Dissecting cell type-specific genetic programming of islet (dys)function in type 2 diabetes

Jackson Laboratory, Bar Harbor ME

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Abstract

PROJECT SUMMARY Type 2 diabetes (T2D) is a complex genetic disease that occurs when pancreatic islets cannot secrete sufficient insulin to overcome peripheral tissue insulin resistance. Genome-wide association studies (GWAS) have identified DNA sequence variants associated with T2D (T2D SNVs) in >600 regions of the human genome. T2D SNVs are enriched in islet cis-regulatory elements (CREs), implicating them as putative causal and functional variants that contribute to T2D by altering islet transcriptional regulation and function. Focused variant-to-function efforts are critical to understand the biological significance of these observations and identify novel therapeutic targets in T2D. In recent work, we nominated 145 T2D SNVs as high-confidence functional variants that significantly alter in vivo islet cis-regulatory element (CRE) chromatin accessibility (islet caQTL), but only a subset of these variants has been linked by us or others to their candidate target genes. Based on published and preliminary data, we hypothesize that these T2D SNVs alter cell type-specific (e.g., alpha, beta, and/or delta) CRE use, activity, and target gene expression to contribute to islet (dys)function in T2D risk and progression. In Aim 1, we will define how each of these T2D SNVs alters islet cell type-specific CRE use via chromatin accessibility quantitative trait locus (caQTL) analysis of single nucleus ATAC-seq profiles from an 80-donor islet cohort and CRE activity using massively parallel reporter assays (MPRA) in human islets. In Aim 2, we will determine the target genes of our nominated T2D-overlapping CREs with cell type-specific resolution using innovative and complementary computational (chromatin co-accessibility), genomic (CRISPR-QTL), and genetic (expression QTL) approaches in primary human islets. In Aim 3, we will assess how T2D-associated changes in islet cell type-specific target gene expression and composition affect islet (dys)function using both CRISPR/Cas9 and conventional knock-down and overexpression approaches to engineer human pseudoislets. This study will significantly advance our understanding of the islet cell type-specific determinants of T2D risk and pathophysiology. With complementary genetic, computational, and cell engineering approaches, we will identify and validate T2D target genes as high-priority therapeutic candidates for detailed mechanistic studies, preclinical models, and drug screening. Importantly, the development of ad hoc genomic tools to modulate and monitor cell type-specific gene regulation and output in primary human islets will enhance cell-type-oriented studies in primary and stem cell-derived human islets for disease modeling. Together, the novel tools and knowledge will catalyze progress in T2D risk assessment, prevention, and treatment.