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: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.
Project description:Heterochromatin constitutes a fundamental aspect of genomes that is crucial for maintaining genome stability. In flowering plants, maintenance of heterochromatin relies on a positive feedback loop involving the histone 3 lysine nine methyltransferase (H3K9), KRYPTONITE (KYP), and the DNA methyltransferase, CHROMOMETHYLASE3 (CMT3). An H3K9 demethylase, INCREASED IN BONSAI METHYLATION 1 (IBM1), has evolved to modulate the activity of KYP-CMT3 within transcribed genes. The absence of IBM1 activity results in aberrant methylation of gene bodies, which is deleterious. This study demonstrates extensive genetic and gene expression variations in KYP, CMT3, and IBM1 within and between flowering plant species. IBM1 activity in Arabidopsis thaliana is uniquely regulated by the abundance of H3K9me2 in a repetitive sequence within an intron preceding the histone demethylase domain. This mechanism enables IBM1 to monitor global levels of H3K9me2. We discovered that the methylated intron is prevalent across flowering plants, however, its underlying sequence exhibits dynamic evolution. Its absence in species lacking gene body DNA methylation suggests its primary role in sensing H3K9me2 and preventing its integration into these constitutively expressed genes. Furthermore, our investigation uncovered Arabidopsis thaliana accessions resembling weak ibm1 mutants, several Brassicaceae species with reduced IBM1 expression, and a potential IBM1 deletion. Evolution towards reduced IBM1 activity in some flowering plants could explain the frequent natural occurrence of diminished or lost CMT3 activity, as cmt3 mutants in A. thaliana mitigate the deleterious effects of IBM1.
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. Total RNA from leaves was isolated using the RNeasy Plant Mini kit (Qiagen) and was treated with DNase I (TAKARA). Double-stranded cDNA was synthesized using the SuperScript Double Stranded cDNA Synthesis kit (Invitrogen) and oligo (dT)20 primer. cDNA was labeled by Cy3, hybridized to 4 x 72k array, and scanned according to manufacture's instructions (www.nimblegen.com).
Project description:We generated and sequenced ChIP libraries for the meiotic cohesin subunit REC8 and four histone modifications (H3K4me1, H3K4me2, H3K9me2 and H3K27me1) to investigate their relationships with meiotic chromosome architecture and recombination in Arabidopsis thaliana. REC8 and H3K9me2 ChIP-seq were performed using meiotic-stage floral buds from wild type (Col-0) and non-CG DNA methylation/H3K9me2 pathway mutant (kyp/suvh4 suvh5 suvh6 or cmt3) plants to examine the role of heterochromatin assembly in meiotic cohesin distribution.
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: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:The intent of the experiment was to infer heat-induced transcriptional changes in epigenetically-disturbed Arabidopsis thaliana backgrounds. For this, we performed Illumina 50 bp unstranded single-end RNA-seq in wild-type, met1-1, drm2-2/cmt3-11 (dc) and mom1-2 mutants, under control and heat stress.