Administrative supplement for Intersection of signaling pathways and transcription factors regulating islet development

PROJECT SUMMARY Pancreatic beta cell mass is critical for glucose homeostasis and mutations that impact beta cell mass can lead to neonatal or adult-onset diabetes. The parent project aims to understand the signaling and transcriptional control of pancreatic and beta cell development with the hopes of better understanding rare diseases such as pancreas agenesis and leveraging this knowledge to establish how beta cell development could impact diabetes disease progression. This supplement will test the hypothesis that non-coding single nucleotide polymorphisms (SNP)s associated with type 2 diabetes (T2D) can act via modulation of pancreatic development, leading to decreased beta cell mass. Therefore, in the context of insulin resistance this smaller pool of beta cells would be less able to compensate by increased insulin secretion and lead to diabetes progression. Variants within the CDKAL1 locus are some of the most highly associated with T2D susceptibility. We have identified a novel transcriptional start site (TSS) within intron 5 of the CDKAL1 gene that overlaps the region where the majority of CDKAL1 locus T2D susceptibility variants are present. This short CDKAL1 isoform is expressed specifically at the pancreatic endocrine progenitor stage during beta cell development. We will test the requirement of this novel TSS functionally by deleting it using genome editing in pluripotent stem cells and testing the impact on beta cell development using in vitro differentiation. Multiple risk variants are found within 250 base pairs of the novel TSS. We will test the activity of these variants on the expression of the short CDKAL1 isoform using luciferase-based gene regulation assays in a cell line that utilizes the novel CDKAL1 TSS, NCI- H82. We will then go on perform experiments to examine how the variants influence transcription factor binding as a potential mechanism of action. Overall, these studies will investigate if modulation of beta cell develop can be a mechanism of action of some T2D risk variants.