The SNF2 and RING domain proteins FRG1 and 2 are required for RNA-directed DNA methylation in Arabidopsis
Ontology highlight
ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
SUBMITTER: Hume Stroud
PROVIDER: E-GEOD-62801 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
SNF2 chromatin remodeler-family proteins FRG1 and -2 are required for RNA-directed DNA methylation.
Groth Martin M Stroud Hume H Feng Suhua S Greenberg Maxim V C MV Vashisht Ajay A AA Wohlschlegel James A JA Jacobsen Steven E SE Ausin Israel I
Proceedings of the National Academy of Sciences of the United States of America 20141125 49
DNA methylation in Arabidopsis thaliana is maintained by at least four different enzymes: DNA methyltransferase1 (MET1), chromomethylase3 (CMT3), domains rearranged methyltransferase2 (DRM2), and chromomethylase2 (CMT2). However, DNA methylation is established exclusively by the enzyme DRM2, which acts in the RNA-directed DNA methylation (RdDM) pathway. Some RdDM components belong to gene families and have partially redundant functions, such as the endoribonucleases dicer-like 2, 3, and 4, and i ...[more]
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Project description:DNA methylation in Arabidopsis thaliana is maintained by at least four different enzymes: MET1, CMT3, DRM2, and CMT2. However, DNA methylation is established exclusively by the enzyme DRM2, which acts in the RNA-directed DNA methylation (RdDM) pathway. Some RdDM components belong to gene families and have partially redundant functions, such as DICER-LIKE 2, 3 and 4, and IDN2- LIKE 1 and 2. Traditional mutagenesis screens usually fail to detect genes if they are redundant, since the loss of one gene can be compensated by a related gene. In an effort to circumvent this issue, we utilized co-expression data to identify closely related genes that are co-regulated with genes in the RdDM pathway. Here we report the discovery of two redundant proteins, SNF2-RING-HELICASE-LIKE1 and 2 (FRG1 and 2) that are putative chromatin-related paralogous proteins. Analysis of genomeM-bM-^@M-^Swide bisulfite sequencing shows that simultaneous mutations of FRG1 and 2 cause broad defects in methylation at RdDM targeted loci. We also show that FRG1 stably associates with Su(var)3-9-related SUVR2, a known RdDM component, in vivo. Combined, our results identify FRG1 and FRG2 as novel components of the RdDM machinery. Small RNA profiles of Columbia 0 wild type, frg1-1 frg2-1 double, frg1 to 5 quintuple, suvr2-1, dcl3-1 and drm1-1 drm2-2 double mutants, 3 biological replicates each.
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Project description:DNA methylation in Arabidopsis thaliana is maintained by at least four different enzymes MET1, CMT3, DRM2 and CMT2. However, new methylation patterns are established exclusively by the enzyme DRM2, which acts within a pathway termed RNA-directed DNA methylation (RdDM). Some RdDM components belong to gene families and have partially redundant functions (i.e. DICER LIKE 2, 3 and 4 and IDN2 PARALOG 1 and 2). Traditional screens, which usually affect single genes, usually fail to detect genes if they are redundant, since the loss of one gene is compensated by a related gene. In an effort to circumvent this caveat, we utilized co-expression data to identify closely related genes that are co-regulated with genes in the RdDM pathway. We report the discovery of two redundant proteins, SNF2-RING-HELICASE1 and 2 (FRINGE1 and 2) that are putative chromatin-related paralogous proteins. Analysis of genome–wide bisulfite sequencing shows that simultaneous mutations of FRINGE1 and 2 cause broad defects in methylation at RdDM targeted loci. We also show that FRINGE1 stably associates with Su(var)3-9-related SUVR2, a known RdDM component, in vivo. Combined our results identify FRINGE1 and FRINGE2 as novel components of the RdDM machinery. One fringe 1/2 sample analyzed.
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Project description:DNA methylation in Arabidopsis thaliana is maintained by at least four different enzymes: MET1, CMT3, DRM2, and CMT2. However, DNA methylation is established exclusively by the enzyme DRM2, which acts in the RNA-directed DNA methylation (RdDM) pathway. Some RdDM components belong to gene families and have partially redundant functions, such as DICER-LIKE 2, 3 and 4, and IDN2- LIKE 1 and 2. Traditional mutagenesis screens usually fail to detect genes if they are redundant, since the loss of one gene can be compensated by a related gene. In an effort to circumvent this issue, we utilized co-expression data to identify closely related genes that are co-regulated with genes in the RdDM pathway. Here we report the discovery of two redundant proteins, SNF2-RING-HELICASE-LIKE1 and 2 (FRG1 and 2) that are putative chromatin-related paralogous proteins. Analysis of genome–wide bisulfite sequencing shows that simultaneous mutations of FRG1 and 2 cause broad defects in methylation at RdDM targeted loci. We also show that FRG1 stably associates with Su(var)3-9-related SUVR2, a known RdDM component, in vivo. Combined, our results identify FRG1 and FRG2 as novel components of the RdDM machinery. mRNA profiles of 14 days old seedlings of Columbia 0 wild type and frg1-1 frg2-1 double mutants, 3 biological replicates each.
2014-11-25 | E-GEOD-62799 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below.
2014-11-25 | GSE62801 | GEO
Project description:DNA methylation in Arabidopsis thaliana is maintained by at least four different enzymes: MET1, CMT3, DRM2, and CMT2. However, DNA methylation is established exclusively by the enzyme DRM2, which acts in the RNA-directed DNA methylation (RdDM) pathway. Some RdDM components belong to gene families and have partially redundant functions, such as DICER-LIKE 2, 3 and 4, and IDN2- LIKE 1 and 2. Traditional mutagenesis screens usually fail to detect genes if they are redundant, since the loss of one gene can be compensated by a related gene. In an effort to circumvent this issue, we utilized co-expression data to identify closely related genes that are co-regulated with genes in the RdDM pathway. Here we report the discovery of two redundant proteins, SNF2-RING-HELICASE-LIKE1 and 2 (FRG1 and 2) that are putative chromatin-related paralogous proteins. Analysis of genome–wide bisulfite sequencing shows that simultaneous mutations of FRG1 and 2 cause broad defects in methylation at RdDM targeted loci. We also show that FRG1 stably associates with Su(var)3-9-related SUVR2, a known RdDM component, in vivo. Combined, our results identify FRG1 and FRG2 as novel components of the RdDM machinery.
2014-11-25 | GSE62799 | GEO
Project description:DNA methylation in Arabidopsis thaliana is maintained by at least four different enzymes: DNA methyltransferase1 (MET1), chromomethylase3 (CMT3), domains rearranged methyltransferase2 (DRM2), and chromomethylase2 (CMT2). However, DNA methylation is established exclusively by the enzyme DRM2, which acts in the RNA-directed DNA methylation (RdDM) pathway. Some RdDM components belong to gene families and have partially redundant functions, such as the endoribonucleases dicer-like 2, 3, and 4, and involved in de novo2 (IDN2) interactors IDN2-like 1 and 2. Traditional mutagenesis screens usually fail to detect genes if they are redundant, as the loss of one gene can be compensated by a related gene. In an effort to circumvent this issue, we used coexpression data to identify closely related genes that are coregulated with genes in the RdDM pathway. Here we report the discovery of two redundant proteins, SNF2-ring-helicase-like1 and -2 (FRG1 and -2) that are putative chromatin modifiers belonging to the SNF2 family of helicase-like proteins. Analysis of genome-wide bisulfite sequencing shows that simultaneous mutations of FRG1 and -2 cause defects in methylation at specific RdDM targeted loci. We also show that FRG1 physically associates with Su(var)3-9-related SUVR2, a known RdDM component, in vivo. Combined, our results identify FRG1 and FRG2 as previously unidentified components of the RdDM machinery.
| S-EPMC4267348 | biostudies-literature
Project description:DNA methylation in Arabidopsis thaliana is maintained by at least four different enzymes: MET1, CMT3, DRM2, and CMT2. However, DNA methylation is established exclusively by the enzyme DRM2, which acts in the RNA-directed DNA methylation (RdDM) pathway. Some RdDM components belong to gene families and have partially redundant functions, such as DICER-LIKE 2, 3 and 4, and IDN2- LIKE 1 and 2. Traditional mutagenesis screens usually fail to detect genes if they are redundant, since the loss of one gene can be compensated by a related gene. In an effort to circumvent this issue, we utilized co-expression data to identify closely related genes that are co-regulated with genes in the RdDM pathway. Here we report the discovery of two redundant proteins, SNF2-RING-HELICASE-LIKE1 and 2 (FRG1 and 2) that are putative chromatin-related paralogous proteins. Analysis of genome–wide bisulfite sequencing shows that simultaneous mutations of FRG1 and 2 cause broad defects in methylation at RdDM targeted loci. We also show that FRG1 stably associates with Su(var)3-9-related SUVR2, a known RdDM component, in vivo. Combined, our results identify FRG1 and FRG2 as novel components of the RdDM machinery.
2014-11-25 | GSE62800 | GEO
Project description:RNA-directed DNA methylation (RdDM) in Arabidopsis thaliana depends on the synthesis of non-coding RNAs by nuclear RNA polymerase E (NRPE or Pol V) 1-3, but the mechanism by which Pol V is targeted is unknown. Here we show that genome-wide Pol V association with chromatin redundantly requires the SU(VAR)3-9 homologs, SUVH2 and SUVH9. These proteins resemble histone methyltransferases, however a crystal structure reveals that SUVH9 lacks a peptide-substrate binding cleft and lacks a properly formed S-Adenosyl methionine (SAM) binding pocket necessary for normal catalysis, consistent with a lack of methyltransferase activity for these proteins. SUVH2 and SUVH9 both contain SET- and RING-ASSOCIATED (SRA) domains capable of binding methylated DNA, suggesting that they function to recruit Pol V through DNA methylation. Consistent with this model, mutation of the DNA METHYLTRANSFERASE 1, MET1, causes losses of DNA methylation, a nearly complete loss of Pol V at its normal locations, and redistribution of Pol V to sites that become hypermethylated. By tethering SUVH2 with a zinc finger to an unmethylated epiallele of the homeodomain transcription factor FWA, we demonstrate that SUVH2 is sufficient to both recruit Pol V and establish DNA methylation and gene silencing. Our results suggest that Pol V is recruited to DNA methylation through the methyl-DNA binding SUVH2 and SUVH9 proteins, and our mechanistic findings suggest a means for selectively targeting regions of the plant genome for epigenetic silencing. For wild type plants (ecotype Columbia) and suvh2 suvh9 double mutants whole-genome small RNA (sRNA-seq) and bisulfite sequencing (BS-seq) was performed. For the small RNA sequencing nrpd1 and nrpe1 mutant plants were sequenced in parallel as controls. For the bisulfite sequencing data, the wildtype data and that of the suvh2 and suvh9 single mutants was previously published and is thus not submitted here. In addition, two replicates of whole genome chromatin immunoprecipitation (ChIP-seq) was performed on wild type (ecotype Columbia) plants as a negative control with experimentals consiting of wildtype and suvh2 suvh9 mutant plants carrying a C-terminally epitope tagged (3XFLAG) NRPE1. Whole genome ChIP seq was also performed using a gifted endogenous NRPE1 antibody in a wildtype and met1 mutant background. Whole-genome bisulfite sequencing was also performed on fwa-4 epiallele plants transformed with the wild-type SUVH2 protein-coding construct containing a tethering zinc finger targeted to repeats at the fwa gene. For these transgenic libraries a line carrying a FLAG and fwa targeting zinc-finger tagged KRYPTONITE methyltransferase was bisulfite sequenced as a control (“FLAG-ZF-KYP”).
2014-01-01 | E-GEOD-52041 | biostudies-arrayexpress
Project description:In Arabidopsis, CHG DNA methylation is controlled by the H3K9 methylation mark through a self-reinforcing loop between DNA methyltransferase CHROMOMETHYLASE3 (CMT3) and H3K9 histone methyltransferase KRYPTONITE/SUVH4 (KYP). We report on the structure of KYP in complex with methylated DNA, substrate H3 peptide and cofactor SAH, thereby defining the spatial positioning of the SRA domain relative to the SET domain. The methylated DNA is bound by the SRA domain with the 5mC flipped out of the DNA, while the H3(1-15) peptide substrate binds between the SET and post-SET domains, with the epsilon-ammonium of K9 positioned adjacent to bound SAH. These structural insights complemented by in vivo functional data on key mutants of residues lining the 5mC and H3K9-binding pockets within KYP, establish how methylated DNA recruits KYP to the histone substrate. Together, the structures of KYP and previously reported CMT3 complexes provide insights into molecular mechanisms linking DNA and histone methylation. Plants homozygous for null mutations in the KRYPTONITE H3K9 methyltransferase were stably transformed with transgenes encoding the wildtype KYP protein or transgenes carrying induced point mutations in the KYP active site. The resulting lines were assayed for DNA methylation by whole-genome bisulfite sequencing to learn the efficiency with which wildtype and mutant versions of the KYP protein could restore DNA methylation lost in a kyp mutant. Samples 7 and 8 were run as single Illumina lanes and as such were compared to a previous Col sample (GSM881756), this Col sample was realigned to the TAIR10 genome for this study and as such updated processed files are available with this submission. These samples were used to define kyp mutant CHG context DMRs that were complemented upon introduction of the wildtype KYP protein. Samples 1-6 were run as multiplexed samples and were used to assay the degree of complementation for various point mutants. All plants are in the Col ecotype background.
2014-08-01 | E-GEOD-57963 | biostudies-arrayexpress
Project description:The SNF2 and RING domain proteins FRG1 and 2 are required for RNA-directed DNA methylation in Arabidopsis
| PRJNA265182 | ENA