Dissecting the Mechanism Underlying Epigenetic Activation of the Maize <I>Spm</I> Transposon by the Element-Encoded TnpA Protein
Open Access
- Author:
- Cui, Hongchang
- Graduate Program:
- Plant Physiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- April 30, 2003
- Committee Members:
- Nina Fedoroff, Committee Chair/Co-Chair
Jerry L Workman, Committee Member
Hong Ma, Committee Member
Teh Hui Kao, Committee Member - Keywords:
- transposon
Spm
Epigenetic
demethylation
methylation
demethylase
TnpA
transposase
inducible promoter
transcription
chromatin - Abstract:
- The activity of the maize <I>Spm</I> 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 <I>Spm</I> 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 <I>Spm</I> -encoded transposase proteins, as the trans-acting factor that mediates the epigenetic activation of an inactive <I>Spm</I>. <P>To dissect the underlying mechanism, I developed a novel system that permits demethylation of the <I>Spm</I> 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. <P>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 <I>Spm</I> 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 <I>Spm</I> demethylation was observed when transcription through the <I>Spm</I> 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 <I>Spm</I> demethylation. By using a gel mobility shift assay I showed that the binding affinity of TnpA to fully methylated DNA fragments derived from <I>Spm</I> 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 <I>Spm</I> sequence and promotes rapid DNA demethylation by making the <I>Spm</I> sequence accessible to DNA demethylases as a result of transcriptional activation.