. CUT&amp;RUN mapping of HNF1α DNA-binding sites in primary human islets verified that a subset of HNF1α-regulated genes were direct targets. These data provide unprecedented mechanistic links between HNF1A loss and diabetic phenotypes in mature human α and β cells.

HNF1α transcriptional activation and repression maintain human islet α and β cell function