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Heritable epigenetic inactivation of the maize Suppressor-mutator (Spm) transposon is associated with promoter methylation, and its reversal is mediated by the transposon-encoded TnpA protein. We have developed an assay that permits demethylation of the Spm sequence to be controlled by inducing the expression of TnpA in plant cells. Using this assay, we show that demethylation is a rapid, active process. TnpA is a weak transcriptional activator, and deletions that abolish its transcriptional activity also eliminate its demethylation activity. We show that cell cycle and DNA synthesis inhibitors interfere with TnpA-mediated Spm demethylation. We further show that TnpA has a much lower affinity for fully methylated than for hemimethylated or unmethylated DNA fragments derived from Spm termini. Based on these observations, we suggest that TnpA binds to the postreplicative, hemimethylated Spm sequence and promotes demethylation either by creating an appropriate demethylation substrate or by itself participating in or recruiting a demethylase.
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