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Comparing the targets of distinct RNA Directed DNA Methylation mechanisms in Arabidopsis thaliana

ABSTRACT: The diversity of small RNA-directed DNA methylation (RdDM) mechanisms have been underestimated due to the nearly complete transcriptional silencing of transposable elements (TEs) in the wt Col ecotype of Arabidopsis thaliana. In plants mutant for the SWI/SNF2 histone remodeler DDM1, TEs are globally activated due to loss of genome wide heterochromatin condensation. Activated TEs go through additional non-canonical forms of RdDM. However, the global targets of the non-canonical RdDM pathway are unidentified. In an attempt to identify and contrast the targets of canonical and non-canonical RdDM, we sequenced small RNAs from several RdDM mutants in either the TE-silent or the TE-active (ddm1) contexts. Examination of unopened flower bud small RNAs from wild type and various single or double mutant combinations, many of which have biological replicates. Small RNA sequences from wt Col, controls and other mutants shown in the study are available at GSE41755 and GSE57191.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: R. Keith Slotkin

PROVIDER: E-GEOD-79780 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Comparing the targets of distinct RNA Directed DNA Methylation mechanisms in Arabidopsis thaliana

Project description:The diversity of small RNA-directed DNA methylation (RdDM) mechanisms have been underestimated due to the nearly complete transcriptional silencing of transposable elements (TEs) in the wt Col ecotype of Arabidopsis thaliana. In plants mutant for the SWI/SNF2 histone remodeler DDM1, TEs are globally activated due to loss of genome wide heterochromatin condensation. Activated TEs go through additional non-canonical forms of RdDM. However, the global targets of the non-canonical RdDM pathway are unidentified. In an attempt to identify and contrast the targets of canonical and non-canonical RdDM, we sequenced small RNAs from several RdDM mutants in either the TE-silent or the TE-active (ddm1) contexts.

2016-07-22 | GSE79780 | GEO

Full-length autonomous transposable elements are preferentially targeted by expression-dependent forms of RNA-directed DNA methylation

Project description:The diversity of small RNA-directed DNA methylation (RdDM) mechanisms have been underestimated due to the nearly complete transcriptional silencing of transposable elements (TEs) in the wild-type reference strains of Arabidopsis thaliana. In plants mutant for the SWI/SNF histone remodeler DDM1, TEs are globally activated due to loss of genome wide heterochromatin condensation. Transcriptionally activated TEs go through additional non-canonical forms of RdDM that are dependent on RNA Polymerase II expression. However, the global targets of the non-canonical RdDM pathway have not been explored. In an attempt to identify and contrast the targets of canonical and expression-dependent non-canonical RdDM, we performed MethylC-seq of genome-wide DNA methylation patterns from several RdDM mutants in either the TE-silent or the TE-active (ddm1) contexts. Arabidopsis wildtype and twenty RdDM pathway mutants

2016-07-22 | E-GEOD-79746 | biostudies-arrayexpress

Arabidopsis small RNAs associated with RDR6-dependent RNA-directed DNA Methylation

Project description:Background: The majority of plant transposable elements (TEs) are found in a silenced state that is epigenetically propagated by the maintenance of symmetrical DNA methylation. TE methylation is established and reinforced by a mechanism of RNA-dependent DNA methylation, which is dependent on transcription of the PolIV RNA polymerase. Recently, a pathway has been described that initiates de novo DNA methylation dependent on components of the RNAi post-transcriptional silencing pathway, independent of PolIV. To define the function of this new pathway, we have focused on the RDR6 protein, which plays a central role in the pathway we refer to as RDR6-dependent RNA-directed DNA Methylation (RDR6-RdDM). Methods: We have sequenced total small RNAs from wild-type and three mutant genotypes: ddm1, ddm1/rdr6, and rdr6. Conclusions: We demonstrate that RDR6-RdDM utilizes 21 and 22 nucleotide siRNAs to de novo methylate actively transcribing TEs. In addition, we find that the RDR6-RdDM pathway functions in the efficient initiation and re-establishment of TE transcriptional silencing, while it is not necessary to maintain trans-generational epigenetic silencing. Examination of flower bud small RNAs from wild-type and 3 mutant genotypes

2013-04-11 | E-GEOD-41755 | biostudies-arrayexpress

Full-length autonomous transposable elements are preferentially targeted by expression-dependent forms of RNA-directed DNA methylation

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2016-07-22 | GSE79746 | GEO

Pathway conversion enables a double-lock mechanism to maintain DNA methylation and genome stability

Project description:The CMT2 and RNA-directed DNA methylation (RdDM) pathways have been proposed to separately maintain CHH methylation in specific regions of the Arabidopsis thaliana genome. Here, we show that dysfunction of the chromatin remodeller DDM1 causes hundreds of genomic regions to switch from CMT2-dependency to RdDM-dependency in DNA methylation. These converted loci are enriched at the edge regions of long transposable elements (TEs). Furthermore, we found that blocking the pathway switch by disrupting both DDM1 and RdDM causes strong reactivation of TEs and a burst of TE transposition in the first generation of mutant plants, indicating that the pathway conversion is critical to maintaining TE repression and protecting genomic stability. Our findings reveal the existence of a novel pathway conversion-based backup mechanism to guarantee the maintenance of DNA methylation and genome integrity.

2021-08-11 | GSE165877 | GEO

DNA Topoisomerase 1a Promotes RNA-directed DNA Methylation and Histone Lysine 9 Dimethylation at Transposable Elements in Arabidopsis [RNA-Seq]

Project description:RNA-directed DNA methylation (RdDM) is a transcriptional silencing mechanism mediated by small and long noncoding RNAs produced by the plant-specific RNA polymerases Pol IV and Pol V, respectively. Through a chemical genetics screen with a luciferase-based DNA methylation reporter, LUCL, we found that camptothecin, a compound with anti-cancer properties that targets DNA topoisomerase 1a (TOP1a) was able to de-repress LUCL by reducing its DNA methylation and H3K9 dimethylation (H3K9me2) levels. Further studies with Arabidopsis top1a mutants showed that TOP1a promotes RdDM by facilitating the production of Pol V-dependent long non-coding RNAs, AGONAUTE4 recruitment and H3K9me2 deposition at transposable elements (TEs). 5 small RNA libraries were sequenced

2014-07-08 | E-GEOD-50719 | biostudies-arrayexpress

Arabidopsis small RNAs associated with RDR6-dependent RNA-directed DNA Methylation

2013-04-11 | GSE41755 | GEO

DDM1 and RdDM are the major regulators of transposon DNA methylation in Arabidopsis

Project description:Eukaryotic DNA methylation is found in silent transposable elements and active genes. Nucleosome remodelers of the DDM1/Lsh family are thought to be specifically required to maintain transposon methylation, but the reason for this is unknown. Here, we find that a chromatin gradient that extends from the most heterochromatic transposons to euchromatic genes determines the requirement of DDM1 for methylation maintenance in all sequence contexts. We also show that small RNA-directed DNA methylation (RdDM) is inhibited by heterochromatin and absolutely requires the nucleosome remodeler DRD1. DDM1 and RdDM independently mediate nearly all transposon methylation, which is catalyzed by the methyltransferases MET1 (CG), CMT3 (CHG), DRM2 (CHH) and CMT2 (CHH), and collaborate to repress transposition and regulate the methylation and expression of genes. Our results indicate that the Arabidopsis genome is defined by a heterochromatic continuum that governs the access of DNA methyltransferases and potentially all DNA binding proteins. Examination of DNA methylation, transcription and nucleosomes in Arabidopsis wild-type and/or ddm1, RdDM and DNA methylase mutants.

2013-04-17 | E-GEOD-41302 | biostudies-arrayexpress

Small RNA accumulation in Transposable Element-silent and Transposable Element-active epigenomes

Project description:Background: The small RNAs that Transposable Elements generate are vastly different when they are transcriptionally silenced compared to when they are transcriptionally activated. We performed the deep sequencing of small RNAs in a number of small RNA biogenesis mutants in both Transposable Element-silenced and Transposable Element-active epigenome backgrounds. Results: We found that Transposable Elements generate large amounts of 21-22nt siRNAs only when they are transcriptionally active. These 21-22nt siRNAs are incorporated into the AGO6 protein. Conclusion: Ago6 is the key protein that bridges the post-transcriptional degradation of Transposable Element mRNAs and the establishment of DNA methylation. Examination of flower bud small RNAs from wild type and 5 single or double mutant combinations, many of which have biological replicates. In addition, IP purification of the AGO6 protein (and mock no-antigen controls) followed by sequencing of the incorporated small RNAs. Replicate A for Col and ddm1 are submitted in GSE41755

2014-11-14 | E-GEOD-57191 | biostudies-arrayexpress

Control of agricultural traits by hc-siRNA associated MITEs in rice

Project description:Transposable elements (TEs) and repetitive sequences comprise over 40% of rice genome. Different TEs are tightly regulated by distinct epigenetic mechanisms. For example, the activities of LTR retrotransposon Tos17 and non-LTR retrotransposon LINE element Karma are uniquely regulated by histone H3K9 methylation and histone H3K4 demethylation, respectively. Miniature inverted repeat transposable elements (MITEs) are one of the most high-copy-number DNA transposons, which are interspersed around rice genome and might influence nearby gene expression. In plants, 24-nucleotide (24-nt) heterochromatic small interfering RNAs (hc-siRNAs) derived from repeats and TEs. To what extent hc-siRNA associated TEs affect gene expression and therefore contribute to agricultural traits in rice remains elusive. Here, we show that OsDCL3a, one of Dicer-Like 3 (DCL3) homolog, is primarily responsible for 24-nt hc-siRNA processing in rice. Impaired OsDCL3a displayed altered important agricultural traits in rice. We found that genome-wide (281,563) 24-nt hc-siRNA clusters were OsDCL3a-dependent, among which MITEs were significantly enriched. Impaired OsDCL3a caused significant overlapping between reduced hc-siRNAs from MITEs and elevated nearby gene expression. Intriguingly, genes involved in Gibberellin and Brassinosteroid homeostasis were identified as direct targets of OsDCL3a, which may attribute to dwarfism and enlarged flag leaf angle upon OsDCL3a deficiency. Our work uncovers OsDCL3a-dependent hc-siRNAs derived from MITEs as broad spectrum of regulators for fine-tuning gene expression, and this observation may reflect a conserved mechanism in other higher plants with dispersed repeat- or TE-rich genomes. Examination of OsDCL3a-dependent hc-siRNAs derived from MITEs.

2014-02-04 | E-GEOD-50778 | biostudies-arrayexpress

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