Project description:Using BS-Seq to provide single-base resolution of DNA methylation status in 35S-SUC2 WT and antisilencing mutants (arp6, pie1, h2a.z, idm1, and ros1)
Project description:To survey transcriptome changes by the mutations of a DNA demethylase ROS1 responding to a phytohormone abscisic acid, we performed the Next-gen sequencing (NGS) associated RNA-seq analysis. Two ROS1 knockout lines (ros1-3, ros1-4; Penterman et al. 2007 [PMID: 17409185]) with the wild-type Col line (wt) were subjected. Three samples (ros1-3, ros1-4 and wt), biological triplicates, ABA or mock treatment, using Illumina HiSeq 2500 system
Project description:Using MethylC-Seq to provide single-base resolution of DNA methylation status in WT, nrpd1-3, nrpe1-11, ros1-4 single mutants and ros1-4nrpd1-3 double mutant
Project description:Arabidopsis ROS1 is the first genetically characterized DNA demethylase in eukaryotes. Dysfunction of ROS1 leads to increase in DNA methylation level at thousands of genomic loci. However, the features of ROS1 targets are not well understood. In this study, we identified and characterized ROS1 target loci in Arabidopsis Col-0 and C24 ecotypes. Most ROS1 targets are transposable elements (TEs) and intergenic regions. Compared to other TEs, ROS1-targeted TEs are closer to protein coding genes, suggesting a role for ROS1 in preventing the spreading of DNA methylation from highly methylated TEs to nearby genes. Interestingly, we found that unlike general TEs, ROS1 targets are associated with an enrichment of H3K18ac and H3K27me3, and depletion of H3K27me and H3K9me2. We investigated the antagonism between ROS1 and RNA-directed DNA methylation (RdDM) by identifying and characterizing thousands of genomic regions regulated by both ROS1 and RdDM. Unexpectedly, we uncovered thousands of previously unidentified RdDM targets by analyzing the DNA methylome of ros1/nrpd1 double mutant plants. In addition, we show that ROS1 also antagonizes RdDM-independent DNA methylation at more than a thousand genomic loci. Our results provide significant insights into the genome-wide effects of both ROS1-mediated active DNA demethylation and RNA-directed DNA methylation as well as their interaction in plants. Using small RNA-Seq(sRNA-Seq) to get small RNA profiling of WT, ros1-4, nrpd1 single mutants and ros1-4/nrpd1doubble mutant
Project description:To investigate the influence of the AtChz1A/B and ARP6 in H2A.Z incorporation, we analysed genome-wide H2A.Z density in the mutant and wild-type by ChIP-seq. We then performed H2A.Z occupancy analysis using data obtained from ChIP-seq of 3 different plants including mutants and wild-type.
Project description:To survey transcriptome changes by the mutations of a DNA demethylase ROS1 responding to a phytohormone abscisic acid, we performed the Next-gen sequencing (NGS) associated RNA-seq analysis. Two ROS1 knockout lines (ros1-3, ros1-4; Penterman et al. 2007 [PMID: 17409185]) with the wild-type Col line (wt) were subjected.
Project description:The SWR1 chromatin remodeling complex, which deposits the histone variant H2A.Z into nucleosomes, has been characterized in yeast and animals but had not been purified from plants. We used the conserved SWR1 subunit ACTIN RELATED PROTEIN 6 (ARP6) as bait in tandem affinity purification experiments to isolate associated proteins from Arabidopsis thaliana. We identified all 11 subunits found in yeast SWR1 and the homologous mammalian SRCAP complexes, demonstrating that this complex is conserved in plants. We also identified several additional proteins not previously associated with SWR1, including Methyl-CpG-BINDING DOMAIN 9 (MBD9). Since mbd9 mutant plants were phenotypically similar to arp6 mutants, we further explored a potential role for MBD9 in H2A.Z deposition. We found that MBD9 is required for proper H2A.Z incorporation at thousands of discrete sites, which represent a subset of the regions normally enriched with H2A.Z. Genetic analyses showed that arp6;mbd9 double mutants have far more severe phenotypes than either single mutant. In conjunction with the finding that MBD9 does not appear to be a core subunit of the Arabidopsis SWR1 complex, this suggests that MBD9 also has important roles beyond H2A.Z deposition. Our data establish the SWR1 complex as being conserved across eukaryotes and also provide new insights into the mechanisms that target H2A.Z to chromatin.
Project description:Deposition of histone variant H2A.Z by the SWR1 chromatin-remodeling complex is critical for the appropriate expression of many genes in eukaryotes, yet, despite its importance, the composition of the Arabidopsis SWR1 complex has not been thoroughly analyzed. Here we have identified the interacting partners of a conserved Arabidopsis SWR1 subunit, actin-related protein 6 (ARP6). We isolated 9 predicted components, identifying subunits implicated in histone acetylation and interacting partners implicated in chromatin biology. We found that the methyl-CpG-binding domain 9 (MBD9) subunit functioned synergistically with ARP6 to control flowering time. MBD9, in combination with ARP6, was involved in the SWR1-mediated incorporation of the majority of H2A.Z. MBD6 was further required for deposition of H2A.Z at a distinct subset of loci. MBD9 was preferentially bound to nucleosome-depleted regions at the 5’ of genes containing high levels of activating histone marks. Our data suggests a model for MBD9 in recruiting the SWR1 complex to open chromatin of actively transcribing genes.