Abstract 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, though it is not known how FACT targets DME sites. Here, we investigated whether H2AX is involved in DME- and FACT-mediated DNA demethylation during Arabidopsis reproduction. We show that h2a.x mutants are more sensitive to genotoxic stress, consistent with previous reports. H2A.X fused to the Green Fluorescent Protein (GFP) gene under the H2A.X promoter was highly expressed in newly developing Arabidopsis tissues, including in male and female gametophytes. We examined DNA methylation in h2a.x developing seeds using whole genome bisulfite sequencing, and found that CG DNA methylation in the developing endosperm, but not the embryo, is decreased genome-wide in h2a.x mutants, predominately in transposons and intergenic DNA. Hypomethylated sites overlapped 62 % with canonical DME loci. These data indicate that H2A.X is not required for DME function, but is important for DNA methylation homeostasis in the unique chromatin environment of Arabidopsis endosperm.

H2A.Xmutants exhibit enhanced DNA demethylation inArabidopsis thaliana