scholarly journals Epigenetic transitions leading to heritable, RNA-mediated de novo silencing in Arabidopsis thaliana

2015 ◽  
Vol 112 (3) ◽  
pp. 917-922 ◽  
Author(s):  
Donna M. Bond ◽  
David C. Baulcombe
Keyword(s):  
Dna Methylation ◽  
Rna Polymerase ◽  
Gene Silencing ◽  
De Novo ◽  
Feedback Mechanism ◽  
Epigenetic Silencing ◽  
Virus Induced Gene Silencing ◽  
Epigenetic Modifiers ◽  
Rna Polymerase Iv ◽  
Genetic Mutants

In plants, RNA-directed DNA methylation (RdDM), a mechanism where epigenetic modifiers are guided to target loci by small RNAs, plays a major role in silencing of transposable elements (TEs) to maintain genome integrity. So far, two RdDM pathways have been identified: RNA Polymerase IV (PolIV)-RdDM and RNA-dependent RNA Polymerase 6 (RDR6)-RdDM. PolIV-RdDM involves a self-reinforcing feedback mechanism that maintains TE silencing, but cannot explain how epigenetic silencing is first initiated. A function of RDR6-RdDM is to reestablish epigenetic silencing of active TEs, but it is unknown if this pathway can induce DNA methylation at naïve, non-TE loci. To investigate de novo establishment of RdDM, we have used virus-induced gene silencing (VIGS) of an active FLOWERING WAGENINGEN epiallele. Using genetic mutants we show that unlike PolIV-RdDM, but like RDR6-RdDM, establishment of VIGS-mediated RdDM requires PolV and DRM2 but not Dicer like-3 and other PolIV pathway components. DNA methylation in VIGS is likely initiated by a process guided by virus-derived small (s) RNAs that are 21/22-nt in length and reinforced or maintained by 24-nt sRNAs. We demonstrate that VIGS-RdDM as a tool for gene silencing can be enhanced by use of mutant plants with increased production of 24-nt sRNAs to reinforce the level of RdDM.

scholarly journals Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Todd Blevins ◽  
Ram Podicheti ◽  
Vibhor Mishra ◽  
Michelle Marasco ◽  
Jing Wang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Cytosine Methylation ◽  
De Novo ◽  
Transferase Activity ◽  
Small Interfering Rnas ◽  
Pol Iv ◽  
Nuclear Rna ◽  
Rna Polymerase Iv ◽  
Nuclear Rna Polymerase

In Arabidopsis thaliana, abundant 24 nucleotide small interfering RNAs (24 nt siRNA) guide the cytosine methylation and silencing of transposons and a subset of genes. 24 nt siRNA biogenesis requires nuclear RNA polymerase IV (Pol IV), RNA-dependent RNA polymerase 2 (RDR2) and DICER-like 3 (DCL3). However, siRNA precursors are mostly undefined. We identified Pol IV and RDR2-dependent RNAs (P4R2 RNAs) that accumulate in dcl3 mutants and are diced into 24 nt RNAs by DCL3 in vitro. P4R2 RNAs are mostly 26-45 nt and initiate with a purine adjacent to a pyrimidine, characteristics shared by Pol IV transcripts generated in vitro. RDR2 terminal transferase activity, also demonstrated in vitro, may account for occasional non-templated nucleotides at P4R2 RNA 3’ termini. The 24 nt siRNAs primarily correspond to the 5’ or 3’ ends of P4R2 RNAs, suggesting a model whereby siRNAs are generated from either end of P4R2 duplexes by single dicing events.

scholarly journals Post-transcriptional gene silencing triggers dispensable DNA methylation in gene body in Arabidopsis

2019 ◽  
Vol 47 (17) ◽  
pp. 9104-9114 ◽  
Author(s):  
Christelle Taochy ◽  
Agnès Yu ◽  
Nicolas Bouché ◽  
Nathalie Bouteiller ◽  
Taline Elmayan ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Gene Silencing ◽  
De Novo ◽  
Transcriptional Silencing ◽  
Transcriptional Gene Silencing ◽  
Gene Body ◽  
Coding Sequence ◽  
Post Transcriptional Gene Silencing ◽  
Methylation Signal ◽  
Rule Out

Abstract Spontaneous post-transcriptional silencing of sense transgenes (S-PTGS) is established in each generation and is accompanied by DNA methylation, but the pathway of PTGS-dependent DNA methylation is unknown and so is its role. Here we show that CHH and CHG methylation coincides spatially and temporally with RDR6-dependent products derived from the central and 3′ regions of the coding sequence, and requires the components of the RNA-directed DNA methylation (RdDM) pathway NRPE1, DRD1 and DRM2, but not CLSY1, NRPD1, RDR2 or DCL3, suggesting that RDR6-dependent products, namely long dsRNAs and/or siRNAs, trigger PTGS-dependent DNA methylation. Nevertheless, none of these RdDM components are required to establish S-PTGS or produce a systemic silencing signal. Moreover, preventing de novo DNA methylation in non-silenced transgenic tissues grafted onto homologous silenced tissues does not inhibit the triggering of PTGS. Overall, these data indicate that gene body DNA methylation is a consequence, not a cause, of PTGS, and rule out the hypothesis that a PTGS-associated DNA methylation signal is transmitted independent of a PTGS signal.

Dynamics in the expression of epigenetic modifiers and histone modifications in perinatal rat germ cells during de novo DNA methylation†

2020 ◽  
Author(s):  
Arlette Rwigemera ◽  
Rhizlane El omri-Charai ◽  
Laetitia L Lecante ◽  
Geraldine Delbes
Keyword(s):  
Dna Methylation ◽  
Germ Cell ◽  
Histone Modifications ◽  
Germ Cells ◽  
Posttranslational Modifications ◽  
Fluorescent Protein ◽  
De Novo ◽  
Expression Patterns ◽  
Dna Methyltransferases ◽  
Epigenetic Modifiers

Abstract Epigenetic reprogramming during perinatal germ cell development is essential for genomic imprinting and cell differentiation; however, the actors of this key event and their dynamics are poorly understood in rats. Our study aimed to characterize the expression patterns of epigenetic modifiers and the changes in histone modifications in rat gonocytes at the time of de novo DNA methylation. Using transgenic rats expressing Green Fluorescent Protein (GFP) specifically in germ cells, we purified male gonocytes by fluorescent activated cell sorting at various stages of perinatal development and established the transcriptomic profile of 165 epigenetic regulators. Using immunofluorescence on gonad sections, we tracked six histone modifications in rat male and female perinatal germ cells over time, including methylation of histone H3 on lysines 27, 9, and 4; ubiquitination of histone H2A on lysine119; and acetylation of histone H2B on lysine 20. The results revealed the dynamics in the expression of ten-eleven translocation enzymes and DNA methyltransferases in male gonocytes at the time of de novo DNA methylation. Moreover, our transcriptomic data indicate a decrease in histone ubiquitination and methylation coinciding with the beginning of de novo DNA methylation. Decreases in H2AK119Ub and H3K27me3 were further confirmed by immunofluorescence in the male germ cells but were not consistent for all H3 methylation sites examined. Together, our data highlighted transient chromatin remodeling involving histone modifications during de novo DNA methylation. Further studies addressing how these dynamic changes in histone posttranslational modifications could guide de novo DNA methylation will help explain the complex establishment of the male germ cell epigenome.

scholarly journals Silencing of Mutator Elements in Maize Involves Distinct Populations of Small RNAs and Distinct Patterns of DNA Methylation

Genetics ◽  
2020 ◽  
Vol 215 (2) ◽  
pp. 379-391 ◽  
Author(s):  
Diane Burgess ◽  
Hong Li ◽  
Meixia Zhao ◽  
Sang Yeol Kim ◽  
Damon Lisch
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Small Rnas ◽  
Cytosine Methylation ◽  
De Novo ◽  
Epigenetic Silencing ◽  
Inverted Repeats ◽  
Causal Relationships ◽  
Terminal Inverted Repeats ◽  
Silencing Pathways

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.

scholarly journals Role of the Arabidopsis DRM Methyltransferases in De Novo DNA Methylation and Gene Silencing

2002 ◽  
Vol 12 (13) ◽  
pp. 1138-1144 ◽  
Author(s):  
Xiaofeng Cao ◽  
Steven E. Jacobsen
Keyword(s):  
Dna Methylation ◽  
Gene Silencing ◽  
De Novo

scholarly journals Roles of RNA polymerase IV in gene silencing

2008 ◽  
Vol 13 (7) ◽  
pp. 390-397 ◽  
Author(s):  
Craig S. Pikaard ◽  
Jeremy R. Haag ◽  
Thomas Ream ◽  
Andrzej T. Wierzbicki
Keyword(s):  
Rna Polymerase ◽  
Gene Silencing ◽  
Rna Polymerase Iv

scholarly journals Reaction mechanisms of Pol IV, RDR2 and DCL3 drive RNA channeling in the siRNA-directed DNA methylation pathway

2019 ◽  
Author(s):  
Jasleen Singh ◽  
Vibhor Mishra ◽  
Feng Wang ◽  
Hsiao-Yun Huang ◽  
Craig S. Pikaard
Keyword(s):  
Dna Methylation ◽  
Rna Polymerase ◽  
Dna Transcription ◽  
Pol Iv ◽  
Methylation Pathway ◽  
Terminal Nucleotide ◽  
Passenger Strand ◽  
Rna Polymerase Iv ◽  
Rna Pathways ◽  
Template Dna

SummaryIn eukaryotes with multiple small RNA pathways the mechanisms that channel RNAs within specific pathways are unclear. Here, we reveal the reactions that account for channeling in the siRNA biogenesis phase of the Arabidopsis RNA-directed DNA methylation pathway. The process begins with template DNA transcription by NUCLEAR RNA POLYMERASE IV (Pol IV) whose atypical termination mechanism, induced by nontemplate DNA basepairing, channels transcripts to the associated RNA-dependent RNA polymerase, RDR2. RDR2 converts Pol IV transcripts into double-stranded RNAs then typically adds an extra untemplated 3’ terminal nucleotide to the second strands. The dicer endonuclease, DCL3 cuts resulting duplexes to generate 24 and 23nt siRNAs. The 23nt RNAs bear the untemplated terminal nucleotide of the RDR2 strand and are underrepresented among ARGONAUTE4-associated siRNAs. Collectively, our results provide mechanistic insights into Pol IV termination, Pol IV-RDR2 coupling and RNA channeling from template DNA transcription to siRNA guide strand/passenger strand discrimination.

scholarly journals A DCL3 dicing code within Pol IV-RDR2 transcripts diversifies the siRNA pool guiding RNA-directed DNA methylation

2021 ◽  
Author(s):  
Andrew Loffer ◽  
Jasleen Singh ◽  
Akihito Fukudome ◽  
Vibhor Mishra ◽  
Feng Wang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Rna Polymerase ◽  
Transferase Activity ◽  
Rna Dependent Rna Polymerase ◽  
Dna Viruses ◽  
Selfish Genetic Elements ◽  
Pol Iv ◽  
Nuclear Rna ◽  
Rna Polymerase Iv ◽  
Nuclear Rna Polymerase

In plants, selfish genetic elements including retrotransposons and DNA viruses are transcriptionally silenced by RNA-directed DNA methylation. Guiding the process are short interfering RNAs (siRNAs) cut by DICER-LIKE 3 (DCL3) from double-stranded precursors of ~30 bp synthesized by NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). We show that Pol IV initiating nucleotide choice, RDR2 initiation 1-2 nt internal to Pol IV transcript ends and RDR2 terminal transferase activity collectively yield a code that influences which end of the precursor is diced and whether 24 or 23 nt siRNAs are generated from the Pol IV or RDR2-transcribed strands. By diversifying the size, sequence, and strand polarity of siRNAs derived from a given precursor, alternative patterns of DCL3 dicing allow maximal siRNA coverage at methylated target loci.

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