A Phosphorylated Histone H2A Variant Displays Properties of Chromatin Insulator Proteins inDrosophila

Abstract

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 (γH2Av), a marker of DNA double strand breaks. Gypsy insulator components colocalize with γH2Av throughout the genome. Mutation of insulator components prevents stable H2Av phosphorylation in polytene chromatin. Phosphatase inhibition strengthens the association between insulator components and γH2Av and rescues γH2Av localization in insulator mutants. We also show that γH2Av 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 γH2Av and TAD boundaries. Together, our results indicate a novel mechanism linking insulator function with a histone H2A variant and with genome stability.

Drosophila insulator proteins exhibit in-vivo liquid-liquid phase separation properties

“…However, the full complement of biomolecules in the insulator bodies is yet to be ascertained. Independent reports suggest that insulator bodies comprise of many unrelated proteins including the EAST protein (102), the gypsy insulator complex proteins, BEAF32, dCTCF (47) and the phosphorylated histone variant γH2Av (49). Interestingly, the mammalian CTCF and cohesin subunits also form clusters of characteristic size of approximately, 200 nm (103, 104).…”

“…Proteins within this group display both weak and strong polyampholyte features enabling them to either collapse or expand depending on the environmental conditions (126). This contest dependency may explain why insulator proteins coalesce into bodies during salt stress that dissolve when isosmotic conditions are restored (47, 49).…”

“…We previously demonstrated the reliance of insulator bodies on the phosphorylation of the Drosophila histone variant, H2Av (49). In this work, inhibition of dephosphorylation significantly decreased the number of insulator bodies (figure 7A and 7B).…”

“…More recently, results from our lab show that the phosphorylated histone variant H2Av (γH2Av) interacts with IBPs at insulator sites genome-wide and that γH2Av is also a critical component of insulator bodies (47,49).…”

“…Though results from these experiments suggested that insulator proteins contribute to long-range interactions in the genome, we showed that the large foci known as insulator bodies are only induced as a response to osmotic stress and are not significantly attached to chromatin (47, 48). More recently, results from our lab show that the phosphorylated histone variant H2Av (γH2Av) interacts with IBPs at insulator sites genome-wide and that γH2Av is also a critical component of insulator bodies (47, 49).…”