The role of mammalian DNA methyltransferases in the regulation of temporal order of DNA replication [microarray]

Multiple epigenetic pathways underlie the temporal order of DNA replication (replication timing) in the context of development and disease. DNA methylation by DNA methyltransferases (DNMTs) and downstream chromatin reorganization and transcriptional changes are thought to impact DNA replication, yet this remains to be comprehensively tested. Using cell biological and genome-wide approaches to measure replication timing, we identified a number of genomic regions undergoing subtle but reproducible replication timing changes in various DNMT-mutant mouse ES cell lines that include a line with a drug-inducible DNMT3a2 expression system. Replication timing within pericentromeric heterochromatin (PH) correlates with redistribution of H3K27me3 induced upon DNA hypomethylation: later replicating PH coincides with H3K27me3 enriched regions. In contrast, this relationship with H3K27me3 was not evident at chromosomal arm regions that undergo either early-to-late (EtoL) or late-to-early (LtoE) replication timing switching upon loss of DNMTs. Interestingly, transcriptional up- and down-regulation frequently coincide with earlier and later shifts in replication timing of chromosomal arm regions, respectively. Our study revealed previously unrecognized complex and diverse roles of DNMTs in shaping the mammalian DNA replication landscape. We investigated the effect of Dnmt3a2 expression in the transcription profile of Dnmt3a and 3b double knockout (DKO) ES cells. Dnmt3a−/−Dnmt3b−/− DKO ES cell clones stably expressing RU486 (mifepristone, a progesterone receptor antagonist)-inducible DNMT3a2 were generated by electroporation-mediated introduction of an expression plasmid for DNMT3a2 fused with the ligand-binding domain of the human progesterone receptor driven by the CAG promoter, followed by selection with blasticidin. To induce DNMT3a2 protein, obtained ES cells (clone 1723) were cultured in ES medium containing 1 µM RU486 for 14 days on feeder MEFs. As a control, cells were treated with ethanol. RNA was isolated from these cells and hybridized to Affymetrix microarrays