Single-Cell Sequencing of Mouse Thymocytes Reveals Mutational Landscape Shaped by Replication Errors, Mismatch Repair, and H3K36me3

DNA mismatch repair (MMR) corrects replication errors and is recruited by the histone mark H3K36me3, enriched in exons of transcriptionally active genes. To dissect in vivo the mutational landscape shaped by these processes, we employed single-cell exome sequencing on T cells of wild-type and MMR-deficient (Mlh1(−/−)) mice. Within active genes, we uncovered a spatial bias in MMR efficiency: 3′ exons, often H3K36me3-enriched, acquire significantly fewer MMR-dependent mutations compared with 5′ exons. Huwe1 and Mcm7 genes, both active during lymphocyte development, stood out as mutational hotspots in MMR-deficient cells, demonstrating their intrinsic vulnerability to replication error in this cell type. Both genes are H3K36me3-enriched, which can explain MMR-mediated elimination of replication errors in wild-type cells. Thus, H3K36me3 can boost MMR in transcriptionally active regions, both locally and globally. This offers an attractive concept of thrifty MMR targeting, where critical genes in each cell type enjoy preferential shielding against de novo mutations.