Project description:10-day-old seedlings from Col and h2a.w mutants (h2a.w.6, h2a.w.7, h2a.w.6,7, h2a.w.6,7,12) were harvested. Nuclei extracted using Honda buffer was fragmented with mirococcal nuclease (MNase, NEB M0247S). The fragmented DNA was purified by phenol/chloroform/isoamyl alcohol (24:24:1) extraction, followed by ethanol precipitation and 2% agarose gel separation and gel extraction of ~145-150 bp DNA. Purified DNA sent to BGI (Hong Kong) for library preparation and sequencing.
Project description:How histone intrinsic sequence variation or regulatory modifications regulate nucleosome interactions with transcription remain unclear. To clarify this question, we examined how histone variants and histone modifications assemble in the Arabidopsis thaliana genome, identifying a limited number of chromatin states that divide euchromatin and heterochromatin in biologically significant subdomains. We showed that histone variants were as significant as histone modifications to determine the composition of chromatin states. The loss of function of the chromatin remodeler DECREASED IN DNA METHYLATION (DDM1) prevented the exchange between the histone variants H2A.Z and H2A.W over transposons resulting in their enrichment in chromatin states found only on proteins coding genes in the wild type. Hence, the dynamics of histone H2A variants exchange impacted the definition and distribution of chromatin states. We propose that dynamics of histone variants control the organization of histone modifications into chromatin states to achieve landmarks that signify the ability for transcription. Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for histone H2A variants and histone modifications in seedlings .
Project description:Col-0 floral stem was grafted on the msh1 mutant (Col-0/msh1); on the dcl2,3,4,msh1 quadruple mutant (Col-0/dcl2,3,4,msh1); on Col-0 (Col-0/Col-0). Seeds were collected from the grafted Col-0 scion after grafts were established. Seed coming from the graft then were grown on the peat mix, leaf tissue was collected at the bolting and used for the total RNA sequencing.
Project description:Silencing of transposons by the chromatin remodeler DDM1 is mediated by the deposition of heterochromatic H2A variants. Transposon mobility and silencing participates in genome evolution but also threaten genome integrity. DECREASED DNA METHYLATION 1 (DDM1) belongs to a conserved family of chromatin remodelers that are required to silence transposons, yet the underlying molecular mechanism has remained unknown. Here we show that DDM1 binds the histone variant H2A.W through two conserved domains that are required to deposit H2A.W and maintain transposon silencing. The mechanism of transcriptional silencing described here is likely shared among chromatin remodelers of the DDM1 family and heterochromatic H2A variants that have evolved in mammals.
Project description:0.3-0.5 mm buds from Col and h2a.w mutants (h2a.w.6,7, h2a.w.6,7,12) were harvested. Nuclei extracted using Honda buffer was fragmented with mirococcal nuclease (MNase, NEB M0247S). The fragmented DNA was purified by phenol/chloroform/isoamyl alcohol (24:24:1) extraction, followed by ethanol precipitation and 2% agarose gel separation and gel extraction of ~145-150 bp DNA. Purified DNA sent to BGI (Hong Kong) for library preparation and sequencing.
Project description:Col-0 floral stem was grafted on the msh1 mutant (Col-0/msh1); on the dcl2,3,4,msh1 quadruple mutant (Col-0/dcl2,3,4,msh1); on Col-0 (Col-0/Col-0). Seeds were collected from the grafted Col-0 scion after grafts were established. Seed coming from the graft then were grown on the peat mix, leaf tissue was collected at the bolting and used for the bisulfite sequencing (methylome). Tissue from the msh1 mutant and dcl2,3,4,msh1 quadruple mutants used as rootstocks was similarly collected at the bolting stage and used for the bisulfite sequencing.
Project description:To investigate the deposition of HTR5 in Arabidopsis, we analysed genome-wide HTR5 density in the wild-type Col-0 by ChIP-seq. We then performed HTR5 occupancy analysis using data obtained from ChIP-seq of 3 different plants including HA-HTR5/Col-0 and Col-0. Col-0 acted as negative control.
