H2A.Xmutants exhibit enhanced DNA demethylation inArabidopsis thaliana

Abstract

AbstractH2A.Xis an H2A variant histone in eukaryotes, unique for its ability to respond to DNA damage, initiating the DNA repair pathway.H2A.Xreplacement 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 inArabidopsis thalianafemale 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 thath2a.xmutants are more sensitive to genotoxic stress, consistent with previous reports.H2A.Xfused to theGreen Fluorescent Protein (GFP)gene under theH2A.Xpromoter was highly expressed in newly developingArabidopsistissues, including in male and female gametophytes. We examined DNA methylation inh2a.xdeveloping seeds using whole genome bisulfite sequencing, and found that CG DNA methylation in the developing endosperm, but not the embryo, is decreased genome-wide inh2a.xmutants, 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 ofArabidopsisendosperm.