Abstract A009: Direct multi-omics for the masses: Linking DNA methylation to chromatin targets via CUT&RUN-EM

Abstract Localized DNA methylation (DNAme), particularly 5-methylcytosine (5mC), contributes to the regulation of gene expression, imprinting, and the silencing of parasitic DNA elements, with dysregulation implicated in multiple diseases including cancer. Understanding how DNAme signaling drives physiological dysfunction has great potential to reveal novel biomarkers for therapeutic strategies. Despite growing evidence that DNAme is functionally linked to various histone post translational modifications (PTMs), these entities are generally independently genomic mapped, with the resulting data at best correlative. Further, the traditional approach to measure 5mC relies on bisulfite conversation, a harsh chemical treatment that introduces DNA breaks and systemic biases. To address these limitations, we have developed CUT&RUN-EM. This multi-omic genomic mapping technology delivers high resolution DNAme profiles at epitope-defined chromatin features (histone PTMs or chromatin associated proteins (CAPs)), thus interrogating the potential for a direct molecular link. The ultra-sensitive workflow seamlessly integrates CUT&RUN with enzymatic methyl-seq (EM-seq) and incorporates in situ standards to monitor specificity and efficiency at each step. CUT&RUN uses antibodies to locally tether protein A/G-micrococcal nuclease (pAG-MNase) to chromatin within intact cells or nuclei, followed by controlled nuclease activation to cleave nearby DNA. EM-seq then enzymatic converts 5mC/5hmC in sub-ng amounts of enriched DNA to generate single-base resolution, unbiased DNAme profiles. To determine the capabilities and limitations of CUT&RUN-EM, we tested multiple chromatin targets and cell types. With in situ standards throughout the workflow, we demonstrate that CUT&RUN-EM is highly reproducible, specific, and efficient (<10% off-target binding, >90% enzymatic conversion of DNAme, <0.5% conversion of unmethylated DNA). The targeted approach is also highly-sensitive, using <500k cells (depending on PTM/CAP abundance) and <5M short-read sequences per assay (10-50x less than whole-genome 5mC-sequencing). To demonstrate potential applications and the mechanistic insight provided by true-multiomics, we have explored the impact of 5mC within DNA motifs on transcription factor binding; the recruitment of de novo methytransferase DNMT3A1 to PTM enriched chromatin; and the DNAme binding preference of MeCP2 alleles linked to neurodegenerative Rett syndrome. Citation Format: Vishnu Sunitha Kumary, Allison Hickman, Bryan J Venters, Anup Vaidya, James Anderson, Jonathan M Burg, Louise Williams, Chaithanya Ponnaluri, Pierre Esteve, Issac Meek, Zu-Wen Sun, Martis W Cowles, Sriharsa Pradhan, Michael-Christopher Keogh. Direct multi-omics for the masses: Linking DNA methylation to chromatin targets via CUT&RUN-EM [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr A009.