Programming the Transcriptional State of Replicating Methylated DNA

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 replicationcoupled 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.CpG methylation is causal for transcriptional silencing, imprinting, and X inactivation (1-4). CpG island hypermethylation at promoter elements of tumor suppressor genes correlates with their repression (5-8). The regulatory mechanisms by which DNA methylation interferes with gene activity have begun to be unraveled (2). Large repressor complexes containing methyl-CpG-binding proteins and histone deacetylases have been characterized (9 -11), directly linking CpG methylation to transcriptional repression. To achieve the precise epigenetic control of gene expression, the mechanisms of maintenance methylation, de novo methylation, and demethylation must be strictly regulated.Several mechanisms have been proposed to keep a promoter in a CpG island methylation-free state (reviewed in Ref. 12). Active demethylation occurs during early mouse development (13). However, the existence of proteins exerting demethylase activity has been controversial and remains obscure (Ref. 14 and references therein). Alternatively, DNA replication might be involved in controlling the methylation state of promoters (15). Replicative processes might provide a window of opportunity for transcription factors to bind to hemimethylated DNA and thereby prevent remethylation and subsequent gene repression. Transcription factor binding might interfere with the remethylating activity of DNA methyltransferases at the replication fork (16,17).Transcription factor binding during the replication of DNA leads to subsequent demethylation concomitant with DNA replication (18). In Xenopus eggs it has been shown that replication, together with factor binding but not ongoing transcription, results in partial locus-specific demethylation (15). Alternatively, nonmethylated CpG islands may result from a transcriptionally active gene, the sequences of which also function as an origin of DNA replication at a very early developmental stage (19).All enzymatic activities and co-factors necessary for efficient DNA replication...

The p16-Specific Reactivation and Inhibition of Cell Migration Through Demethylation of CpG Islands by Engineered Transcription Factors

“…This result implies that demethylation maybe a necessary step for reactivation of methylated p16 by p16ATFs. As transient p16ATF transfection alone does not lead to demethylation of p16 CpG islands, the process of stable p16ATF transfection inducing p16 demethylation may be a passive demethylation through inhibition of remethylation of the daughter DNA strands by DNMT1 at the replication forks, as observed in Xenopus oocytes by others (Matsuo et al, 1998;Stü nkel et al, 2001). The decreased binding of DNMT1 to p16 CpG island in the p16ATF transfected H1299 cells supports this hypothesis.…”

Demethylase Activity Is Directed by Histone Acetylation

“…This model has recently gained experimental support (33). However, more recent data have shown that passive demethylation is not simply dependent on the presence of DNA binding transcriptional activators (34). Yet still, other experiments have demonstrated site-and cell-specific active demethylation in transient transfection assays (35), suggesting the presence of site-and cell-specific demethylation machinery (36).…”