Silencing ofMutatorElements in Maize Involves Distinct Populations of Small RNAs and Distinct Patterns of DNA Methylation

Abstract

Transposable elements (TEs) are a ubiquitous feature of plant genomes. Because of the threat they post to genome integrity, most TEs are epigenetically silenced. However, even closely related plant species often have dramatically different populations of TEs, suggesting periodic rounds of activity and silencing. Here, we show that the process of de novo methylation of an active element in maize involves two distinct pathways, one of which is directly implicated in causing epigenetic silencing and one of which is the result of that silencing. Epigenetic changes involve changes in gene expression that can be heritably transmitted to daughter cells in the absence of changes in DNA sequence. Epigenetics has been implicated in phenomena as diverse as development, stress response, and carcinogenesis. A significant challenge facing those interested in investigating epigenetic phenomena is determining causal relationships between DNA methylation, specific classes of small RNAs, and associated changes in gene expression. Because they are the primary targets of epigenetic silencing in plants and, when active, are often targeted for de novo silencing, TEs represent a valuable source of information about these relationships. We use a naturally occurring system in which a single TE can be heritably silenced by a single derivative of that TE. By using this system it is possible to unravel causal relationships between different size classes of small RNAs, patterns of DNA methylation, and heritable silencing. Here, we show that the long terminal inverted repeats within Zea mays MuDR transposons are targeted by distinct classes of small RNAs during epigenetic silencing that are dependent on distinct silencing pathways, only one of which is associated with transcriptional silencing of the transposon. Further, these small RNAs target distinct regions of the terminal inverted repeats, resulting in different patterns of cytosine methylation with different functional consequences with respect to epigenetic silencing and the heritability of that silencing.

Analysis of CACTA transposase genes unveils the mechanism of intron loss and distinct small RNA silencing pathways underlying divergent evolution of Brassica genomes

“…Interestingly, when we focused on a ~1.5‐kb region, which covers ORF2 in B. oleracea , there are a large number of 21‐ and 22‐nt small RNAs enriched in this region, with the ratio of 10.4:1.0 for 21‐ and 22‐nt to 24‐nt small RNAs (Figures 7c and S15). These 21‐ and 22‐nt small RNAs may be produced by one locus directly or through a transitive process that can trigger conversion of a target transcript by RDR6 into dsRNA, which is then cleaved into 21‐ and 22‐nt small RNAs (Choudhary et al ., 2019; Burgess et al ., 2020). These data suggest that both the Pol IV‐RDR2 and Pol II‐RDR6 pathways functioned at different regions of the Bot1 elements in B. oleracea , and most likely only Pol IV‐RDR2 based maintenance of heritable silencing is functional in the elements in B. rapa .…”

“…The second pathway involves Pol II‐RDR6‐dependent 21‐ and 22‐nt small RNAs that are corresponding to the transcribed proportion of the Bot1 elements, mainly a predicted ORF (ORF2) that is adjacent to the TNP2 ‐like transposase gene (Figure 7). These 21‐ and 22‐nt small RNAs were recently found to be processed from inverted repeats that are generated by duplication of a proportion of the original TE element (Burgess et al ., 2020; Wang et al ., 2020). These inverted repeats were called ‘ Mu killer ’ and ‘ Ac killer ’, which are transcribed by nearby promoters, produce 21‐ and 22‐nt small RNAs, act in trans to trigger CG and CHG DNA methylation, and heritably silence an active element.…”

RNA-directed DNA methylation prevents rapid and heritable reversal of transposon silencing under heat stress in Zeamays

“…Even more unexpected is our observation that, once mudrA becomes silenced, in mop1 mutants there appears to be reciprocal relationship between DNA methylation of TIRA and H3K9me2 enrichment. Based on previous experiments, our expectation was that mop1 would eliminate cytosine methylation in the 5’ end of TIRA, which is unrelated to transcriptional gene silencing of mudrA , but that it would not elimination of DNA methylation in the 3’ portion of TIRA, which is primarily in the CG and CHG contexts and is specifically associated with silencing of this gene [76]. In fact, we find that methylation in all three sequence context is eliminated throughout TIRA in mop1 mutants, but this does not result in reactivation of mudrA .…”

“…Methylation at the 5' and 3' portions of this TIR have distinctive causes and consequences. The 5' end of the TIR is readily methylated in the absence of the transposase, but this methylation does not induce transcriptional silencing of mudrA [76]. Methylation in this end of TIRA is readily eliminated in the presence of functional transposase.…”

“…These experiments were performed as previously described [76]. In brief, genomic DNA was isolated and digested with RNase A (Thermo Fisher Scientific).…”