Genetic programming of human islet metabolic and endoplasmic reticulum (ER) stress responses in diabetes

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PROJECT SUMMARY Type 2 diabetes (T2D) results from failure of pancreatic islets to secrete sufficient insulin to compensate for increased insulin resistance in peripheral tissues. Precise understanding of the molecular mechanisms underlying genetic and environmental contributions to islet failure is essential to develop new, targeted approaches to prevent and treat T2D. Endoplasmic reticulum (ER) and (gluco)lipotoxic stress responses are central (patho)physiologic processes that contribute to islet dysfunction and failure. Our overall objective in this proposal is to elucidate the genetic regulation of islet stress responses and to determine how genetic variants, including SNPs associated with T2D and other metabolic traits (T2D SNPs), modulate these responses to contribute to islet dysfunction and T2D pathogenesis. Based on previous studies and preliminary data, we hypothesize that T2D SNPs alter human islet stress responses by changing islet regulatory element (RE) use/function and expression of their target genes to contribute to islet dysfunction and T2D. In Aim 1, we will test this hypothesis by characterizing islet ER and (gluco)lipotoxic stress responses at the level of gene expression and identifying genetic variants altering human islet stress responses. For this, we will use bulk and single-cell RNA-Seq as well as computational analyses to discover response expression quantitative trait loci (reQTL). In Aim 2, we will take a complementary epigenomic approach to elucidate genetic effects on stress response regulatory element (RE) use in human islets. From the same islets as in Aim 1, we will determine the genome-wide location of REs at steady state and after exposure to stressors using the assay for transposase accessible chromatin-sequencing (ATAC-seq). Using these data, we will computationally identify transcription factors binding to these REs, identify genetic variants altering stress-responsive RE use by chromatin accessibility quantitative trait locus (caQTL) analyses, and test allelic effects on stress-responsive RE activity using massively parallel reporter assays (MPRA) in beta cell lines. Finally, we will experimentally manipulate these islet stress-responsive genes in human EndoC-ßH3 beta cells using CRISPR/Cas9 (epi)genome editing to determine their functions in beta cell proliferation, function, and survival (Aim 3). This study will provide mechanistic insight into how human genetic variation modulates these T2D-relevant stress responses in human islets. By delineating the genes and pathways that modulate islet (gluco)lipotoxic and ER stress responses and experimentally validating their effects on islet/beta cell resilience, this study will reveal novel therapeutic targets and guide strategies for subsequent studies manipulating these responses to prevent or treat islet failure and T2D.