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 genome-wide 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.
Highlights
Transposable elements (TEs) comprise large percentages of both animal and plant genomes
We have focused on RDR6, a protein identified as a necessary component of the Polymerase IV (Pol IV)-RNA-directed DNA methylation (RdDM)-independent DNA methylation of trans-acting small interfering RNAs (siRNAs) (TAS) loci as well as the single Copia and Helitron elements (Pontier et al, 2012; Wu et al, 2012)
In this report we used the AtENSPM6 TE to demonstrate that methylation can be established by RDR6-dependent 21-22 nt siRNAs through a separate pathway from Pol IVRdDM
Summary
Transposable elements (TEs) comprise large percentages of both animal and plant genomes. In the Arabidopsis genome, most TEs are found in a transcriptionally silenced state (Lippman et al, 2004) This transcriptional gene silencing (TGS) is maintained by symmetrical DNA methylation, which is propagated through mitotic cell divisions (reviewed in Law and Jacobsen, 2010). In addition to the maintenance of symmetrical methylation, methylation of TEs is continually reinforced through a process of RNA-directed DNA methylation (RdDM)(reviewed in Law and Jacobsen, 2010; Haag and Pikaard, 2011). This pathway involves RNA Polymerase IV (Pol IV), a plant-specific DNA-dependent RNA polymerase that transcribes heterochromatic regions such as TEs into non-protein coding transcripts. The Pol IV-RDR2-DCL3-AGO4/6-POL VDRM2 pathway (from this point forward referred to as the Pol IV-RdDM pathway) acts as a loop reinforcing methylation states at regions of heterochromatin and silenced TEs
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