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Analysis of the Argonaute 4-associated non-coding RNA in Arabidopsis thaliana sheds novel insights into gene regulation through RNA-directed DNA methylation

ABSTRACT: RNA-directed DNA methylation (RdDM) is a de novo DNA methylation mechanism in plants that plays a fundamental role in plant defence against invasive DNA and in maintaining genome stability by silencing transposons and repetitive sequences. Using nuclear RNA immunoprecipitation, we constructed a highly enriched library and obtained sequences of ncRNAs specifically associated with Argonaute 4 (AGO4), a key component of RNA-directed DNA methylation. A negative immunoprecipation (IP) library was prepared using IP RNA from tissues that don't express the target protein. A nuclear RNA library was also prepared using nuclei isolated RNA. Three libraries (1X FLAGAGO4 IP, 1X negative IP, 1X nuclear RNA). RNA-IP or nuclear RNA seq cDNA libraries were prepared using the template-switch cDNA library preparation method derived from Zhao et al (2010), and subjected to Illumina GAIIx single end sequencing (100bp)

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Phil Au

PROVIDER: E-GEOD-48617 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Analysis of the Argonaute 4-associated non-coding RNA in Arabidopsis thaliana sheds novel insights into gene regulation through RNA-directed DNA methylation

Project description:RNA-directed DNA methylation (RdDM) is a de novo DNA methylation mechanism in plants that plays a fundamental role in plant defence against invasive DNA and in maintaining genome stability by silencing transposons and repetitive sequences. Using nuclear RNA immunoprecipitation, we constructed a highly enriched library and obtained sequences of ncRNAs specifically associated with Argonaute 4 (AGO4), a key component of RNA-directed DNA methylation. A negative immunoprecipation (IP) library was prepared using IP RNA from tissues that don't express the target protein. A nuclear RNA library was also prepared using nuclei isolated RNA.

2016-07-01 | GSE48617 | 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

Patterns of Population Epigenomic Diversity in Arabidopsis thaliana (RNA-Seq)

Project description:Natural epigenetic variation provides a source for the generation of phenotypic diversity, but to understand its contribution to phenotypic diversity, its interaction with genetic variation requires further investigation. Here, we report population-wide DNA sequencing of genomes, transcriptomes, and methylomes of wild Arabidopsis thaliana accessions. Single cytosine methylation polymorphisms are unlinked to genotype. However, the rate of linkage disequilibrium decay amongst differentially methylated regions targeted by RNA-directed DNA methylation is similar to the rate for single nucleotide polymorphisms. Association analyses of these RNA-directed DNA methylation regions with genetic variants identified 2,372 methylQTL, which revealed the first population estimate of genetically dependent methylation variation. Analysis of invariably methylated transposons and genes across this population indicates that loci targeted by RNA-directed DNA methylation are epigenetically reactivated during male gametogenesis, which facilitates their silencing across generations. RNA-seq from naturally-occurring Arabidopsis accessions

2013-03-05 | E-GEOD-43858 | biostudies-arrayexpress

RNA_Seq of AGOI_IP long (16:8), short (8:16) day heads

Project description:Input and IP (AGO1_ab) RNA_seq libraries were prepared from long (2 libraries) and short (1 library) day heads

2021-12-04 | GSE189992 | GEO

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

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. 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.

2016-07-22 | E-GEOD-79780 | biostudies-arrayexpress

Genome-wide profiling of DNA methylation in two Arabidopsis ecotypes and their reciprocal hybrids - mRNA-seq

Project description:Heterosis is a fundamental biological phenomenon characterized by the superior performance of a hybrid over its parents in many traits, but the underlying molecular basis remains elusive. To investigate whether DNA methylation plays a role in heterosis, we compared at single base-pair resolution the DNA methylomes of Arabidopsis Ler and C24 parental lines and their reciprocal F1 hybrids that exhibited heterosis for many quantitative traits. Both hybrids displayed increased DNA methylation across their entire genomes, especially in transposable elements. Interestingly, we found that increased methylation of the hybrid genomes predominantly occurred in regions that were differentially methylated in the two parents and covered by small RNAs (sRNAs), implying that the RNA-directed DNA methylation (RdDM) pathway may direct DNA methylation in hybrids. In addition, we found that 77 genes sensitive to remodeling of DNA methylation were transcriptionally repressed in both reciprocal hybrids, including genes involved in flavonoid biosynthesis and two circadian oscillator genes, CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL. Moreover, growth vigor of F1 hybrids was compromised by treatment with an agent that demethylates DNA, and by abolishing production of functional small RNAs due to mutations in Arabidopsis RNA methyltransferase HUA ENHANCER1. Together, our data suggest that genome-wide remodeling of DNA methylation directed by the RdDM pathway may play a role in hybrid vigor. Examination of mRNA sequencing in 2 Arabidopsis ecotypes and their reciprocal hybrids.

2011-12-22 | E-GEOD-34655 | biostudies-arrayexpress

The role of m6A methylation modification in CPB2 toxin treatment of porcine small intestinal epithelial cells

Project description:In this experiment, m6A-seq sequencing technology was used to study the functional role of methylated molecules in the process of CPB2 processing porcine small intestinal epithelial cells. In this experiment, an IP library and an input library were built together. The IP library was enriched with m6A specific antibodies to generate methylated RNA, and the influence of m6A methylation modification on its expression was analyzed by bioinformatics. We finally concluded that m6A methylation modification may play a very important role after CPB2 toxin treats small intestinal epithelial cells.

2021-10-05 | GSE167267 | GEO

Genome-wide profiling of DNA methylation in two Arabidopsis ecotypes and their reciprocal hybrids - small RNA-seq

Project description:Heterosis is a fundamental biological phenomenon characterized by the superior performance of a hybrid over its parents in many traits, but the underlying molecular basis remains elusive. To investigate whether DNA methylation plays a role in heterosis, we compared at single base-pair resolution the DNA methylomes of Arabidopsis Ler and C24 parental lines and their reciprocal F1 hybrids that exhibited heterosis for many quantitative traits. Both hybrids displayed increased DNA methylation across their entire genomes, especially in transposable elements. Interestingly, we found that increased methylation of the hybrid genomes predominantly occurred in regions that were differentially methylated in the two parents and covered by small RNAs (sRNAs), implying that the RNA-directed DNA methylation (RdDM) pathway may direct DNA methylation in hybrids. In addition, we found that 77 genes sensitive to remodeling of DNA methylation were transcriptionally repressed in both reciprocal hybrids, including genes involved in flavonoid biosynthesis and two circadian oscillator genes, CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL. Moreover, growth vigor of F1 hybrids was compromised by treatment with an agent that demethylates DNA, and by abolishing production of functional small RNAs due to mutations in Arabidopsis RNA methyltransferase HUA ENHANCER1. Together, our data suggest that genome-wide remodeling of DNA methylation directed by the RdDM pathway may play a role in hybrid vigor. Examination of small RNA sequencing in 2 Arabidopsis ecotypes and their reciprocal hybrids.

2011-12-22 | E-GEOD-34656 | 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

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