Mutations in the gene encoding the transcription factor regulatory factor X-box binding 6 (RFX6) are associated with human diabetes. Within pancreatic islets, RFX6 expression is most abundant in islet α-cells, and α-cell RFX6 expression is altered in diabetes. However, the roles of RFX6 in regulating gene expression, glucagon output, and other crucial human adult α-cell functions are not yet understood. We developed a method for selective genetic targeting of human α-cells and assessed RFX6-dependent α-cell function. RFX6 suppression with RNA interference led to impaired α-cell exocytosis and dysregulated glucagon secretion in vitro and in vivo. By contrast, these phenotypes were not observed with RFX6 suppression across all islet cells. Transcriptomics in α-cells revealed RFX6-dependent expression of genes governing nutrient sensing, hormone processing, and secretion, with some of these exclusively expressed in human α-cells. Mapping of RFX6 DNA-binding sites in primary human islet cells identified a subset of direct RFX6 target genes. Together, these data unveil RFX6-dependent genetic targets and mechanisms crucial for regulating adult human α-cell function.

Article Highlights
  • RFX6 is expressed in all islet endocrine cell types and is dysregulated in multiple forms of diabetes, but its function has not yet been delineated in α-cells.

  • We used specific targeting of shRNA-mediated suppression of RFX6 in primary human α-cells to unveil glucagon secretion phenotypes.

  • RFX6 is required in adult human α-cells to maintain gene regulation and hallmark functions, including regulated glucagon secretion.

  • RNA-sequencing and cleavage under targets and release using nuclease studies reveal distinct RFX6 genetic targets in adult human α- and β-cells.

This article contains supplementary material online at https://doi.org/10.2337/figshare.24615675.

R.J.B. is currently affiliated with Diabetes, Obesity, and Metabolism Institute at Icahn School of Medicine, Mount Sinai, New York, NY.

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