An siRNA-guided AGO protein recruits Polymerase V to initiate RNA-directed DNA methylation (BSA-seq dataset)
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ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
PROVIDER: GSE165573 | GEO | 2021/09/02
REPOSITORIES: GEO
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Project description:Foreign DNA is silenced in plants by small RNA-directed DNA methylation (RdDM). RNA Polymerase V (Pol V) is the key protein required to recruit DNA methyltransferases to a RdDM target region. However, the mechanism required for the initial recruitment of Pol V to a targeted locus remains a mystery. In this study, we used first generation (T1) transgene transformations to elucidate a novel mechanism by which Pol V is recruited to both new transgenic and endogenous loci. This study is heavily dependent on analyzing the DNA methylation pattern and small RNAs produced from specific loci. All small RNA sequences (sRNA-seq) and bisulfite converted amplicon sequencing data (BSAS) used in this study are submitted as part of this data series.
2021-09-02 | GSE165574 | GEO
Project description:Small RNA-dependent pairing of AGO/PIWI-containing effector complexes to chromatin-bound nascent transcripts has been proposed as a universal mechanism for guiding RNA-mediated TGS in eukaryotes. Likewise, Pol V-dependent transcripts have been implicated in the targeting of AGO4 to chromatin in RNA-directed DNA methylation (RdDM) in plants. Here, we show that the AGO hook platforms of PolV and SPT5L, another component of the transcriptional complex, are functionally redundant yet essential for RdDM at a genome-wide level. Synthesis of Pol V transcripts is uncoupled from AGO4 recruitment in AGO hook-minus plants, challenging the prevailing RNA-based mechanism of AGO4 targeting to chromatin in RdDM. Transcription is essential to lock the PolV transcription complex into a stable and productive DNA-bound state potentiating interactions of AGO4 with DNA. Consistent with this idea, laser UV-assisted crosslinking shows specific AGO4-DNA interaction at RdDM loci, suggesting a revised model for Pol V-mediated DNA methylation in plants, which explains the exquisite specificity of methylation.
2015-06-01 | GSE67216 | GEO
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
Project description:DNA methylation is an important epigenetic mark in many eukaryotic organisms. De novo DNA methylation in plants can be achieved by the RNA-directed DNA methylation (RdDM) pathway, where the plant-specific DNA-dependent RNA polymerase Pol IV transcribes target sequences to initiate 24-nt siRNA production and action. The Arabidopsis DTF1/SHH1 has been shown to associate with Pol IV and is required for 24-nt siRNA accumulation and transcriptional silencing at several RdDM target loci. However, the extent and mechanism of DTF1 function in RdDM is unclear. We show here that DTF1 is necessary for the accumulation of the majority of Pol IV-dependent 24-nt siRNAs. It is also required for a large proportion of Pol IV-dependent de novo DNA methylation. Interestingly, there is a group of RdDM target loci where 24-nt siRNA accumulation but not DNA methylation is dependent on DTF1. Taken together, our results show that DTF1 is a core component of the RdDM pathway. Our results also suggest the involvement of DTF1 in an important negative feedback mechanism for DNA methylation at some RdDM target loci. Examination of whole-genome DNA methylation and small RNA in 12-day-old Col-0, dtf1-2, nrpd1-3 and nrpe1-11 seedlings
2013-06-03 | E-GEOD-44209 | 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.
2012-06-11 | GSE36424 | GEO
Project description:DNA methylation is an important epigenetic mark in many eukaryotic organisms. De novo DNA methylation in plants can be achieved by the RNA-directed DNA methylation (RdDM) pathway, where the plant-specific DNA-dependent RNA polymerase Pol IV transcribes target sequences to initiate 24-nt siRNA production and action. The Arabidopsis DTF1/SHH1 has been shown to associate with Pol IV and is required for 24-nt siRNA accumulation and transcriptional silencing at several RdDM target loci. However, the extent and mechanism of DTF1 function in RdDM is unclear. We show here that DTF1 is necessary for the accumulation of the majority of Pol IV-dependent 24-nt siRNAs. It is also required for a large proportion of Pol IV-dependent de novo DNA methylation. Interestingly, there is a group of RdDM target loci where 24-nt siRNA accumulation but not DNA methylation is dependent on DTF1. Taken together, our results show that DTF1 is a core component of the RdDM pathway. Our results also suggest the involvement of DTF1 in an important negative feedback mechanism for DNA methylation at some RdDM target loci.
2013-06-03 | GSE44209 | GEO
Project description:DNA methylation can be established by RNA-directed DNA methylation (RdDM) in plants. The association of RNA polymerase V (Pol V) with chromatin is a critical step for RdDM. While the SRA-domain-containing proteins SUVH2 and SUVH9 and the DDR complex are known to be required for the association of Pol V with chromatin, it is unknown whether the association of Pol V with chromatin requires other unidentified regulators. Here we found that SUVH9 is able to interact with a conserved histone-interaction protein, FVE, and a previously uncharacterized RRM domain-containing protein, which we named RRM1. We demonstrated that FVE facilitates the association of Pol V with chromatin and thus contributes to DNA methylation at a substantial subset of RdDM target loci, while RRM1 is only slightly involved in RdDM. FVE-dependent RdDM target loci are more abundant in gene-rich chromosome arms than FVE-independent RdDM target loci. FVE was previously shown to be a shared subunit of the RPD3-type histone deacetylase complex and the polycomb-type histone H3K27 trimethyltransferase complex, both of which are involved in transcriptional repression. This study reveals a previously uncharacterized role of FVE in RdDM and suggests that FVE may coordinate RdDM, histone deacetylation, and H3K27 trimethylation, thus ensuring transcriptional silencing of TEs in gene-rich chromosome arms to protect genes from harmful effects of potentially transcribed TEs.
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Project description:DNA methylation can be established by RNA-directed DNA methylation (RdDM) in plants. The association of RNA polymerase V (Pol V) with chromatin is a critical step for RdDM. While the SRA-domain-containing proteins SUVH2 and SUVH9 and the DDR complex are known to be required for the association of Pol V with chromatin, it is unknown whether the association of Pol V with chromatin requires other unidentified regulators. Here we found that SUVH9 is able to interact with a conserved histone-interaction protein, FVE, and a previously uncharacterized RRM domain-containing protein, which we named RRM1. We demonstrated that FVE facilitates the association of Pol V with chromatin and thus contributes to DNA methylation at a substantial subset of RdDM target loci, while RRM1 is only slightly involved in RdDM. FVE-dependent RdDM target loci are more abundant in gene-rich chromosome arms than FVE-independent RdDM target loci. FVE was previously shown to be a shared subunit of the RPD3-type histone deacetylase complex and the polycomb-type histone H3K27 trimethyltransferase complex, both of which are involved in transcriptional repression. This study reveals a previously uncharacterized role of FVE in RdDM and suggests that FVE may coordinate RdDM, histone deacetylation, and H3K27 trimethylation, thus ensuring transcriptional silencing of TEs in gene-rich chromosome arms to protect genes from harmful effects of potentially transcribed TEs.
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Project description:DNA methylation can be established by RNA-directed DNA methylation (RdDM) in plants. The association of RNA polymerase V (Pol V) with chromatin is a critical step for RdDM. While the SRA-domain-containing proteins SUVH2 and SUVH9 and the DDR complex are known to be required for the association of Pol V with chromatin, it is unknown whether the association of Pol V with chromatin requires other unidentified regulators. Here we found that SUVH9 is able to interact with a conserved histone-interaction protein, FVE, and a previously uncharacterized RRM domain-containing protein, which we named RRM1. We demonstrated that FVE facilitates the association of Pol V with chromatin and thus contributes to DNA methylation at a substantial subset of RdDM target loci, while RRM1 is only slightly involved in RdDM. FVE-dependent RdDM target loci are more abundant in gene-rich chromosome arms than FVE-independent RdDM target loci. FVE was previously shown to be a shared subunit of the RPD3-type histone deacetylase complex and the polycomb-type histone H3K27 trimethyltransferase complex, both of which are involved in transcriptional repression. This study reveals a previously uncharacterized role of FVE in RdDM and suggests that FVE may coordinate RdDM, histone deacetylation, and H3K27 trimethylation, thus ensuring transcriptional silencing of TEs in gene-rich chromosome arms to protect genes from harmful effects of potentially transcribed TEs
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Project description:DNA methylation can be established by RNA-directed DNA methylation (RdDM) in plants. The association of RNA polymerase V (Pol V) with chromatin is a critical step for RdDM. While the SRA-domain-containing proteins SUVH2 and SUVH9 and the DDR complex are known to be required for the association of Pol V with chromatin, it is unknown whether the association of Pol V with chromatin requires other unidentified regulators. Here we found that SUVH9 is able to interact with a conserved histone-interaction protein, FVE, and a previously uncharacterized RRM domain-containing protein, which we named RRM1. We demonstrated that FVE facilitates the association of Pol V with chromatin and thus contributes to DNA methylation at a substantial subset of RdDM target loci, while RRM1 is only slightly involved in RdDM. FVE-dependent RdDM target loci are more abundant in gene-rich chromosome arms than FVE-independent RdDM target loci. FVE was previously shown to be a shared subunit of the RPD3-type histone deacetylase complex and the polycomb-type histone H3K27 trimethyltransferase complex, both of which are involved in transcriptional repression. This study reveals a previously uncharacterized role of FVE in RdDM and suggests that FVE may coordinate RdDM, histone deacetylation, and H3K27 trimethylation, thus ensuring transcriptional silencing of TEs in gene-rich chromosome arms to protect genes from harmful effects of potentially transcribed TEs.
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