epigenetics

M.A. Abdurashitov, A.G. Akishev, S.Kh. Degtyarev SibEnzyme Ltd, Novosibirsk Corresponding author: M.A. Abdurashitov, SibEnzyme Ltd., 2/12 Ak. Timakova Street,Novosibirsk 630117, Russia; Tel.: +7 (383) 333-4991; Fax: +7 (383) 333-6853; E-mail: abd@sibenzyme.ru Abstract The 5-methylcytosine-dependent DNA endonuclease GlaI specifically recognizes and cleaves methylated DNA sites of the sequence 5′-R(5mC)GY-3′ / 3′-YG(5mC)R-5′, which are generated de novo in the human genome by DNMT3 DNA methyltransferases. Due to this specificity, GlaI can be effectively used for assessing the methylation status of RCGY sites in mammalian genomes. Genome-wide mapping of R(5mC)GY sites can be achieved by next-generation sequencing (NGS) of GlaI DNA hydrolysates, whereas targeted analysis of individual genomic regions can be performed using GlaI-PCR analysis. In the present study, we compared the results of NGS analysis of GlaI DNA hydrolysates with data obtained by GlaI-PCR analysis for a set of regulatory regions of tumor suppressor genes in human cell lines L68 (normal lung fibroblasts), Raji (Burkitt lymphoma), and U937 (histiocytic lymphoma). The majority of analyzed regulatory regions were unmethylated in L68 cells, while extensive hypermethylation was observed in malignant cell lines Raji and U937. For almost all analyzed genomic fragments, the methylation status determined by GlaI-PCR analysis fully correlated with the number of R(5mC)GY sites detected by NGS. A single discrepancy was observed for the RASSF1A regulatory region in L68 cells, which was resolved by additional GlaI-PCR analysis in real-time, demonstrating allele-specific methylation of this fragment. Taking allelic methylation into account resulted in full concordance between NGS and GlaI-PCR data. Overall, the results demonstrate that NGS analysis of GlaI DNA hydrolysates and GlaI-PCR analysis are complementary and mutually validating approaches for assessing DNA methylation at RCGY sites. Combined application of these methods enables reliable identification of aberrantly methylated genomic regions in normal and malignant human cells and can be effectively used in epigenetic studies and DNA-based diagnostics. Keywords: 5-methylcytosine; DNA methylation; methyl-dependent restriction endonucleases;GlaI; NGS analysis; GlaI-PCR analysis; epigenetic DNA diagnostics; allele-specific methylation. DOI: 10.26213/3034-4298.2025.6.1.001 Citation: M.A. Abdurashitov, A.G. Akishev, S.Kh. Degtyarev (2025) Determination of the Methylation Status of RCGY Sites in the Human Genome: A Comparison of NGS Analysis of GlaI DNA Hydrolysates and GlaI-PCR Data, Epigenetic DNA diagnostics, vol 2025(1), DOI: 10.26213/3034-4298.2025.6.1.001 This work is available at Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International Введение 5-метилцитозин-зависимая ДНК-эндонуклеаза GlaI узнает и расщепляет в ДНК человека сайты…