2667-P: Exciting Patterns of 5mC and 5hmC in Alpha and Beta Cells of Pancreatic Islets

Introduction and Objective: An important aspect of the epigenetics of gene regulation is methylation of cytosines. Our recent manuscript (https://doi.org/10.1101/2025.07.02.662814 final review Nature Metabolism) identifies changes in DNA methylation that occur with age and T2D in pancreatic islet alpha and beta cells, using bisulfite or enzymatic conversion for DNA sequencing. However, these techniques do not distinguish between 5mC and 5hmC and report the sum of the two. Oxford Nanopore Technology sequencing offers the ability to measure the standard four DNA bases as well as provide separate 5mC and 5hmC status. We seek to disentangle these modifications across pancreatic cell types and identify their different roles in gene regulation. Methods: We performed whole genome Nanopore sequencing of alpha (N=17), beta (N=15), and exocrine (N=13) sorted cells from control donors in the Human Pancreas Analysis Program (HPAP) collection to generate DNA sequence and 5mC and 5hmC modifications. CpG modification levels were tabulated and compared on a variety of genomic and islet gene-regulatory features and related to gene expression levels. Results: Analysis results include segmenting the genome into regions of nearly constant levels of 5mC or 5hmC, revealing that 5mC domains are typically smaller (median=360bp, IQR=530bp) than 5hmC domains (median=1344bp, IQR=1236bp) suggesting that different mechanisms govern these modifications. In active islet enhancers, 5hmC constitutes about 50% of the total 5(h+m)C level in endocrine cells and only 10-15% in exocrine cells, suggesting that activation of islet enhancers involves the oxidation of 5mC to 5hmC. On gene bodies in endocrine cells, 5hmC is about 2x higher than exocrine cells. Conclusion: The higher levels of 5hmC on gene bodies of expressed genes in endocrine cells is reminiscent of the modification levels seen in the brain, and their localization to islet enhancers suggest a role in gene activation. These results support further examination of the relationship of 5hmC and true 5mC with aging and diabetes in islet endocrine cells. Disclosure J. Schug: None. E. Manduchi: None. T. Da: None. S. Dashti: None. C. liu: None. A. Naji: None. K. Kaestner: None.