Mechanistic role of ARGONAUTE4 in RNA-directed DNA methylation in Arabidopsis thaliana

Epigenetic gene silencing through DNA methylation is one of several mechanisms available to control gene expression. Numerous studies have linked components of the RNA interference (RNAi) pathway to gene silencing. One such component is the protein Argonaute which binds 21-24 nucleotide long RNAs called small interfering RNAs (siRNAs). In Arabidopsis thaliana, ARGONAUTE4 (AGO4) was shown to be exclusively involved in transcriptional gene silencing in the phenomenon termed RNA-directed DNA methylation (RdDM), where siRNAs cause DNA methylation at complementary sequences. From mutant analysis, the loss of AGO4 affected both DNA methylation and histone modifications at certain loci. However, the exact mechanism of how AGO4 functions to affect DNA methylation and/or histone methylation leading to transcriptional silencing is still unclear.;This study focuses on the mechanistic role of AGO4 in gene silencing by examining the subnuclear localization and the various interacting partners of AGO4. Results from this work include the dynamic localization of AGO4 to two distinct populations of nuclear bodies that either colocalizes with the Cajal body or is immediately adjacent to the 45S ribosomal DNA loci. Other gene silencing factors which colocalize with the 45S-adjacent AGO4 nuclear body include the RNA Polymerase IV subunits NRPD1b and NRPD2, and the DNA methyltransferase DRM2. Consistent with their colocalization, AGO4 interacts with NRPD1b in vivo and copurifies with the C-terminal domain of NRPD1b in vitro. Surprisingly, AGO4 also associates with the N-terminal tail of histone H3 in vitro.;The findings from this work reveal an intricate regulation of AGO4 localization to chromatin and within Arabidopsis nuclei. While targeting to specific genes requires siRNAs, the stable association of AGO4 at chromatin may require interaction with NRPD1b and the tail of histone H3. On a subcellular level, the spatial distribution of AGO4 within the nucleus may reflect active sites of RdDM (such as at the 45S loci), or sites of siRNA-loading or priming of AGO4 for RdDM action (possibly at the Cajal body). This study provides a novel insight into the regulation of AGO4 within the nucleus, and further elucidates the mechanism of DNA methylation that is AGO4 dependent.