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In mammalian cells, DNA methylation is associated with heritable and stable gene repression, mediated in part by methyl-CpG-binding domain (MBD) proteins that recruit corepressors to modify chromatin. MBD1 protein, a member of the MBD family, forms a complex with SETDB1 histone methylase to silence transcription at target promoters by methylation of lysine 9 of histone H3. How MBD1-mediated transcriptional repression is regulated is currently unknown. Here we show that MBD1 is a target for sumoylation by PIAS1 (Protein Inhibitors of Activated STAT 1) and PIAS3 E3 SUMO (small ubiquitin-like modifier)-ligases, at two conserved lysine residues within the C-terminus of MBD1. Although sumoylated MBD1 binds to methylated DNA, it does not incorporate into a complex with SETDB1 and does not efficiently repress transcription of a target gene, p53BP2, in HeLa cells. Our data suggest that transcriptional silencing by MBD1 is regulated by a PIAS-mediated conjugation of SUMO1, which antagonizes the formation of a repressive complex with SETDB1.

DNA methylation is necessary for normal embryogenesis in animals. Here we show that loss of the maintenance methyltransferase, xDnmt1p, triggers an apoptotic response during Xenopus development, which accounts for the loss of specific cell populations in hypomethylated embryos. Hypomethylation-induced apoptosis is accompanied by a stabilization in xp53 protein levels after the mid-blastula transition. Ectopic expression of HPV-E6, which promotes xp53 degradation, prevents cell death, implying that the apoptotic signal is mediated by xp53. In addition, inhibition of caspase activation by overexpression of Bcl-2 results in the development of cellular masses that resemble embryonic blastomas. Embryonic tissue explant experiments suggest that hypomethylation alters the developmental potential of early embryo cells and that apoptosis is triggered by differentiation. Our results imply that loss of DNA methylation in differentiated somatic cells provides a signal via p53 that activates cell death pathways.

In Escherichia coli, adenine methylation at the sequence GATC allows coupling of cellular processes to chromosome replication and the cell cycle. The transient presence of hemimethylated DNA after replication facilitates post-replicative mismatch repair, induces transcription of some genes and allows transposition of mobile elements. We were interested in estimating the half-life of hemimethylated DNA behind the replication fork in plasmid molecules and in determining whether Dam methyltransferase restores N6 adenine methylation simultaneously on both replicative arms. We show that remethylation takes place asynchronously on the leading and lagging daughter strands shortly after replication. On the leading arm the fully methylated adenine is restored approximately 2000 bp (corresponding to 2 s) behind the replication fork, while remethylation takes twice as long (at 3500-4000 bp or approximately 3.5-4 s) on the lagging replicative arm. This observation suggests that Dam remethylation of the lagging arm requires ligated Okazaki fragments.