Phosphorylated histone variant γH2Av is associated with chromatin insulators in Drosophila

Chromatin insulators are responsible for orchestrating long-range interactions between enhancers and promoters throughout the genome and align with the boundaries of Topologically Associating Domains (TADs). Here, we demonstrate an association between gypsy insulator proteins and the phosphorylated histone variant H2Av (gamma H2Av), normally a marker of DNA double strand breaks. Gypsy insulator components colocalize with gamma H2Av throughout the genome, in polytene chromosomes and in diploid cells in which Chromatin IP data shows it is enriched at TAD boundaries. Mutation of insulator components su(Hw) and Cp190 results in a significant reduction in gamma H2Av levels in chromatin and 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, but not H2Av, is a component of insulator bodies, which are protein condensates that form during osmotic stress. Phosphatase activity is required for insulator body dissolution after stress recovery. Together, our results implicate the H2A variant with a novel mechanism of insulator function and boundary formation. Author summary The DNA in eukaryotic genomes is folded into domains called Topologically Associating Domains (TADS), which promote gene specific transcription regulation. Insulator proteins are DNA binding proteins that bind at the boundaries between adjacent TADs. Loop extrusion is a mechanism by which insulators promote TAD formation in vertebrate genomes, but the mechanism by which insulator proteins facilitate the formation of boundaries in Drosophila is not well understood. In this wok we show that there is an association between Drosophila Gypsy insulator proteins and the phosphorylated version of the histone variant H2Av (gamma H2Av). gamma H2Av has been traditionally linked to the mechanism of DNA repair, but our data shows that Gypsy insulator components colocalize with gamma H2Av throughout the genome, and that gamma H2Av is also enriched at TAD boundaries. Mutation of genes encoding insulator proteins su(Hw) and Cp190 results in a significant reduction in gamma H2Av levels, and inhibition of the phosphatase activity that removes phosphate from gamma H2Av strengthens the association between insulator proteins and gamma H2Av. We also show that gamma H2Av is a component of insulator bodies, which are protein condensates that form during osmotic stress. Together, our results implicate the H2A variant with a novel mechanism of insulator function and boundary formation.

Automated summary

Insulator proteins and phosphorylated histone variant H2Av are associated with each other and play a role in gene transcription regulation. The study found that in Drosophila, insulator components colocalize with the phosphorylated H2Av throughout the genome and are enriched at TAD boundaries, suggesting their important role in boundary formation.