Project description:Plants bearing a sulfurea epiallele (TAB2sulf) were crossed with cmt3 mutants in Solanum Lycopersicum. 6 plants of a F2 progeny segregating both for TAB2sulf and cmt3 mutation were sequenced. 4 plants bear the cmt3 mutation and 2 plants have CMT3 WT alleles. the aim of the experiment was to compare sRNA accumulation between cmt3 and WT plants with particular interest on SlTAB2 (Solyc02g005200) which has been previously associated with paramutation in the sulfurea background. The WT plants had methylation levels consistent with the TAB2sulf epiallele and the cmt3 plants had methylation levels consistent with TAB2+ (low DNA methylation). cmt3 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.
Project description:Whole genome bisulfite sequencing was done in F3 plants resulting from a cross between Solanum lycopersicum plants bearing the sulfurea epiallele and cmt3 mutants. 6 F3 plants were sequenced: 3 WT and 3 cmt3. The F3 WT plants had methylation levels (quantified with McrBC-qPCR) consistent with the TAB2sulf epiallele and the cmt3 plants had methylation levels consistent with TAB2+ (low DNA methylation). cmt3 mutants from the F2 generation were backcrossed with WT cv M82 plants to restore CMT3 activity and followed for 2 generations (BC2). 4 BC2 plants were sequenced 2 WT and 2 cmt3. The aim of the experiment was to compare DNA methylation levels in all contexts between cmt3 and WT F3 plants with particular interest on SlTAB2 (Solyc02g005200) which has been previously associated with paramutation in the sulfurea background. The stability of DNA methylation upon CMT3 reintroduction was assessed in the BC2 plants. WT cv M82, cmt3 and sulfurea controls were added in single replicates.
Project description:This SuperSeries is composed of the following subset Series: GSE22953: Genome-wide analysis of H3K9me2 in ibm1, kyp, and cmt3 mutants of Arabidopsis thaliana GSE22957: Genome-wide expression analysis in kyp and cmt3 mutants of Arabidopsis thaliana Refer to individual Series
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.
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.
Project description:RNA-seq of seedlings of four tomato species Solanum habrochaites, Solanum lycopersicum, Solanum pimpinelliolium, and Solanum pennellii. An additional panel of samples include many tissues from Solanum lycopersicum and Solanum pennellii in two light conditions
Project description:DNA methylation occurs at preferred sites in eukaryotes, although the basis for preference is not known. We use a microarray-based profiling method to explore the involvement of Arabidopsis CMT3 and DRM DNA methyltransferases, a histone H3 lysine-9 methyltransferase (KYP) and an Argonaute-related RNA silencing component (AGO4) in methylating target loci. We find that KYP targets are also CMT3 targets, suggesting that histone methylation maintains CNG methylation genome-wide. CMT3 and KYP targets show similar proximal distributions that corresponds to the overall distribution of transposable elements of all types, whereas DRM targets are distributed more distally along the chromosome. We find an inverse relationship between element size and loss of methylation in ago4 and drm mutants. Our results suggest that RNA-directed DNA methylation is required to silence isolated elements that may be too small to be maintained in a silent state by a chromatin-based mechanism. Thus, parallel pathways would be needed to maintain silencing of transposable elements. Keywords: Methylation profiling using Msp I enzyme
Project description:This study evaluates the transcriptional response of Solanum lycopersicum cv. Fla. 8000 (wild-type and dmr6 mutants) to the pathogen Xanthomonas gardneri 153.
Project description:Investigation of genome-wide expression in the mutant of histone H3K9 methyltransferase KRYPTONITE (KYP) or DNA methyltransferase CHROMOMETHYLASE3 (CMT3) in Arabidopsis. These mutants showed decrease in H3K9 methylation and DNA methylation levels, and transcriptional activation at transposons and repeats. Using NimbleGen DNA microarray, global pattern of expression of genes and transposons were examined in these mutants.