Single-cell sequencing of mouse thymocytes reveals mutational landscape shaped by replication errors, mismatch repair and H3K36me3

ABSTRACT Background DNA mismatch repair (MMR) safeguards genome stability by correcting errors made during DNA replication. In vitro evidence indicates that the MMR machinery is recruited to chromatin via H3K36me3, a histone mark enriched in 3’ exons of genes and associated with transcriptional activity. To dissect how replication errors, abundance of H3K36me3 and MMR together shape the mutational landscape in normal mammalian cells, we applied single-cell exome sequencing to thymic T cells isolated from MMR-proficient ( Mlh1 +/+ ) and MMR-deficient ( Mlh1 −/− ) mice. Results Using single-cell exome sequencing we identified short deletions as sensitive and quantifiable reporters of MMR-dependent mutations. We found H3K36me3-enriched Huwe1 and Mcm7 genes to be mutational hotspots exclusive to Mlh1 −/− T cells. In Mlh1 +/+ cells, exons of H3K36me3-enriched genes had a lower mutation frequency compared to H3K36me3-depleted genes. Moreover, within transcriptionally active genes, 3’ exons, often H3K36me3-enriched, rather than 5’ exons had significantly fewer MMR-dependent mutations, indicating that MMR operates more efficiently within 3’ exons in Mlh1 +/+ T cells. Conclusions Our results provide evidence that H3K36me3 confers preferential MMR-mediated protection from transcription-associated deleterious replication errors. This offers an attractive concept of thrifty MMR targeting, where genes critical for the development of given cell type are preferentially shielded from de novo mutations by H3K36me3-guided MMR.