Dataset Information

The SET domain proteins SUVH2 and SUVH9 are required for Pol V occupancy at RNA-directed DNA methylation loci.

ABSTRACT: RNA-directed DNA methylation (RdDM) is required for transcriptional silencing of transposons and other DNA repeats in Arabidopsis thaliana. Although previous research has demonstrated that the SET domain-containing SU(VAR)3-9 homologs SUVH2 and SUVH9 are involved in the RdDM pathway, the underlying mechanism remains unknown. Our results indicated that SUVH2 and/or SUVH9 not only interact with the chromatin-remodeling complex termed DDR (DMS3, DRD1, and RDM1) but also with the newly characterized complex composed of two conserved Microrchidia (MORC) family proteins, MORC1 and MORC6. The effect of suvh2suvh9 on Pol IV-dependent siRNA accumulation and DNA methylation is comparable to that of the Pol V mutant nrpe1 and the DDR complex mutant dms3, suggesting that SUVH2 and SUVH9 are functionally associated with RdDM. Our CHIP assay demonstrated that SUVH2 and SUVH9 are required for the occupancy of Pol V at RdDM loci and facilitate the production of Pol V-dependent noncoding RNAs. Moreover, SUVH2 and SUVH9 are also involved in the occupancy of DMS3 at RdDM loci. The putative catalytic active site in the SET domain of SUVH2 is dispensable for the function of SUVH2 in RdDM and H3K9 dimethylation. We propose that SUVH2 and SUVH9 bind to methylated DNA and facilitate the recruitment of Pol V to RdDM loci by associating with the DDR complex and the MORC complex.

SUBMITTER: Liu ZW

PROVIDER: S-EPMC3898904 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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The SET domain proteins SUVH2 and SUVH9 are required for Pol V occupancy at RNA-directed DNA methylation loci.

Liu Zhang-Wei ZW Shao Chang-Rong CR Zhang Cui-Jun CJ Zhou Jin-Xing JX Zhang Su-Wei SW Li Lin L Chen She S Huang Huan-Wei HW Cai Tao T He Xin-Jian XJ

PLoS genetics 20140122 1

RNA-directed DNA methylation (RdDM) is required for transcriptional silencing of transposons and other DNA repeats in Arabidopsis thaliana. Although previous research has demonstrated that the SET domain-containing SU(VAR)3-9 homologs SUVH2 and SUVH9 are involved in the RdDM pathway, the underlying mechanism remains unknown. Our results indicated that SUVH2 and/or SUVH9 not only interact with the chromatin-remodeling complex termed DDR (DMS3, DRD1, and RDM1) but also with the newly characterized ...[more]

PMID: 24465213

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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”).

Dataset Information

The SET domain proteins SUVH2 and SUVH9 are required for Pol V occupancy at RNA-directed DNA methylation loci.

Publications

The SET domain proteins SUVH2 and SUVH9 are required for Pol V occupancy at RNA-directed DNA methylation loci.

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