Programming the Transcriptional State of Replicating Methylated DNA

Walter Stünkel,Slimane Ait-Si-Ali,Peter L Jones,Alan P Wolffe

doi:10.1074/jbc.m010967200

Abstract

CpG methylation is maintained in daughter chromatids by the action of DNA methyltransferase at the replication fork. An opportunity exists for transcription factors at replication forks to bind their cognate sequences and thereby prevent remethylation by DNA methyltransferase. To test this hypothesis, we injected a linearized, methylated, and partially single-stranded reporter plasmid into the nuclei of Xenopus oocytes and followed changes in the transcriptional activity after DNA replication. We find that dependent on Gal4-VP16, the action of DNA methyltransferase, and replication-coupled chromatin assembly DNA replication provides a window of time in which regulatory factors can activate or repress gene activity. Demethylation in the promoter region near the GAL4 binding sites of the newly synthesized DNA did not occur even though the Gal4 binding sites were occupied and transcription was activated. We conclude that "passive" demethylation at the replication fork is not simply dependent on the presence of DNA binding transcriptional activators.

Highlights

Several mechanisms have been proposed to keep a promoter in a CpG island methylation-free state
We examine whether transcription factors compete with the DNA methylation maintenance machinery on CpG-methylated templates directly at the replication fork and whether this competition can determine the transcriptional fate of a reporter gene
Transcriptional activators or repressors are capable of programming the transcriptional state in the context of methylated CpG dinucleotides within regulatory sites during DNA synthesis

Summary

Programming the Transcriptional State of Replicating Methylated DNA*

An opportunity exists for transcription factors at replication forks to bind their cognate sequences and thereby prevent remethylation by DNA methyltransferase To test this hypothesis, we injected a linearized, methylated, and partially single-stranded reporter plasmid into the nuclei of Xenopus oocytes and followed changes in the transcriptional activity after DNA replication. The modified DNA is microinjected into frog oocytes Using this partially single-stranded methylated template, we find that the stable binding of transcriptional activators or repressors during the synthesis of the second DNA strand programs the gene to remain either active or repressed. This programming is, under our experimental conditions, independent of complete demethylation near the binding site. Factor binding during the time window of second strand synthesis might tag the sites for complete demethylation once replication is completed

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION