Abstract 5018: Multiomic integration of DNA methylation, DNA hydroxymethylation, and gene expression in clear cell renal cell carcinoma

Abstract Background: DNA methylation (5-methylcytosine -5mC) and DNA hydroxymethylation (5-hydroxymethylcytosine -5hmC) are critical epigenetic modifications for understanding gene expression in clear cell renal cell carcinoma (ccRCC). While 5mC and 5hmC modifications may influence promoter and enhancer regions differently, their distinct roles in gene regulation remain unclear. Integrating RNA sequencing (RNA-seq) data with 5mC and 5hmC analyses enables a multiomic understanding of gene regulation, revealing critical changes associated with ccRCC progression. This study integrates 5mC, 5hmC, and RNA-seq data to identify genes and transcriptional regulators driving ccRCC by comparing tumor and normal samples. Methods: We analyzed 188 ccRCC samples from the Dartmouth Renal Tumor Biobank using the Infinium MethylationEPIC BeadChip to differentiate 5-methylcytosine (5mC) from 5-hydroxymethylcytosine (5hmC) via bisulfite and oxidative bisulfite tandem conversion. Whole RNAseq data was obtained from the same samples. Briefly, we used ENmix to process and perform quality control of the 5hmC/5mC data. RNA-seq data was processed using Nextflow rnaseq using the STAR/Salmon alignment option. The distal and proximal genomic associations between 5mC/5hmC and gene expression were analyzed using Enhancer Linking by Methylation/Expression Relationship (ELMER). The supervised mode was used because the two groups of comparisons were specified. For each differentially methylated or hydroxymethylated probes, the CpGs were mapped to nearby genes (10 upstream and 10 downstream), and enriched motifs and associated transcription factors (TFs) were identified. Results/Discussion: Motifs associated with transcription factors ZBTB14 and ZNF148 were enriched in regions of both hypo-hydroxymethylation and hypo-methylation at enhancers, implicating their roles in kidney cancer development1, 2. Both ZBTB14 and ZNF148 have been associated with negative regulation of transcription by RNA polymerase II, while ZNF148 has also been involved in negative regulation of gene expression1, 2. Conclusion: The initial analysis of DNAm and DNAhydroxym after integrating RNAseq in ccRCC allows us to find the essential genes that could contribute to kidney cancer development. Next, we hope to integrate a robust analysis at the transcript level to explore the implications of epigenetic regulation on alternative splicing in ccRCC. Further research is warranted to explore additional regulatory genes and epigenetic alterations in ccRCC progression. Reference 1) doi:10.7554/elife.807602 2) doi:10.1158/0008-5472.can-15-3150 Citation Format: Yiping Li, Brock C. Christensen, Lucas A. Salas. Multiomic integration of DNA methylation, DNA hydroxymethylation, and gene expression in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5018.