CRISPR screening uncovers a long-range enhancer for ONECUT1 in pancreatic differentiation and links a diabetes risk variant

• DOI: 10.1016/j.celrep.2024.114640
• Publication Date: 2024-08-23T06:51:38Z
• Authors: Samuel Joseph Kaplan; Wilfred Wong; Jielin Yan; Julian Pulecio; Hyein S. Cho; Qianzi Li; Jiahui Zhao; Jayanti Leslie-Iyer; Jonathan Kazakov; Dylan Murphy; Renhe Luo; Kushal K. Dey; Effie Apostolou; Christina S. Leslie; Danwei Huangfu
• Keywords: Pluripotent Stem Cells; 0301 basic medicine; QH301-705.5; Cell Differentiation; Article; 03 medical and health sciences; Enhancer Elements, Genetic; Diabetes Mellitus, Type 2; GATA6 Transcription Factor; Humans; CP: Molecular biology; Clustered Regularly Interspaced Short Palindromic Repeats; CP: Developmental biology; Biology (General); CRISPR-Cas Systems; Pancreas

AbstractFunctional enhancer annotation is a valuable first step for understanding tissue-specific transcriptional regulation and prioritizing disease-associated non-coding variants for investigation. However, unbiased enhancer discovery in physiologically relevant contexts remains a major challenge. To discover regulatory elements pertinent to diabetes, we conducted a CRISPR interference screen in the human pluripotent stem cell (hPSC) pancreatic differentiation system. Among the enhancers uncovered, we focused on a long-range enhancer ∼664 kb from theONECUT1promoter, since coding mutations inONECUT1cause pancreatic hypoplasia and neonatal diabetes. Homozygous enhancer deletion in hPSCs was associated with a near-complete loss ofONECUT1gene expression and compromised pancreatic differentiation. This enhancer contains a confidently fine-mapped type 2 diabetes associated variant (rs528350911) which disrupts a GATA motif. Introduction of the risk variant into hPSCs revealed substantially reduced binding of key pancreatic transcription factors (GATA4, GATA6 and FOXA2) on the edited allele, accompanied by a slight reduction ofONECUT1transcription, supporting a causal role for this risk variant in metabolic disease. This work expands our knowledge about transcriptional regulation in pancreatic development through the characterization of a long-range enhancer and highlights the utility of enhancer discovery in disease-relevant settings for understanding monogenic and complex disease.