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 ...

In plant genomes the vast majority of transposable elements (TEs) are found in a transcriptionally silenced state that is epigenetically propagated from generation to generation. Although the mechanism of this maintenance of silencing has been well studied, it is now clear that the pathways responsible for maintaining TEs in a silenced state differ from the pathways responsible for initially targeting the TE for silencing. Recently, attention in this field has focused on investigating the molecular mechanisms that initiate and establish TE silencing. Here we review the current models of how TEs are triggered for silencing, the data supporting each model, and the key future questions in this fast moving field.

ORCID ID: 0000-0001-9582-3533 (R.K.S.)The propagation of epigenetic marks has received a great deal of attention, yet the initiation of epigenetic silencing of a new transgene, virus, or transposable element (TE) remains enigmatic. The overlapping and simultaneous function of multiple silencing mechanisms has obscured this area of investigation. Here, we revealed two broad mechanisms that can initiate silencing independently: identity-based and expression-dependent silencing. We found that identity-based silencing is targeted by 21-to 22-nucleotide or 24-nucleotide small interfering RNAs (siRNAs) generated from previously silenced regions of the genome. By transforming exogenous TEs into Arabidopsis thaliana, we circumvented identity-based silencing, allowing us to isolate and investigate the molecular mechanism of expression-dependent silencing. We found that several siRNAgenerating mechanisms all trigger de novo expression-dependent RNA-directed DNA methylation (RdDM) through RNA Polymerase V. In addition, while full-length TEs quickly progress beyond RdDM to heterochromatin formation and the final maintenance methylation state, TE fragments stall at the RdDM phase. Lastly, we found that transformation into a mutant genotype followed by introgression into the wild type does not result in the same level of silencing as direct transformation into the wild type. This demonstrates that the plant genotype during a narrow window of time at TE insertion (or transgene transformation) is key for establishing the transgenerational extent of epigenetic silencing.

Large regions of nearly identical repeats, such as the 45S ribosomal RNA (rRNA) genes of Nucleolus Organizer Regions (NORs), can account for major gaps in sequenced genomes. To assemble these regions, ultra-long sequencing reads that span multiple repeats have the potential to reveal sets of repeats that collectively have sufficient sequence variation to unambiguously define that interval and recognize overlapping reads. Because individual repetitive loci typically represent a small proportion of the genome, methods to enrich for the regions of interest are desirable. Here we describe a simple method that achieves greater than tenfold enrichment of Arabidopsis thaliana 45S rRNA gene sequences among ultra-long Oxford Nanopore Technology sequencing reads. This method employs agarose-embedded genomic DNA that is subjected to restriction endonucleases digestion using a cocktail of enzymes predicted to be non-cutters of rRNA genes. Most of the genome is digested into small fragments that diffuse out of the agar plugs, whereas rRNA gene arrays are retained. In principle, the approach can also be adapted for sequencing other repetitive loci for which gaps exist in a reference genome.

Arabidopsis thaliana has two ribosomal RNA gene loci, nucleolus organizer regions NOR2 and NOR4, whose complete sequences remain unknown. Using ultra-long DNA sequencing technology, we generated 5.5 and 3.9 Mbp sequence assemblies for NOR2 and NOR4 (in the reference strain, Col-0), revealing their distinctive gene subtype compositions. RNA sequencing, and gene identification within flow-sorted nucleoli, in both wild-type and histone deacetylase 6 mutants, shows that most, but not all, NOR4 genes are active whereas most, but not all, NOR2 genes are epigenetically silenced. Long intervals of low CG and CHG methylation overlap regions of gene activity and gene subtype homogenization. Collectively, the data illuminate the genetic and epigenetic landscapes of the NORs and implicate transcription in rRNA gene concerted evolution.

SUMMARYNucleolus organizer regions (NORs) are eukaryotic chromosomal loci where ribosomal RNA (rRNA) genes are clustered, typically in hundreds to thousands of copies. Transcription of these rRNA genes by RNA polymerase I and processing of their transcripts results in the formation of the nucleolus, the sub‐nuclear domain in which ribosomes are assembled. Approximately 90 years ago, cytogenetic observations revealed that NORs inherited from the different parents of an interspecific hybrid sometimes differ in morphology at metaphase. Fifty years ago, those chromosomal differences were found to correlate with differences in rRNA gene transcription and the phenomenon became known as nucleolar dominance. Studies of the past 30 years have revealed that nucleolar dominance results from selective rRNA gene silencing, involving repressive chromatin modifications, and occurs in pure species as well as hybrids. Recent evidence also indicates that silencing depends on the NOR in which an rRNA gene is located, and not on the gene's sequence. In this perspective, we discuss how our thinking about nucleolar dominance has shifted over time from the kilobase scale of individual genes to the megabase scale of NORs and chromosomes and questions that remain unanswered in the search for a genetic and biochemical understanding of the off switch.

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Bioinformatic alignment of the assembled genomes of Col-0 and Sha ecotypes of Arabidopsis thalianarevealed ~ 10,000 genome-wide structural variants involving simple insertions or deletions and repeat contractions or expansions. Using some of these structural variants, we developed new, rapid, and low-cost PCR-based molecular markers that are genetically linked to the nucleolus organizer regions (NORs). A. thaliana has two NORs, one each on chromosome 2 (NOR2) and chromosome 4 (NOR4). Both NORs are ~ 4 Mb each, and hundreds of 45S ribosomal RNA (rRNA) genes are tandemly arrayed at these loci. Using previously characterized recombinant inbred lines (RILs) derived from Col-0 x Sha crosses, we validated the utility of the newly developed NOR-linked markers in genetically mapping rRNA genes and the associated telomeres to either NOR2 or NOR4. Lastly, we sequenced Sha genome using Oxford Nanopore Technology (ONT) and used the data to obtain sequences of NOR-telomere junctions, and with the help of RILs, we mapped them as new genetic markers to their respective NORs (NOR2-TEL2N and NOR4-TEL4N). The structural variants obtained from this study would serve as valuable data for genome-wide association studies (GWAS), developing synteny, and to rapidly design more genome-wide genetic (molecular) markers for new gene/trait mapping purposes.