Abstract
In Arabidopsis, the 24-nucleotide (nt) small interfering RNAs (siRNAs) mediates RNA-directed DNA methylation (RdDM) and transcriptional gene silencing (TGS) of transposable elements (TEs). In the present study, we examined genome-wide changes in DNA methylation and siRNA accumulation in Arabidopsis induced by expression of the Cucumber mosaic virus silencing suppressor protein 2b known to directly bind to both the 21/24-nt siRNAs as well as their associated Argonaute proteins. We demonstrated a genome-wide reduction of CHH and CHG methylation in the 2b-transgenic plants. We found that 2b suppressed RdDM not only at the previously annotated loci directed by 24-nt siRNAs but also a new set of loci associated with 21/22-nt siRNAs. Further analysis showed that the reduced methylation of TEs and coding genes targeted by 21/22-nt siRNAs was associated with sequestration of the duplex siRNAs by the 2b protein but not with changes in either siRNA production or transcription. Notably, we detected both the deletion and/or the transposition of multicopy TEs associated with 2b-induced hypomethylation, suggesting potential TE reactivation. We propose that the silencing of many TEs in Arabidopsis is controlled by the 24- and 21-nt endogenous siRNAs analogous to Drosophila TE silencing by PIWI-interacting RNAs and siRNAs.
Highlights
The methylation of cytosines in nuclear DNA is a conserved epigenetic silencing mechanism and controls many important biological processes, including defense against transposon proliferation, control of genomic imprinting, and the regulation of gene expression, which imparts an additional layer of heritable information upon the DNA code[1,2,3,4]
We found that tandem repeat, non-codingRNA, pseudogenes and transposons were highly methylated in both Col-0 and 2b-3 plants (Fig. 1A)
The DNA methylation levels in CG, CHG and CHH of the expressed genes showed a tendency to decrease at transcription start sites (TSS), but the methylation level of CG to increase at gene bodies (Fig. 1B), which was consistent with previous reports for the whole genome methylation sequencing results in Col-031,32 and 2b-3 (Figures S1 and S2)
Summary
The methylation of cytosines in nuclear DNA is a conserved epigenetic silencing mechanism and controls many important biological processes, including defense against transposon proliferation, control of genomic imprinting, and the regulation of gene expression, which imparts an additional layer of heritable information upon the DNA code[1,2,3,4]. Comprehensive methylome analyses of many Arabidopsis silencing mutants have shown that among all of the mutants that are not involved in canonical 24-nt-siRdDM, the rdr[1] mutant was even stronger than the rdr[6] mutant in reducing the methylation of all CG, CHG, and CHH contexts in chromosome 116 in regions that were more likely to be associated with genes and 21-22-nt siRNAs in wild-type Arabidopsis[17] These data suggest that proteins other than RDR6, such as RDR1, that generate 21-22-nt siRNAs play a role and affect more loci in DNA methylation. Genome-wide 21-22-nt siRNA-related methylation loci ( referred to as 21/22-nt-siRdDM to distinguish the canonical 24-nt-siRdDM) remain to be elucidated
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