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Genome-wide bisulphite sequencing of Arabidopsis thaliana embryo and endosperm

ABSTRACT: The DEMETER (DME) DNA glycosylase mediates genome-wide DNA demethylation and is required for endosperm genomic imprinting and embryo viability. Targets of DME-demethylation are small, AT-rich transposons and boundaries of large heterochromatic transposons, but how DME interacts with chromatin is unclear. To investigate the interaction between DME and chromatin, we analyzed DNA methylation in Arabidopsis seeds and pollen deficient in the chromatin remodeler FAcilitates Chromatin Transactions (FACT) complex. We find that FACT co-localizes with nuclear DME, and is required for DME activity in chromatin domains with high nucleosome occupancy and histone modifications associated with heterochromatin, which comprise over half of DME target loci. We also demonstrate that heterochromatin-associated linker histone H1 mediates the requirement for FACT at a subset of DME-target loci. However, FACT is not required for DME demethylation of targets in euchromatic regions. Thus, chromatin structure determines the degree to which FACT facilitates access of DME to its targets.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE105000 | GEO | 2018/04/17

REPOSITORIES: GEO

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Project description:H2A.X is an H2A variant histone in eukaryotes, unique for its ability to respond to DNA damage, initiating the DNA repair pathway. H2A.X replacement within the histone octamer is mediated by the FAcilitates Chromatin Transactions (FACT) complex, a key chromatin remodeler. FACT is required for DEMETER (DME)-mediated DNA demethylation at certain loci in Arabidopsis thaliana female gametophytes during reproduction. Here, we sought to investigate whether H2A.X is involved in DME- and FACT-mediated DNA demethylation during reproduction. H2A.X is encoded by two genes in Arabidopsis genome, HTA3 and HTA5. We generated h2a.x double mutants, which displayed a normal growth profile, whereby flowering time, seed development, and root tip organization, S-phase progression and proliferation were all normal. However, h2a.x mutants were more sensitive to genotoxic stress, consistent with previous reports. H2A.X fused to Green Fluorescent Protein (GFP) under the H2A.X promoter was highly expressed especially in newly developing Arabidopsis tissues, including in male and female gametophytes, where DME is also expressed. We examined DNA methylation in h2a.x developing seeds and seedlings using whole genome bisulfite sequencing, and found that CG DNA methylation is decreased genome-wide in h2a.x mutant seeds. Hypomethylation was most striking in transposon bodies, and occurred on both parental alleles in the developing endosperm, but not the embryo or seedling. h2a.x-mediated hypomethylated sites overlapped DME targets, but also included other loci, predominately located in heterochromatic transposons and intergenic DNA. Our genome-wide methylation analyses suggest that H2A.X could function in preventing access of the DME demethylase to non-canonical sites. Alternatively, H2A.X may be involved in recruiting methyltransferases to those sites. Overall, our data show that H2A.X is required to maintain DNA methylation homeostasis in the unique chromatin environment of the Arabidopsis endosperm.

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Genome-wide bisulphite sequencing of Arabidopsis thaliana embryo and endosperm

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