AN EMBRYONIC DEMETHYLATION MECHANISM INVOLVING BINDING OF TRANSCRIPTION FACTORS TO REPLICATING DNA

In vertebrates, transcriptionally active promoters are undermethylated , Since the transcription factor Sp1 and more recently NF-kappa B, hav e been implicated in the demethylation process, we examined tile effec t of transcription factors on demethylation by injecting in vitro meth ylated plasmid DNA into Xenopus fertilized eggs. We found that various transactivation domains, including a strong acidic activation domain from the viral protein VP16, can enhance demethylation of a promoter r egion when fused to a DNA binding domain which recognizes the promoter Furthermore, demethylation occurs only after the midblastula transiti on, when the general transcription machinery of the: host embryo becom es available. Nevertheless, transcription factor binding need not be f ollowed by actual transcription, since demethylation is not blocked by a-amanitin treatment, Finally, replication of the target DNA is a pre requisite for efficient demethylation since only plasmids that carry t he bovine papilloma virus sequences which support plasmid replication after the midblastula transition are demethylated, No demethylation is detectable in the oocyte system where DNA fs not replicated, These re sults suggest that, in the Xenopus embryo, promoters for which transcr iption factors are available are demethylated by a replication-depende nt, possibly passive mechanism.