Identification of genes required for de novo DNA methylation in Arabidopsis

Greenberg, Maxim V. C.; Ausín, Israel; Chan, Simon W. L.; Cokus, Shawn J.; Cuperus, Josh T.; Feng, Suhua; Law, Julie A.; Chu, Carolyn; Pellegrini, Matteo; Carrington, James C.; Jacobsen, Steven E.

doi:10.4161/epi.6.3.14242

Cited by 60 publications

(57 citation statements)

References 47 publications

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“…All known genes affecting de novo methylation are involved in the maintenance of DRM2-mediated methylation at several loci (13,14). Combining methylation-sensitive enzymes and PCR or Southern blot analysis, as well as bisulfite sequencing techniques, we examined the methylation status of known RdDM targets: MEA-ISR, FWA repeats, and the AtSN1 transposon (Fig.…”

Section: Resultsmentioning

confidence: 99%

INVOLVED IN DE NOVO 2-containing complex involved in RNA-directed DNA methylation in Arabidopsis

Ausín

Greenberg

Simanshu

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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At least three pathways control maintenance of DNA cytosine methylation in Arabidopsis thaliana. However, the RNA-directed DNA methylation (RdDM) pathway is solely responsible for establishment of this silencing mark. We previously described INVOLVED IN DE NOVO 2 (IDN2) as being an RNA-binding RdDM component that is required for DNA methylation establishment. In this study, we describe the discovery of two partially redundant proteins that are paralogous to IDN2 and that form a stable complex with IDN2 in vivo. Null mutations in both genes, termed IDN2-LIKE 1 and IDN2-LIKE 2 (IDNL1 and IDNL2), result in a phenotype that mirrors, but does not further enhance, the idn2 mutant phenotype. Genetic analysis suggests that this complex acts in a step in the downstream portion of the RdDM pathway. We also have performed structural analysis showing that the IDN2 XS domain adopts an RNA recognition motif (RRM) fold. Finally, genome-wide DNA methylation and expression analysis confirms the placement of the IDN proteins in an RdDM pathway that affects DNA methylation and transcriptional control at many sites in the genome. Results from this study identify and describe two unique components of the RdDM machinery, adding to our understanding of DNA methylation control in the Arabidopsis genome.genomics | mass spectrometry | siRNAs | epigenetics D NA methylation is a stable epigenetic mark that is associated with the repression of genes and transposable elements. In Arabidopsis thaliana, maintenance of DNA methylation at silent loci is carried out by at least three methyltransferases: METH-YLTRANSFERASE 1 (MET1), CHROMOMETHYLTRANS-FERASE 3 (CMT3), and DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2). However, DRM2 is solely responsible for establishment of DNA methylation-or de novo methylation-of silent elements (1). DRM2 is guided to chromatin by small interfering RNAs (siRNAs) in a process known as RNAdirected DNA methylation (RdDM) (2). It has been proposed that RdDM also requires intergenic noncoding (IGN) transcripts that are synthesized by RNA Polymerase V (Pol V). These transcripts likely serve as platforms for the recruitment of siRNA-loaded ARGONAUTE 4 (AGO4) to methylated loci (3, 4).Recently, we discovered the requirement of INVOLVED IN DE NOVO 2 (IDN2) for RdDM from a forward genetic screen (5). Alleles of this same gene were later reported from another screen for DNA methylation mutants (6). We previously demonstrated that IDN2 binds to double-stranded RNA through its XS domain, which is also observed in the XS-domain-containing protein SUPPRESSOR OF GENE SILENCING 3 (SGS3) (7). We also found that IDN2 was likely to act in a step downstream of initial siRNA biogenesis. The XS domain is conserved throughout the plant kingdom, and XS-domain-containing proteins are involved in a wide range of processes such as viral defense (8) and stress response (9).To gain a better understanding of the in vivo role of IDN2, we performed affinity purifications from complementing transgenic lines expressing epitope-tagged full-length I...

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Section: Resultsmentioning

confidence: 99%

INVOLVED IN DE NOVO 2-containing complex involved in RNA-directed DNA methylation in Arabidopsis

Ausín

Greenberg

Simanshu

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…1E). Although the RdDM pathway is known to be required for epigenetic silencing of some transgenes and numerous endogenous loci, including many transposon repeats (22)(23)(24)(25), our forward genetic screen here identified the RdDM as an antisilencing pathway. The notion of the RdDM pathway also having an antisilencing role is consistent with a previous report (17).…”

Section: Rddm Mutations Cause the Silencing Of 35s::suc2 And 2 × 35s:mentioning

confidence: 99%

Regulatory link between DNA methylation and active demethylation in Arabidopsis

Lei

Zhang

Julian

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

214

207

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De novo DNA methylation through the RNA-directed DNA methylation (RdDM) pathway and active DNA demethylation play important roles in controlling genome-wide DNA methylation patterns in plants. Little is known about how cells manage the balance between DNA methylation and active demethylation activities. Here, we report the identification of a unique RdDM target sequence, where DNA methylation is required for maintaining proper active DNA demethylation of the Arabidopsis genome. In a genetic screen for cellular antisilencing factors, we isolated several REPRESSOR OF SILENCING 1 (ros1) mutant alleles, as well as many RdDM mutants, which showed drastically reduced ROS1 gene expression and, consequently, transcriptional silencing of two reporter genes. A helitron transposon element (TE) in the ROS1 gene promoter negatively controls ROS1 expression, whereas DNA methylation of an RdDM target sequence between ROS1 5′ UTR and the promoter TE region antagonizes this helitron TE in regulating ROS1 expression. This RdDM target sequence is also targeted by ROS1, and defective DNA demethylation in loss-of-function ros1 mutant alleles causes DNA hypermethylation of this sequence and concomitantly causes increased ROS1 expression. Our results suggest that this sequence in the ROS1 promoter region serves as a DNA methylation monitoring sequence (MEMS) that senses DNA methylation and active DNA demethylation activities. Therefore, the ROS1 promoter functions like a thermostat (i.e., methylstat) to sense DNA methylation levels and regulates DNA methylation by controlling ROS1 expression.NA methylation is a conserved epigenetic mark important for development and stress responses in plants and many animals (1-4). Genome-wide DNA methylation patterns are dynamically regulated by establishment, maintenance, and removal activities (4, 5). In plants, de novo DNA methylation is controlled by the RNA-directed DNA methylation (RdDM) pathway, in which complementary pairing between long noncoding RNAs and siRNAs mediates cytosine methylation in a sequence-specific manner (2, 4, 6, 7). Best characterized in Arabidopsis, RdDM involves a complex array of regulators and mainly targets heterochromatic regions that are enriched with transposon elements (TEs) and other DNA repeat sequences (8, 9). Once established, Arabidopsis DNA methylation is maintained via different mechanisms depending on the cytosine contexts [i.e., CG, CHG, CHH (H represents A, T, or C)]. CG and CHG methylation is maintained by MET1 and CMT3, respectively (2), whereas CHH methylation within pericentromeric long TEs can be catalyzed by CMT2 and CHH methylation at other loci is established de novo by DRM2 during every cell cycle (2, 10). In contrast to DNA methyltransferases that establish and/or maintain cytosine methylation, plant 5-methylcytosine DNA glycosylases initiate a base excision repair pathway that erases DNA methylation, thereby generating, together with the establishment and maintenance activities, a dynamic landscape of DNA methylation (11, 12).The Arabidopsis ...

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“…When transformed into Arabidopsis, a naive FWA transgene with a TE in its promoter is de novo silenced by the activity of the Pol IV-RdDM pathway (Chan et al, 2004;Greenberg et al, 2011;Kinoshita et al, 2007;Cao and Jacobsen, 2002). To determine and differentiate how the Pol IV-RdDM pathway functions in the silencing of TE transgenes, we transformed the Athila LTR and Athila LTR Dpro transgenes directly into the pol IV (nrpd1a) mutant background.…”

Section: Mechanism Of Identity-based Silencingmentioning

confidence: 99%

“…To focus solely on the initiation and establishment phases of silencing, and separate these mechanisms from the maintenance of epigenetic marks, investigation has concentrated on transformation or infection of epigenetically naïve (chromatin modification-free) DNA into plants (Greenberg et al, 2011;Bond and Baulcombe, 2015;Jackel et al, 2016). Although transgenesis might be considered an artificial approach to investigate silencing, evidence suggests that transgenesis can occur naturally (Kyndt et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

Exogenous Transposable Elements Circumvent Identity-Based Silencing, Permitting the Dissection of Expression-Dependent Silencing

Fultz

Slotkin

2017

Plant Cell

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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.

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Identification of genes required for de novo DNA methylation in Arabidopsis

Cited by 60 publications

References 47 publications

INVOLVED IN DE NOVO 2-containing complex involved in RNA-directed DNA methylation in Arabidopsis

INVOLVED IN DE NOVO 2-containing complex involved in RNA-directed DNA methylation in Arabidopsis

Regulatory link between DNA methylation and active demethylation in Arabidopsis

Exogenous Transposable Elements Circumvent Identity-Based Silencing, Permitting the Dissection of Expression-Dependent Silencing

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