The Initiation of Epigenetic Silencing of Active Transposable Elements Is Triggered by RDR6 and 21-22 Nucleotide Small Interfering RNAs    

Abstract

Transposable elements (TEs) are mobile fragments of DNA that are repressed in both plant and animal genomes through the epigenetic inheritance of repressed chromatin and expression states. The epigenetic silencing of TEs in plants is mediated by a process of RNA-directed DNA methylation (RdDM). Two pathways of RdDM have been identified: RNA Polymerase IV (Pol IV)-RdDM, which has been shown to be responsible for the de novo initiation, corrective reestablishment, and epigenetic maintenance of TE and/or transgene silencing; and RNA-dependent RNA Polymerase6 (RDR6)-RdDM, which was recently identified as necessary for maintaining repression for a few TEs. We have further characterized RDR6-RdDM using a genomewide search to identify TEs that generate RDR6-dependent small interfering RNAs. We have determined that TEs only produce RDR6-dependent small interfering RNAs when transcriptionally active, and we have experimentally identified two TE subfamilies as direct targets of RDR6-RdDM. We used these TEs to test the function of RDR6-RdDM in assays for the de novo initiation, corrective reestablishment, and maintenance of TE silencing. We found that RDR6-RdDM plays no role in maintaining TE silencing. Rather, we found that RDR6 and Pol IV are two independent entry points into RdDM and epigenetic silencing that perform distinct functions in the silencing of TEs: Pol IV-RdDM functions to maintain TE silencing and to initiate silencing in an RNA Polymerase II expression-independent manner, while RDR6-RdDM functions to recognize active Polymerase II-derived TE mRNA transcripts to both trigger and correctively reestablish TE methylation and epigenetic silencing.Transposable elements (TEs) constitute large percentages of both animal and plant genomes. TEs are major targets of multiple endogenous gene-silencing pathways that act to limit their expression and ability to generate new insertions and mutations (for review, see Girard and Hannon, 2008). To study TE silencing, the process has been divided into three distinct mechanisms: the de novo initiation/triggering of silencing, the corrective reestablishment of silencing of TEs that were recently transcriptionally reactivated, and the epigenetic maintenance of TE silencing.In the Arabidopsis (Arabidopsis thaliana) genome, nearly all TEs are found in a transcriptionally silenced state (Lippman et al., 2004). This transcriptional gene silencing is maintained by symmetrical DNA methylation, which is propagated through mitotic cell divisions (for review, see Law and Jacobsen, 2010). In contrast to animals, plants do not erase the DNA methylation patterns of their gametes; therefore, CG and CHG (where H = A, T, or C) symmetrical DNA methylation patterns established in one generation are inherited and act to maintain TE silencing in the next generation through a process termed transgenerational epigenetic inheritance (Mathieu et al., 2007;Becker et al., 2011). In addition to the maintenance of symmetrical methylation, methylation of TEs is continually reinforced through a process of ...

RNAi in Plants: An Argonaute-Centered View

“…In rice, 24-nucleotide lmiRNAs generated by coordinated actions of DCL1 and DCL3 are associated with AGO4 to direct DNA methylation in trans at the miRNA target genes, resulting in TGS in some cases (Wu et al, 2010). In addition to AGO4 that functions in the canonical RdDM pathway, AGO2 and AGO6 can also mediate de novo DNA methylation by recruiting 21-to 22-nucleotide siRNAs, which are processed from Pol II/RDR6-dependent dsRNAs by DCL2 and DCL4 Marí-Ordóñez et al, 2013;Nuthikattu et al, 2013).…”

“…It was also shown that AGO4 and AGO6 differ in their subnuclear colocalization and in interactions with Pol II and Pol IV, and it was proposed that they may act sequentially to mediate DNA methylation . AGO6 also binds RDR6-dependent 21-to 22-nucleotide siRNAs generated from transcriptionally active TEs and is thought to trigger the initiation of de novo TE DNA methylation (Nuthikattu et al, 2013;McCue et al, 2015), which may provide a link between PTGS and TGS of TEs.…”

Genome-Wide Characterization of Maize Small RNA Loci and Their Regulation in the required to maintain repression6-1 (rmr6-1) Mutant and Long-Term Abiotic Stresses

“…Pol IV-dependent siRNAs are often produced from transposable elements (TEs), TE-like sequences, and other repeats (Zhang et al, 2007;Mosher et al, 2008). They contribute to the reinforcement of TE silencing (Slotkin et al, 2005;Marí-Ordóñez et al, 2013;Nuthikattu et al, 2013) and in some cases are also essential to control the expression of proteincoding genes in cis or in trans (Liu et al, 2004;Kinoshita et al, 2007;McCue et al, 2013).…”

DNA sequence properties that predict susceptibility to epiallelic switching

“…This suggests that a threshold regulation is involved in epigenetic switches at E-TELs. In contrast, transposable element families naturally targeted by RDR6-RdDM, such as AtREP10C (part of the HELITRON superfamily) and ROMANIAT5 (Nuthikattu et al, 2013), were consistently found among R-TELs in our analyses (Appendix Figs S11 and S12). This opens the possibility that TEs naturally able to produce RDR6-dependent sRNAs cannot stably switch to demethylated epialleles and would thus be in the R-TELs category.…”

“…That E-TELs might be stimulated to acquire DNA methylation when particular mechanisms involved in restoration of "silent locus identity" are locally reinforced is an interesting concept. Recent studies have revealed an alternative RdDM pathway involving 21-to 22-nt sRNAs and RNA-dependent RNA polymerase 6 (RDR6), known as RDR6-RdDM, that separates initiation of de novo DNA methylation and silencing from its maintenance (Nuthikattu et al, 2013;Bond & Baulcombe, 2015). It has been demonstrated that the lost DNA methylation at the FWA promoter, a typical and frequently studied E-TEL, can be reversed when additional 21-to 22-nt sRNAs are provided through a viral vector (Bond & Baulcombe, 2015).…”