Phosphorylated histone variant γH2Av is associated with chromatin insulators in Drosophila

Abstract

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 (γH2Av), normally a marker of DNA double strand breaks. Gypsy insulator components colocalize with γH2Av 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 γH2Av levels in chromatin and phosphatase inhibition strengthens the association between insulator components and γH2Av and rescues γH2Av localization in insulator mutants. We also show that γH2Av, 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.

Interactions Between the Histone Variant H2Av and the Suppressor of Hairy wing Insulator Influence the DNA Damage Response and Lymph Gland Development inDrosophila

“…To determine if Su(Hw) is involved in the cellular response to ionizing radiation, third instar larvae were subjected to a 10 Gy dose of X-rays and allowed to recover for four hours. Immunostaining of diploid cells from the larval brain of unexposed control samples revealed a significantly lower degree of H2Av phosphorylation in the su(Hw) e04061 mutant as compared to wild type (Figure 3 A, B), a finding consistent with previous results in polytene chromatin (Simmons et al, 2022). However, upon X-ray irradiation, phosphorylated H2Av immunostaining levels increased significantly in both the wild type larvae and the su(Hw) e04061 mutant (Figure 3 A, B).…”

“…As we previously explored the relationship between Su(Hw) and H2Av phosphorylation in insulator protein mutants, we next questioned how insulator activity and binding are affected by mutation of His2Av. Heterozygous mutation of His2Av in a heterozygous su(Hw) mutant background partially rescues defects observed in the wing margins of ct 6 mutants (Simmons et al, 2022). Based on this, and on previous immunostains showing extensive colocalization between Su(Hw) insulator components and γH2Av (Simmons et al, 2022), we examined binding of insulator proteins to polytene chromatin in the His2Av 810 null background.…”

“…Heterozygous mutation of His2Av in a heterozygous su(Hw) mutant background partially rescues defects observed in the wing margins of ct 6 mutants (Simmons et al, 2022). Based on this, and on previous immunostains showing extensive colocalization between Su(Hw) insulator components and γH2Av (Simmons et al, 2022), we examined binding of insulator proteins to polytene chromatin in the His2Av 810 null background.…”

“…Chromatin insulators also help mitigate the response to DNA damage in human cells (Hilmi et al, 2017) and previous work by our lab characterized the relationship between the phosphorylated form of the H2A variant in Drosophila, γH2Av, and insulator proteins. A close association was observed between Su(Hw) insulator proteins and γH2Av throughout the genome by a combination of immunostaining and ChIP-seq analysis (Simmons et al, 2022). Mutation of any component of the Su(Hw) insulator complex was enough to disrupt the interaction of this complex with γH2Av, and su(Hw) mutants displayed significantly less γH2Av in polytene chromatin than wild type.…”

“…The association between insulator proteins and γH2Av was strengthened by treatment with an inhibitor of protein phosphatase 2A (PP2A). Further experiments revealed that γH2Av is a component of insulator bodies that form during osmotic stress and that recovery of the bodies after stress can be prevented by inhibition of PP2A (Simmons et al, 2022). Mutation of His2Av suppressed the ct 6 phenotype in a sensitized su(Hw) heterozygous background, demonstrating that H2Av and Su (Hw) function in the same developmental pathway in the wing.…”