A Phosphorylated Histone H2A Variant Displays Properties of Chromatin Insulator Proteins in Drosophila

Chromatin insulators are responsible for mediating long-range interactions between enhancers and promoters throughout the genome and align with the boundaries of topologically associating domains (TADs). Here, we demonstrate an interaction between proteins that associate with the gypsy insulator and the phosphorylated histone variant H2Av ({gamma}H2Av), a marker of DNA double strand breaks. Gypsy insulator components colocalize with {gamma}H2Av throughout the genome. Mutation of insulator components prevents stable H2Av phosphorylation in polytene chromatin. Phosphatase inhibition strengthens the association between insulator components and {gamma}H2Av and rescues {gamma}H2Av localization in insulator mutants. We also show that {gamma}H2Av is a component of insulator bodies, and that phosphatase activity is required for insulator body dissolution after recovery from osmotic stress. We further demonstrate a tight association between {gamma}H2Av and TAD boundaries. Together, our results indicate a novel mechanism linking insulator function with a histone H2A variant and with genome stability.