Xuehua Zhong, Christopher J. Hale, Minh Nguyen, Israel Ausin, Martin Groth, Jonathan Hetzel, Ajay A. Vashisht, Ian R. Henderson, James A. Wohlschlegel, and Steven E. Jacobsen

Significance

DNA methylation, a chemical mark on chromatin, while not affecting DNA's primary sequence, plays important roles in silencing “bad DNA” that would become deleterious to cells if abnormally expressed. This DNA methylation-mediated silencing system against bad DNA is tightly regulated to prevent the misplacement of methylation on “good DNA.” In Arabidopsis thaliana, DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2) controls RNA-directed DNA methylation in a pathway that also involves the plant-specific RNA Polymerase V (Pol V). The Arabidopsis genome also encodes an evolutionarily conserved but catalytically inactive methyltransferase, DRM3. Here, we investigate the molecular mechanism of DRM3 action on DNA methylation and its dynamic regulation of Pol V transcription. Together, this study sheds further light on the mechanism of RNA-directed DNA methylation.

Abstract

DNA methylation is a mechanism of epigenetic gene regulation and genome defense conserved in many eukaryotic organisms. In Arabidopsis, the DNA methyltransferase DOMAINS REARRANGED METHYLASE 2 (DRM2) controls RNA-directed DNA methylation in a pathway that also involves the plant-specific RNA Polymerase V (Pol V). Additionally, the Arabidopsis genome encodes an evolutionarily conserved but catalytically inactive DNA methyltransferase, DRM3. Here, we show that DRM3 has moderate effects on global DNA methylation and small RNA abundance and that DRM3 physically interacts with Pol V. In Arabidopsis drm3 mutants, we observe a lower level of Pol V-dependent noncoding RNA transcripts even though Pol V chromatin occupancy is increased at many sites in the genome. These findings suggest that DRM3 acts to promote Pol V transcriptional elongation or assist in the stabilization of Pol V transcripts. This work sheds further light on the mechanism by which long noncoding RNAs facilitate RNA-directed DNA methylation.

See: http://www.pnas.org/content/112/3/911.abstract.html?etoc

PNAS January 20, 2015 vol. 112 no. 3 911-916  

Fig. 3.

DRM3 is associated with Pol V in vivo. (A) Complementation test of drm3 mutant with pDRM3::HA-DRM3 at the MEA-ISR locus by restriction digestion with methylation-sensitive MspI and Southern blot. Me, methylated DNA; unMe, unmethylated DNA. (B) Coimmunoprecipitation assays confirming DRM3–NRPE1 interaction. Input lanes confirm the expression of the epitope-tagged proteins in the parental lines and F1. F1 represents the first generation from a cross between the two parental lines.