DNMT1 loss leads to hypermethylation of a subset of late replicating domains by DNMT3A

Loss of DNA methylation is a hallmark of cancer that is proposed to promote carcinogenesis through gene expression alterations, retrotransposon activation and induction of genomic instability. Cancer-associated hypomethylation does not occur across the whole genome but leads to the formation of partially methylated domains (PMDs). However, the mechanisms underpinning PMD formation remain unclear. PMDs replicate late in S-phase leading to the hypothesis that they become hypomethylated due to incomplete re-methylation by the maintenance methyltransferase DNMT1 during cell division. Here we investigate the role of DNMT1 in shaping the cancer methylome by conducting whole genome bisulfite sequencing (WGBS), repli-seq and ChIP-seq on DNMT1 knockout HCT116 colorectal cancer cells (DNMT1 KO cells). We find that DNMT1 loss leads to preferential hypomethylation in late replicating, heterochromatic PMDs marked by the constitutive heterochromatic mark H3K9me3 or the facultative heterochromatic mark H3K27me3. However, we also observe that a subset of H3K9me3-marked PMDs gain methylation in DNMT1 KO cells. We find that, in DNMT1 KO cells, these hypermethylated PMDs remain late replicating but DNMT3A localises to them. This is accompanied by loss of heterochromatic H3K9me3, specific gain of euchromatic H3K36me2 and some gene upregulation. These same domains also have more variable DNA methylation than other PMDs in colorectal tumours in vivo. Our observations suggest that hypermethylated PMDs lose their heterochromatic state, enabling their methylation by DNMT3A and the establishment of a hypermethylated, non-PMD state, despite their late replication timing. More generally, our findings suggest that differential de novo DNMT activity plays a key role in establishing domain level DNA methylation patterns in cancer cells.