Poster: Chromatin Remodeling & Epigenetics
Abs #
1029: DNA demethylation of the Spm sequence as a consequence of transcriptional activation
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Presenter: |
Cui, Hongchang , hxc167@psu.edu |
Authors | Cui, Hongchang (A) Fedoroff, Nina V. (A) | | Affiliations: |
(A): Biology Department, Plant Physiology Program and Huck Institute of Life Sciences, Penn State University
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| Web Site: | http://www.personal.psu.edu/hxc167 | |
The maize Spm transposon is epigenetically regulated. Its sequence is methylated when the transposon is inactive and unmethylated when the element is active. Spm can be activated by the element-encoded TnpA protein and this is accompanied by demethylation of the Spmpromoter and its downstream GC-rich sequence. In previous studies (Cui and Fedoroff, 2002. Plant Cell, 14(11): 2883-99), we demonstrated that TnpA-mediated Spm demethylation is an active process. We also showed that TnpA is a transcriptional activator and this activity is required for TnpAÕs ability to promote Spm demethylation.
The Spmpromoter, but not the GC-rich sequence, contains multiple copies of TnpA binding site. TnpA could promote active DNA demethylation if it has DNA demethylase activity or could recruit such an enzyme to the Spm sequence. To distinguish these possibilities, we first developed an in vitro DNA demethylase assay aimed at sensitive detection of TnpA-associated DNA demethylase activity. We detected a DNA demethylase activity in nuclear extracts prepared from wildtype tobacco suspension cultured cells. We then purified plant-expressed TnpA protein by immunoprecipitation, however, our results did not reveal direct association between TnpA and DNA demethylase activity. We further generated transgenic tobacco calli that contain the Spmsequence under the control of a glucocorticoid inducible promoter. Rapid DNA demethylation of the Spm sequence was observed after induction. These results taken together ruled out the possibility that TnpA itself is a sequence-specific DNA demethylase, and strongly suggest that recruitment of DNA demethylase to the Spm sequence is a consequence, rather than a cause, of transcription activation.
