Unknown,Transcriptomics,Genomics,Proteomics

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SmallRNA-seq of Solanum lycopersicum bearing the sulfurea epiallele in nrpe1 versus WT

ABSTRACT: Plants bearing a sulfurea epiallele (TAB2sulf) were crossed with nrpe1 (largest polV subunit) mutants in Solanum Lycopersicum. 7 plants of a F3 progeny expected to be 100% TAB2sulf and segregating for the nrpe1 mutation were sequenced. 4 plants bear the nrpe1 mutation and 3 plants have NRPE1 WT alleles. The aim of the experiment was to compare sRNA accumulation between nrpe1 and WT F3 plants with particular interest on SlTAB2 (Solyc02g005200) which has been previously associated with paramutation in the sulfurea background. Both WT and nrpe1 F3 plants had methylation levels consistent with the TAB2sulf epiallele. nrpe1 and sulfurea parental controls are added in single replicates and a WT cv M82 plant is also sequenced as control. More replicates of WT and M82 are sequenced in a related experiment.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Solanum lycopersicum

SUBMITTER: Claudia Martinho

PROVIDER: E-MTAB-10565 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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

SmallRNA-seq of Solanum lycopersicum bearing the sulfurea epiallele in nrpe1 versus WT

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