DNA methylation is required to maintain both DNA replication timing precision and 3D genome organization integrity.

DNA replication timing and three-dimensional (3D) genome organization are associated with distinct epigenome patterns across large domains. However, whether alterations in the epigenome, in particular cancer-related DNA hypomethylation, affects higher-order levels of genome architecture is still unclear. Here, using Repli-Seq, single-cell Repli-Seq, and Hi-C, we show that genome-wide methylation loss is associated with both concordant loss of replication timing precision and deregulation of 3D genome organization. Notably, we find distinct disruption in 3D genome compartmentalization, striking gains in cell-to-cell replication timing heterogeneity and loss of allelic replication timing in cancer hypomethylation models, potentially through the gene deregulation of DNA replication and genome organization pathways. Finally, we identify ectopic H3K4me3-H3K9me3 domains from across large hypomethylated domains, where late replication is maintained, which we purport serves to protect against catastrophic genome reorganization and aberrant gene transcription. Our results highlight a potential role for the methylome in the maintenance of 3D genome regulation. [Display omitted] • DNA hypomethylation is associated with single-cell replication timing heterogeneity • Hypomethylation induces loss of allelic replication at cancer-related gene loci • Hypomethylation alters higher-order genome architecture of PMD boundaries • Non-canonical H3K4me3-H3K9me3 domains form to protect silent late replication Du et al. demonstrate the importance of DNA methylation in the maintenance of 3D genome regulation. They show that hypomethylation, a hallmark of cancer, is associated with the disruption of 3D genome compartmentalization, gain in replication timing heterogeneity, and loss of allelic replication, potentially through the deregulation of DNA replication and genome organization pathways. [ABSTRACT FROM AUTHOR]