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 ...

The epigenetic activity of transposable elements (TEs) can influence the regulation of genes; though, this regulation is confined to the genes, promoters, and enhancers that neighbor the TE. This local cis regulation of genes therefore limits the influence of the TE's epigenetic regulation on the genome. TE activity is suppressed by small RNAs, which also inhibit viruses and regulate the expression of genes. The production of TE heterochromatin-associated endogenous small interfering RNAs (siRNAs) in the reference plant Arabidopsis thaliana is mechanistically distinct from gene-regulating small RNAs, such as microRNAs or trans-acting siRNAs (tasiRNAs). Previous research identified a TE small RNA that potentially regulates the UBP1b mRNA, which encodes an RNA–binding protein involved in stress granule formation. We demonstrate that this siRNA, siRNA854, is under the same trans-generational epigenetic control as the Athila family LTR retrotransposons from which it is produced. The epigenetic activation of Athila elements results in a shift in small RNA processing pathways, and new 21–22 nucleotide versions of Athila siRNAs are produced by protein components normally not responsible for processing TE siRNAs. This processing results in siRNA854's incorporation into ARGONAUTE1 protein complexes in a similar fashion to gene-regulating tasiRNAs. We have used reporter transgenes to demonstrate that the UPB1b 3′ untranslated region directly responds to the epigenetic status of Athila TEs and the accumulation of siRNA854. The regulation of the UPB1b 3′ untranslated region occurs both on the post-transcriptional and translational levels when Athila TEs are epigenetically activated, and this regulation results in the phenocopy of the ubp1b mutant stress-sensitive phenotype. This demonstrates that a TE's epigenetic activity can modulate the host organism's stress response. In addition, the ability of this TE siRNA to regulate a gene's expression in trans blurs the lines between TE and gene-regulating small RNAs.

Transposable elements (TEs) generate mutations and chromosomal instability when active. To repress TE activity, eukaryotic cells evolved mechanisms to both degrade TE mRNAs into small interfering RNAs (siRNAs) and modify TE chromatin to epigenetically inhibit transcription. Since the populations of small RNAs that participate in TE post-transcriptional regulation differ from those that establish RNA-directed DNA methylation (RdDM), the mechanism through which transcriptionally active TEs transition from post-transcriptional RNAi regulation to chromatin level control has remained unclear. We have identified the molecular mechanism of a plant pathway that functions to direct DNA methylation to transcriptionally active TEs. We demonstrated that 21-22 nucleotide (nt) siRNA degradation products from the RNAi of TE mRNAs are directly incorporated into the ARGONAUTE 6 (AGO6) protein and direct AGO6 to TE chromatin to guide its function in RdDM. We find that this pathway functions in reproductive precursor cells to primarily target long centromeric high-copy transcriptionally active TEs for RdDM prior to gametogenesis. This study provides a direct mechanism that bridges the gap between the posttranscriptional regulation of TEs and the establishment of TE epigenetic silencing.

Transposable elements (TEs) are known to influence the regulation of neighboring genes through a variety of mechanisms. Additionally, it was recently discovered that TEs can regulate non-neighboring genes through the trans-acting nature of small interfering RNAs (siRNAs). When the epigenetic repression of TEs is lost, TEs become transcriptionally active, and the host cell acts to repress mutagenic transposition by degrading TE mRNAs into siRNAs. In this study, we have performed a genome-wide analysis in the model plant Arabidopsis thaliana and found that TE siRNA-based regulation of genic mRNAs is more pervasive than the two formerly characterized proof-of-principle examples. We identified 27 candidate genic mRNAs that do not contain a TE fragment but are regulated through partial complementarity by the accumulation of TE siRNAs and are therefore influenced by TE epigenetic activation. We have experimentally confirmed several gene targets and demonstrated that they respond to the accumulation of specific 21 nucleotide TE siRNAs that are incorporated into the Arabidopsis Argonaute1 protein. Additionally, we found that one TE siRNA specifically targets and inhibits the formation of a host protein that acts to repress TE activity, suggesting that TEs harbor and potentially evolutionarily select short sequences to act as suppressors of host TE repression.

Vascular cognitive impairment (VCI), a form of dementia caused by cerebrovascular disease, accounts for nearly 20% of cognitive dysfunction in the United States, and yet our understanding of the cerebrovascular disease processes underlying this dysfunction remains limited (1). Our understanding of vascular pathology in atherosclerotic cardiovascular disease (ASCVD) and the systemic circulation is more advanced and suggests important avenues of investigation, given the overlap of risk factors and epidemiology between ASCVD and cerebrovascular disease (2).Epidemiological evidence suggests a diet high in saturated fat and cholesterol negatively affects the health of the vasculature and contributes to the detrimental inflammatory injury that occurs in ASCVD and cerebrovascular disease (3-8). Studies also suggest that high levels of saturated fats and cholesterol contribute to the risk of developing VCI (9-14). Chronic intermittent vascular injury, as occurs over the course of decades of consumption of highfat meals, may significantly contribute to the long-term