Genome-wide Identification of Non-coding Transcription by RNA Polymerase V and Its Involvement in Transcriptional Gene Silencing

Abstract

RNA-mediated transcriptional gene silencing is a conserved process where non-coding RNAs target transposons and other sequences for repression by establishing repressive chromatin modifications. A central element of this process is long non-coding RNAs (lncRNAs), which in Arabidopsis thaliana are produced by a specialized RNA polymerase known as Pol V. These lncRNAs recruit small interfering RNAs (siRNAs) and a series of proteins that lead to the establishment of RNA-directed DNA methylation (RdDM) on transposable elements. Transposable elements extant in eukaryotic genomes pose a constant risk of disrupting the integrity of the genome via random integration events and are targeted for silencing by the RdDM machinery. The RdDM pathway results in de novo DNA methylation and it has been quite extensively researched, however, questions about the mechanism of recruitment of Pol V to RdDM loci and subsequent interplay between chromatin modifications and the downstream mechanism of gene silencing are still less understood.

In this work, I have utilized high-throughput molecular sequencing data to expand our understanding of the transcriptional gene silencing pathway. First, I addressed and expanded our understanding of Pol V transcription at RdDM loci. I have successfully identified and annotated Pol V transcribed RdDM loci throughout the genome. I have further shown how Pol V transcription is controlled by preexisting chromatin modifications located within the transcribed regions. I observed that Pol V transcribes into transposons in a non-strand specific manner and the DNA methylation targeted to these transposons also occur on both strands and is tightly restricted to the Pol V transcribed regions. I further show that the preferential enrichment of Pol V transcription and downstream DNA methylation at the edges of transposons depicts a possible role of Pol V in determining heterochromatin boundaries.

Second, my research helped us better understand the mechanism of Pol V transcription. I have shown that Pol V transcription is not restricted to RdDM loci but is much more pervasive. Through my research, I show how at already established RdDM targets, Pol V and siRNA work together to maintain silencing. In contrast, some euchromatic sequences do not give rise to siRNA but are covered by low levels of Pol V transcription, which is needed to establish RdDM de novo, if a transposon is reactivated. Through this study, I show that Pol V surveils the genome to make it competent to silence newly activated transposons, making it essential for maintaining the integrity of the genome.

Third, I address the effect of Pol V transcription on downstream repressive chromatin modifications and gene silencing. I show that RdDM affects nucleosomes through recruitment of the SWI/SNF chromatin remodeling complex. Next, I address the relationship between the two chromatin modifications showing that despite DNA methylation being predominantly enriched at linkers, RdDM target loci show an enrichment of both nucleosomes and DNA methylation. My data further depicts that nucleosome placement by RdDM has no detectable effects on the pattern of DNA methylation. Instead, I show that DNA methylation by RdDM affects nucleosome positioning, suggesting that DNA methylation directs nucleosomes and they both coordinately bring about gene and transposon silencing at the RdDM loci.