Unknown,Transcriptomics,Genomics,Proteomics

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Long non-coding RNA produced by RNA polymerase V determines boundaries of heterochromatin

ABSTRACT: We report genome-wide detection of long noncoding RNA (lncRNA) generate by Pol V in transcriptional gene silencing in Arabidopsis thaliana. We further show that most of these transcripts are bound by AGO4. RNA immunoprecipitation followed by high-throughput sequencing (RIP-seq) RNA immunoprecipitation with an anti-AGO4 antibody was performed in one biological replicate and included Col-0, nrpe1, and ago4-1; RNA immunoprecipitation with an anti-NRPE1 (largest subunit of Pol V) was performed in two replicates; replicate 1 included Col-0 and nrpe1, replicated two included Col-0, nrpe1, ago4-1, and idn2-1

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Andrzej Wierzbicki

PROVIDER: E-GEOD-70290 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:DNA methylation is a mechanism of epigenetic gene regulation and genome defense conserved in many eukaryotic organisms. In Arabidopsis, the DNA methyltransferase DRM2 controls RNA-directed DNA methylation in a pathway that also involves the plant specific RNA Polymerase V (Pol V). The Arabidopsis genome also encodes an evolutionarily conserved but catalytically inactive DNA methyltransferase DRM3. Here, we show that DRM3 has moderate effects on global RNA-directed DNA methylation and small RNA abundance throughout the genome, and DRM3 protein physically interacts with Pol V. In drm3 mutants, we observe a lower level of Pol V-dependent transcripts, even though Pol V chromatin occupancy is increased at many sites in the genome. These findings suggest that DRM3 acts to promote Pol V transcriptional elongation or assist in the stabilization of Pol V transcripts, and shed further light on the mechanism of RNA-directed DNA methylation. For wildtype plants as well as drm3, drm2, and nrpe1 mutants ChIP-seq was carried out using an endogenous NRPE1 antibody given to us by the Craig Pikaard lab. Two biological replicates of ChIP-seq were also carried out using anti-Flag resin on wildtype and drm3 plants carrying a Flag epitope tagged version of NRPE1. Small RNA sequencing was carried out on Col, drm3, drm2, and nrpe1 plants. Finally, whole-genome bisulfite sequencing analysis was carried out on previously published datasets (as detailed below) which were realigned using a newer genome version and mapping protocol. As such the updated processed files are part of this submission. Please note that the drm2, drm3, and nrpe1 mutant libraries used in this study were previously published (GSE39901), as were the 2 other Col replicates used (GSE36129) as below and thus duplicated sample records were created for the convenient retrieval of the complete raw data from SRA; Bisulfite_seq-Col_1 - GSM881756 Bisulfite_seq-Col_2 - GSM1193638 Bisulfite_seq-drm3 - GSM981017 Bisulfite_seq-drm2 - GSM981015 Bisulfite_seq-nrpe1- GSM981040

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Long non-coding RNA produced by RNA polymerase V determines boundaries of heterochromatin

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