Dissecting the mechanism underlying epigenetic activation of the maize Spm transposon by the element-encoded TnpA protei

The activity of the maize Spm transposon is epigenetically regulated. The promoter and its downstream GC-rich sequence are extensively methylated when the transposon is inactive and unmethylated when the element is active. A methylated, inactive Spm transposon can be activated by an active element, and the activation is accompanied by loss of methylation in both the promoter and the GC-rich sequence. Previous studies have identified TnpA, one of the Spm-encoded transposase proteins, as the trans-acting factor that mediates the epigenetic activation of an inactive Spm.;To dissect the underlying mechanism, I developed a novel system that permits demethylation of the Spm sequence to be controlled by inducing expression of TnpA in transgenic tobacco calli. I showed that TnpA-mediated DNA demethylation occurs at a rate much faster than that attributable to interference with methylation maintenance during DNA replication, suggesting an enzymatic process. I then developed an in vitro DNA demethylase assay, and detected a DNA demethylase activity in tobacco suspension cultured cells. However, no DNA demethylase activity was detected in the TnpA protein or possible interacting proteins.;In further studies, I showed that TnpA is a weak transcriptional activator and that deletions that disrupt its ability to activate transcription also eliminate its ability to promote DNA demethylation. Moreover, the fusion protein between the truncated protein and the viral VP16 activation domain has stronger DNA demethylation activity as well as transcriptional activity. Furthermore, rapid Spm demethylation was observed when transcription through the Spm sequence was activated from an upstream glucocorticoid-inducible promoter.;Using inhibitors of DNA replication and cell division, I demonstrated that DNA replication is also required for TnpA-mediated Spm demethylation. By using a gel mobility shift assay I showed that the binding affinity of TnpA to fully methylated DNA fragments derived from Spm termini is much lower than its affinity for the same fragments when hemi-methylated or unmethylated. Based on these observations, a two-step DNA demethylation mechanism is suggested where TnpA binds to the post-replicative, hemi-methylated Spm sequence and promotes rapid DNA demethylation by making the Spm sequence accessible to DNA demethylases as a result of transcriptional activation.