SAT-516 - Non-Coding Regulation of ONECUT1 Controls Pancreatic Progenitor Specification and Diabetes Susceptibility

Genetic susceptibility to diabetes spans a wide spectrum, from rare, high-penetrance coding mutations that cause monogenic disease to common non-coding variants that contribute to complex disorders such as type 2 diabetes (T2D). At the ONECUT1 locus—a CUT homeobox transcription factor essential for pancreatic development—both coding mutations and regulatory variants have been linked to syndromic monogenic diabetes and multifactorial T2D, underscoring the importance of non-coding gene regulation in disease pathogenesis.
Here, we used a human pluripotent stem cell (hPSC)–based pancreatic differentiation system to investigate the function of distal non-coding regulation of ONECUT1 during stages of pancreatic progenitor differentiation. A long-range enhancer located approximately 664 kb upstream of the ONECUT1 promoter (ONECUT1e-664kb) was deleted using CRISPR-based genome editing, and transcriptional sequencings were assessed across developmental stages corresponding to the primitive gut tube (GT) and posterior foregut (PFG). Reanalysis of these sequencing data (GSE261330) revealed marked stage-specific effects of enhancer disruption. Principal component analysis (PCA) showed limited separation among wild-type, heterozygous, and homozygous enhancer-deleted cells at the GT stage, whereas clear segregation of homozygous mutants at the PFG stage. Homozygous deletion led to significant downregulation of 35 genes, while heterozygous cells remained largely indistinguishable from wild type, indicating a dosage-sensitive regulatory requirement.
Pathway analysis of downregulated genes revealed coordinated suppression of programs including pancreatic development and endocrine differentiation (ONECUT1, ONECUT2, ONECUT3, INSM1, HES1, CADPS), oxidative stress defense and metabolic regulation (CAT, AMBP, ADH1B, ITIH2), epithelial integrity and tissue architecture (CLDN1, CHL1, NRCAM, DCC, GRIP2, SEZ6L, WDR72), inflammatory and immune signaling (CXCL1, CXCL6, NLRP2, TRPC4), neuroendocrine calcium signaling (CALB1, ST8SIA3, LRATD1), transcriptional and post-transcriptional regulation (ELL2, HIST1H1A, DNAJC22, MPDU1, UBE2QL1). Together, these pathways link with ONECUT1 regulation to integrated control of pancreatic lineage progression, metabolic homeostasis, and inflammatory responses.
In conclusion, these findings establish ONECUT1e-664kb as a critical long-range enhancer required for pancreatic progenitor maturation and endocrine gene activation. This study provides functional evidence connecting non-coding regulatory variation at the ONECUT1 locus to coordinated disruption of pancreatic developmental and metabolic programs, offering a scalable framework for prioritizing diabetes-associated non-coding variants and elucidating mechanisms underlying both monogenic and complex diabetes susceptibility.

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