Similar Datasets
Project description:ChIP-seq of DRD1, DMS3 and RDM1 in Arabidopsis
| PRJNA538858 | ENA
Project description:RNA-directed DNA methylation (RdDM) plays an essential role in transposable element (TE) silencing in plants. In the Arabidopsis RdDM pathway, the DDR complex containing DRD1, DMS3, and RDM1, is necessary for recruiting Pol V to transcribe scaffold RNA. Although the role of DDR is known, the mechanism by which the DDR complex is regulated remains unexplored. Here, we demonstrate that the Anaphase Promoting Complex/Cyclosome (APC/C) monitors the assembly of the DDR complex by targeting DMS3 for degradation. We show that a subset of Pol V-dependent RdDM loci are de-repressed in apc/c mutants, accompanied by defective recruitment and transcription of Pol V. APC/C targets DMS3 for ubiquitination and degradation in a D box-dependent manner, and the D-box-mutated DMS3 fails to complement the dms3 mutant. Competitive binding assays shows that the dosage of DMS3 is critical for the assembly of the DDR complex, and in vivo gel filtration analysis shows that the assembly of both DDR and Pol V is compromised in the apc8 mutant. These findings uncover a safeguard role of APC/C-mediated DMS3 degradation in the assembly of the DDR complex, and provide a direct link between selective protein degradation and RdDM.
2019-01-31 | GSE106970 | GEO
Project description:RNA-directed DNA methylation (RdDM) plays an essential role in transposable element (TE) silencing in plants. In the Arabidopsis RdDM pathway, the DDR complex containing DRD1, DMS3, and RDM1, is necessary for recruiting Pol V to transcribe scaffold RNA. Although the role of DDR is known, the mechanism by which the DDR complex is regulated remains unexplored. Here, we demonstrate that the Anaphase Promoting Complex/Cyclosome (APC/C) monitors the assembly of the DDR complex by targeting DMS3 for degradation. We show that a subset of Pol V-dependent RdDM loci are de-repressed in apc/c mutants, accompanied by defective recruitment and transcription of Pol V. APC/C targets DMS3 for ubiquitination and degradation in a D box-dependent manner, and the D-box-mutated DMS3 fails to complement the dms3 mutant. Competitive binding assays shows that the dosage of DMS3 is critical for the assembly of the DDR complex, and in vivo gel filtration analysis shows that the assembly of both DDR and Pol V is compromised in the apc8 mutant. These findings uncover a safeguard role of APC/C-mediated DMS3 degradation in the assembly of the DDR complex, and provide a direct link between selective protein degradation and RdDM.
2019-01-31 | GSE106971 | GEO
Project description:The plant-specific DNA-dependent RNA polymerase V (Pol V) evolved from Pol II to function in an RNA-directed DNA methylation pathway. Here, we have identified targets of Pol V in Arabidopsis thaliana on a genome-wide scale using ChIP-seq of NRPE1, the largest catalytic subunit of Pol V. We found that Pol V is enriched at promoters and evolutionarily recent transposons. This localization pattern is highly correlated with Pol V-dependent DNA methylation and small RNA accumulation. We also show that genome-wide association of Pol V with chromatin is dependent on all members of a putative chromatin-remodeling complex termed DDR. Our study presents the first genome-wide view of Pol V occupancy and sheds light on the mechanistic basis of Pol V localization. Furthermore, these findings suggest a role for Pol V and RNA-directed DNA methylation in genome surveillance and in responding to genome evolution. For wild type plants (ecotype Columbia) and nrpe1 mutants whole-genome small RNA (sRNA-seq) and bisulfite sequencing (BS-seq) was performed. In addition whole genome chromatin immunoprecipitation (ChIP-seq) was performed on wild type (ecotype Columbia) plants as a negative control with experimentals consiting of wild type plants carrying a C-terminally epitope tagged (2XFLAG) NRPE1, as well as the NRPE1-FLAG construct in drd1, dms3, and rdm1 mutant backgrounds.
2012-08-05 | E-GEOD-39247 | biostudies-arrayexpress
Project description:We performed WGBS of two independent transgenic lines expressing RDM1-FLAG in rdm1 mutant background, as well as WS and rdm1-3 controls.
2019-07-10 | GSE130319 | GEO
Project description:WBGS of WS, rdm1-3 and RDM1-FLAG-2 and RDM1-FLAG-16 transgenic lines
| PRJNA535489 | ENA
Project description:Chemical cross-linking coupled to mass spectrometry was used to study the A. thaliana DDR complex consisting of DMS3, RMD1 and a peptide from the interaction region of DRD1. Cross-linking was performed using different cross-linking chemistries: disuccinimidyl suberate (DSS) and a combination of adipic acid dihydrazide (ADH) and the coupling reagent, DMTMM.
2019-07-15 | PXD013470 | Pride
Project description:FLAG-tagged RDM1 was immunoprecipitated from Arabidopsis and then analyzed by LC-MS/MS on a Q-Exactive mass spectrometer.
2019-07-14 | MSV000084088 | MassIVE
Project description:We identified in a forward genetic screen a novel factor required for RNA-directed DNA methylation (IWR1-like transcription factor). The aim of the study was to identify natural targets of this factor and to compare it to targets of drd1 another major key factor required for the RNA silencing process in plants. We performed transcriptome analysis to identify targets of this factor. For this we used whole plants at seedling stage, isolated total RNA and then used the standard Affymetrix pipeline/chemistry. The mutated transcription factor, dms4-1 was compared to its respective wildtype (EDT, Col-O background). For microarray analysis THREE biological replicates were run.<br><br>For transcriptome comparison we analyzed another factor required for the process RNA-directed DNA methylation in plants, drd1 also identified in a forward genetic screen and previously characterized in depth (Kanno et al. Current Biology, 14, 801-805, (2004), Huettel et al. EMBO J. 25, 2828-2836 (2006)). For transcriptome analysis two alleles of drd1 (drd1-1 and drd1-6) with two biological replicates each was compared to the respective wildtype DT, which served as starting material in this forward genetic screen (DT, Col-0 background). For drd1 mutants and DT wildtype analysis 10 day old seedlings were used as starting matieral while for dms4-1 and EDT 3 week old seedlings were analyzed.
2010-01-10 | E-MEXP-2251 | biostudies-arrayexpress
Project description:In plants, heterochromatin is maintained by a small RNA-based gene silencing mechanism known as RNA-directed DNA methylation (RdDM). RdDM requires the non-redundant functions of two plant-specific DNA-dependent RNA polymerases (Pol) Pol IV and Pol V. Pol IV plays a major role in siRNA biogenesis, while Pol V may recruit DNA methylation machinery to target endogenous loci for silencing. Although small RNA-generating regions which are dependent on both Pol IV and Pol V have been identified previously, the genomic loci targeted Pol V for siRNA accumulation and silencing have not been described extensively. To characterize the Pol V-dependent, heterochromatic siRNA-generating regions in the Arabidopsis genome, we deeply sequenced the small RNA populations of wild-type and Pol V mutant plants. Furthermore, we characterized the siRNA-generating regions which were dependent on RdDM effectors and examined their dependency on Pol V. Small RNA libraries were generated and deeply sequenced from mutant alleles dms4-1, drd1-1, dms3-1, and rdm1-4, along with their control library (“Wt(T+S)”) which has been described previously (Kanno et al. 2004 Current Biology). More than 2,000 small RNA-generating loci were identified which were greatly suppressed in Pol V mutants. The Pol V-dependent, heterochromatic siRNA-generating regions were characterized in the Arabidopsis genome by deep sequencing the small RNA populations of wild-type and Pol V mutant plants. Deep SBS sequencing was used for small RNA profiling of immature inflorescence tissues from RNA polymerase V and RdDM mutants.
2012-06-11 | E-GEOD-36424 | biostudies-arrayexpress