PROJECT SUMMARY/ABSTRACT Pancreatic beta cells secrete insulin in order to maintain blood glucose homeostasis. Insulin secretion is tightly regulated by glucose and modulated by numerous environmental signals, including other nutrients, hormones, and inflammatory cytokines. Exposure of beta cells to environmental signals affects gene regulatory programs within hours, and these signal-dependent changes serve to adapt insulin secretion to changes in organismal states. Genetic variants associated with measures of insulin secretion are strongly enriched in genomic elements active in beta cells, and many of these variants are also associated with risk of diabetes. Beta cells therefore possess characteristics that make them an ideal cellular model for studying signal-dependent gene regulatory processes relevant to human health and disease. However, the specific genomic programs that drive signal- induced state changes in beta cells remain poorly characterized. Recent advances in the development of human pluripotent stem cell (hPSC)-derived multi-cellular islet organoid models by us and others provide a genetically tractable beta cell model for linking genomic activity to cellular phenotypes. Our group has further pioneered the development of numerous single cell assays, including chromatin accessibility, ultra-high-throughput paired chromatin accessibility and gene expression, and paired 3D chromatin interactions and DNA methylation; methods that we have successfully applied to both primary human islets and hPSC-islet organoids. We have further developed machine learning and network-based approaches for variant interpretation including from single cell RNA and epigenetic data. In this proposal we will develop novel gene regulatory network (GRN) models to predict network-level relationships among genomic elements, genes, and phenotypes derived from single cell multiomic maps charting signal- and time-dependent changes in hPSC-islet organoids. In Sections B and C we will measure genomic element activity, gene expression, and insulin secretion in hPSC-islet organoids exposed to ten different secretory signals each across four time points using paired single nucleus accessible chromatin and gene expression and paired single cell DNA methylation and 3D chromatin architecture assays. In Section D we will generate a GRN from these data, use machine learning to infer element-gene and element-phenotype relationships and use the trained models to refine the GRN. From the resulting GRN we will predict the effects of genetic variants in specific genomic elements on target gene expression, gene network activity, and cellular phenotype. In Section E we will validate and refine models by using medium-scale CRISPR interference of genomic elements individually and in combination as well as allele-specific gene editing of selected glucose-associated variants in hPSC-islet organoids and measuring gene expression changes in cis and trans. Together, the results, data, and methods from this project using a model of a cell type which both rapidly responds to environmental signals and has a quantifiable phenotypic output will be widely applicable to the community studying the dynamics of genomic regulation.

PROJECT NARRATIVE Fine-tuned regulation of insulin secretion by pancreatic islet beta cells in response to environmental signals is critical for human health and survival. Our proposal will apply systematic, single cell-resolved multiomic data collection in human pluripotent stem cell-derived islet organoids exposed to secretory signals to predict and validate network-level relationships among genomic elements, genes, and insulin secretion phenotypes.