View Program - 63rd Annual Drosophila Research Conference

Eukaryotic genomes are characterized by a highly orchestrated 3D organization. Binding of insulator proteins to specific DNA sequences through the genome contribute to genome structure by demarcating the boundaries between genome domains. The functional properties of insulator proteins, along with those of other architectural proteins, are under strong scrutiny given that the understanding of genome structure is essential to explain fundamental aspects of genome expression and genome maintenance. Loop extrusion mediated by Cohesin and chromatin-mediated liquid-liquid phase separation (LLPS) are thought to be the major driving forces contributing to domain formation and compartmentalization of the genome. The Drosophila melanogaster genome expresses an array of insulator proteins that constitute diverse insulator complexes targeted to different sequences across the genome, but how these insulator complexes specifically contribute to genome structure is currently unknown. Although insulator proteins function in the context of chromatin and nucleosomes, specific histone proteins have never been directly implicated in insulator function. Here, we demonstrate an interaction between gypsy insulator proteins and the phosphorylated form of the histone variant H2Av (γH2Av). We show that components of the gypsy insulator complex, Su(Hw), Mod(mdg4)67.2 and CP190 colocalize with γH2Av throughout the genome and mutation of insulator components prevents stable association of phosphorylated H2Av with chromosomes. Inhibition of the PP2A phosphatase results in a stronger chromosomal association of γH2Av and insulator proteins. Interestingly, yH2Av but not unphosphorylated H2Av associates to insulator bodies after osmotic stress, and phosphatase activity is required for insulator body dissolution after recovery from stress. Our evidence suggests a model in which phosphorylation of H2Av regulates insulator activity by modulating the LLPS properties of insulators and associated architectural proteins.

The Phosphorylated Histone Variant H2Av Associates With Gypsy Insulator Proteins Through Liquid-Liquid Phase Separation

**The Phosphorylated Histone Variant H2Av Associates With Gypsy Insulator Proteins Through Liquid-Liquid Phase Separation**