Climent-Cantó, PaulaSubirana-Granés, MarcRamos-Rodríguez, MireiaDámaso, EstelaMarín, FátimaVara, CovadongaPérez-González, BeatrizRaurell Vila, HelenaMunté, ElisabetSoto, José LuisAlonso, ÁngelShin, GiWonJi, HanleeHitchins, MeganCapellá, GabrielPasquali, LorenzoPineda, Marta2025-02-202025-02-202024Climent-Cantó P, Subirana-Granés M, Ramos-Rodríguez M, Dámaso E, Marín F, Vara C, et al. Altered chromatin landscape and 3D interactions associated with primary constitutional MLH1 epimutations. Clin Epigenetics. 2024 Dec 31;16(1):193. DOI: 10.1186/s13148-024-01770-31868-7075http://hdl.handle.net/10230/69656Background: Lynch syndrome (LS), characterised by an increased risk for cancer, is mainly caused by germline pathogenic variants affecting a mismatch repair gene (MLH1, MSH2, MSH6, PMS2). Occasionally, LS may be caused by constitutional MLH1 epimutation (CME) characterised by soma-wide methylation of one allele of the MLH1 promoter. Most of these are "primary" epimutations, arising de novo without any apparent underlying cis-genetic cause, and are reversible between generations. We aimed to characterise genetic and gene regulatory changes associated with primary CME to elucidate possible underlying molecular mechanisms. Methods: Four carriers of a primary CME and three non-methylated relatives carrying the same genetic haplotype were included. Genetic alterations were sought using linked-read WGS in blood DNA. Transcriptome (RNA-seq), chromatin landscape (ATAC-seq, H3K27ac CUT&Tag) and 3D chromatin interactions (UMI-4C) were studied in lymphoblastoid cell lines. The MLH1 promoter SNP (c.-93G > A, rs1800734) was used as a reporter in heterozygotes to assess allele-specific chromatin conformation states. Results: MLH1 epimutant alleles presented a closed chromatin conformation and decreased levels of H3K27ac, as compared to the unmethylated allele. Moreover, the epimutant MLH1 promoter exhibited differential 3D chromatin contacts, including lost and gained interactions with distal regulatory elements. Of note, rare genetic alterations potentially affecting transcription factor binding sites were found in the promoter-contacting region of CME carriers. Conclusions: Primary CMEs present allele-specific differential interaction patterns with neighbouring genes and regulatory elements. The role of the identified cis-regulatory regions in the molecular mechanism underlying the origin and maintenance of CME requires further investigation.application/pdfeng© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.info:eu-repo/semantics/articlehttp://dx.doi.org/10.1186/s13148-024-01770-3Cis-regulatory regionsMLH1 promoter methylation3D interactionsChromatin structureConstitutional MLH1 epimutationLynch syndromeinfo:eu-repo/semantics/openAccess