Xiaofeng Cao 1, 2, Werner Aufsatz 3, Daniel Zilberman 2, M. Florian Mette 3, Michael S. Huang 2, Marjori Matzke 3, and Steven E. Jacobsen * 2, 4
1 Institute of Genetics and Developmental Biology, Chinese
Academy of Sciences, 917 Datun Road, Beijing 100101, China
2 Molecular, Cell, and Developmental Biology, University
of California, Los Angeles, CA 90095 USA
3 Institute of Molecular Biology, Austrian Academy of
Sciences, Billrothstrasse 11, A-5020 Salzburg, Austria
4 Molecular Biology Institute, University of California,
Los Angeles, CA 90095 USA
* Correspondence: Steven E. Jacobsen;
(310) 825-0182 (phone); (310) 206-3987 (fax);
E-mail: jacobsen@ucla.edu
Abstract:
RNA interference is a conserved process in which double-stranded
RNA is processed into 21–25 nucleotide
siRNAs that trigger posttranscriptional gene silencing. In addition,
plants
display a phenomenon termed
RNA-directed DNA methylation (RdDM) in which DNA with sequence
identity to silenced RNA is de novo
methylated at its cytosine residues. This methylation is not only
at canonical CpG sites but also at cytosines in
CpNpG and asymmetric sequence contexts. In this report, we study
the role of the DRM and CMT3 DNA
methyltransferase genes in the initiation and maintenance of RdDM.
Neither drm nor cmt3 mutants affected the
maintenance of preestablished RNA-directed CpG methylation. However,
drm
mutants showed a nearly
complete loss of asymmetric methylation and a partial loss of CpNpG
methylation. The remaining asymmetric
and CpNpG methylation was dependent on the activity of CMT3,
showing that DRM and CMT3 act
redundantly to maintain non-CpG methylation. These DNA methyltransferases
appear to act downstream of
siRNAs, since drm1 drm2 cmt3 triple mutants
show a lack of non-CpG methylation but elevated levels of
siRNAs. Finally, we demonstrate that DRM activity is required
for the initial establishment of RdDM in all
sequence contexts including CpG, CpNpG, and asymmetric sites.
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