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

Abstract

Mounting evidence implicates liquid-liquid phase separation (LLPS), the condensation of biomolecules into liquid-like droplets in the formation and dissolution of membraneless intracellular organelles (MLOs). Eukaryotic cells utilize MLOs or condensates for various biological processes, including emergency signaling, spatiotemporal control over steady-state biochemical reactions and heterochromatin formation. Insulator proteins function as architectural elements involved in establishing independent domains of transcriptional activity within eukaryotic genomes. In Drosophila, insulator proteins coalesce to form nuclear foci known as insulator bodies in response to osmotic stress and during apoptosis. However, the mechanism through which insulator proteins assemble into bodies and whether these bodies confer any genome function are yet to be fully investigated. Here, we identify signatures of liquid-liquid phase separation by insulator bodies, including high disorder tendency in insulator proteins, scaffold-client dependent assembly, extensive fusion behavior, sphericity, and sensitivity to 1,6-hexanediol. We also show that the cohesin subunit Rad21 is a component of insulator bodies adding to the known insulator proteins and the histone variant γH2Av constituents. Our data suggest a concerted role of cohesin and insulator proteins in insulator body formation and under physiological conditions. We propose a mechanism whereby these architectural proteins modulate 3D genome organization through LLPS.

Phosphorylated histone variant γH2Av is associated with chromatin insulators in Drosophila

“…The mechanism by which γH2Av interacts with Su(Hw) insulator components is still unknown. In addition to direct interactions γH2Av may also interact with insulator proteins through weak multivalent interactions [ 89 ].…”

“…A direct interaction between the phosphorylated H2Av, but not the unphosphorylated form, with one or more gypsy insulator proteins could explain the findings presented here, both at the chromatin level and insulator bodies. Alternatively, research from our lab [ 89 ] has revealed that insulator proteins have liquid-liquid phase separation properties in vivo , which suggest the possibility that the association between γH2Av and insulator proteins may be mediated by weak multivalent interactions leading to the formation of insulator protein condensates.…”

Systematic discovery of directional regulatory motifs associated with human insulator sites

“…For another example, although sequence-based predictions of EPIs underestimated actual interactions, an activity-by-contact (ABC) approach, based on a simple distance model, performed well, suggesting that factors in addition to sequence influence EPIs 31,32 . Furthermore, CTCF-mediated chromatin looping induces phase separation between the inner and outer regions of the loops, resulting in the formation of transcriptional condensates and insulator function [33][34][35] . Therefore, structures that result in phase separation, rather than functional differences in DBPs, may be responsible for insulator functions.…”

3D chromatin interactions involvingDrosophilainsulators are infrequent but preferential and arise before TADs and transcription

“…The early discovery of insulator bodies led to the proposal that insulators mediate the formation of stable, rosette-like hubs involving multiple insulator-bound genomic regions [52][53][54][55] . More recently, it was shown that CP190 and Su(HW) insulator bodies formed in cultured-cells under stress conditions exhibit liquid-liquid phase separation properties 56 . This model predicts that genomically-close insulators should interact in space often, nucleating interactions between multiple partners.…”