On the other hand, cohesins are proteins found in all eukaryotes and are traditionally known to mediate sister chromatid cohesion and homologous recombination during cell division, in addition to their role in transcription ( Nasmyth & Haering, 2009). In contrast, CTCF is the only IBP characterized in mammals so far ( Raab et al, 2012 Özdemir & Gambetta, 2019). Most of the insulator proteins, including Suppressor of Hairy wing (Su(Hw)), centrosomal protein 190 (Cp190), modifier of mdg4 67.2 (Mod(mdg4)67.2), and the Drosophila CCTC-binding factor (dCTCF), have been identified in Drosophila ( Raab et al, 2012 Özdemir & Gambetta, 2019). In addition, they serve as physical barriers that prevent heterochromatin spreading to active regions ( Özdemir & Gambetta, 2019). Canonically, IBPs are assembled on DNA elements known as insulators to shield gene promoters from promiscuous interactions with enhancers in a process referred to as enhancer blocking ( Kyrchanova et al, 2013). Insulator-binding proteins (IBPs), lamins, transcription factors, and the cohesin complex notably belong to these architectural proteins ( Matthews & White, 2019 Rowley et al, 2019). Even though the contributions of these processes appear to differ across species, the involvement of certain architectural proteins is crucial and evolutionarily conserved. Suggested mechanisms in the generation of these genomic features include transcription, phase separation, and loop extrusion ( Banigan et al, 2020 Kentepozidou et al, 2020). TAD domains are well conserved and are proposed to delimit regulatory landscapes where functional interactions between gene promoters and distal regulatory elements occur ( Rao et al, 2014 Lupiáñez et al, 2015 Szabo et al, 2020 Torosin et al, 2020). At high resolution, the genome is organized in contiguous regions characterized by high interaction frequencies called topologically associating domains (TADs), which are separated by boundaries that limit the interactions between these domains. The 3D genome organization comprises the distinct nuclear spaces occupied by chromosomes known as chromosome territories, which are in turn made up of active and inactive DNA folds referred to as A and B compartments, respectively. It is becoming increasingly clear that the establishment of independent higher order DNA domains (3D genome organization) in eukaryotes plays a role in important aspects of genome function, including replication, transcription, and DNA damage repair ( Lupiáñez et al, 2015 Ulianov et al, 2016 Stam et al, 2019 Sanders et al, 2020). We propose a mechanism whereby these architectural proteins modulate 3D genome organization through LLPS. Our data suggest a concerted role of cohesin and insulator proteins in insulator body formation and under physiological conditions. We also show that the cohesin subunit Rad21 is a component of insulator bodies, adding to the known insulator protein constituents and γH2Av. Here, we identify signatures of LLPS by insulator bodies, including high disorder tendency in insulator proteins, scaffold–client–dependent assembly, extensive fusion behavior, sphericity, and sensitivity to 1,6-hexanediol. However, the mechanism through which insulator proteins assemble into bodies is yet to be investigated. In Drosophila, insulator proteins form nuclear foci known as insulator bodies in response to osmotic stress. Insulator proteins are architectural elements involved in establishing independent domains of transcriptional activity within eukaryotic genomes. Cells use MLOs or condensates for various biological processes, including emergency signaling and spatiotemporal control over steady-state biochemical reactions and heterochromatin formation. 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).
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