"Mechanism of RNA-directed DNA methylation in plants"
RNA-directed DNA methylation (RdDM) is a process known to influence gene expression and transposon silencing in plants by establishing repressive chromatin modifications. The central element of this process is long non-coding RNAs (lncRNAs), which are produced by a specialized RNA polymerase known as Pol V. These lncRNAs recruit a de novo methyltransferase, DRM2, which brings about DNA methylation to enable silencing of their target loci.
Recent studies have shown that RdDM includes active positioning of nucleosomes on silenced loci. The main goal of this study is to determine if nucleosome positioning determines the pattern of de novo DNA methylation. Genome-wide nucleosome positions were identified using MNase-seq and DNA methylation distribution was determined using Whole Genome Bisulfite Sequencing (WGBS). The data indicate a strong negative correlation between positions of nucleosomes and DNA methylation levels. Preliminary analyses of mutant ATP-dependent chromatin remodelers show that nucleosome positioning does not affect DNA methylation suggesting either DNA methylation controls nucleosome positioning, or both these phenomena occur in parallel.
Since Pol V is the most important factor controlling each of these phenomena, it is important to understand how Pol V activity is regulated. The hypothesis that DNA methylation affects Pol V recruitment to the RdDM loci was tested. RNA-immunoprecipitation was performed with Pol V antibody in DRM2 mutants. Preliminary analysis shows a reduced recruitment of Pol V to the loci in absence of DRM2, suggesting that DRM2 and Pol V are part of a positive feedback loop.