Mechanisms of Genome Organization and Metabolic Implications of Drosophila Insulator Proteins

The 3D genome organization is crucial for gene expression regulation and as such, its aberrations are implicated in a number of disorders including cancer. Chromatin architectural elements such as insulator binding proteins (IBPs) and cohesin are critical in forming the 3D genome structures. However, the principles through which these proteins mediate the spatio-temporal organization of the genome are not fully understood. While an interplay between active loop extrusion and phase separation-mediated compartmentalization processes has been proposed, the extent of their contribution is not adequately explored. We show in this work that the Drosophila IBPs and cohesin subunits exhibit liquid-liquid phase separation (LLPS) features at physiological conditions and in response to osmotic stress, suggesting that LLPS modulates aspects of the Drosophila genome organization.

Further, we harnessed the osmotic stress response to gain insights into differences in the human and Drosophila genome organization principles. Our results show that in response to osmotic stress, architectural proteins in both organisms exit from chromatin leading to a general loss of genome structure. Strikingly, the recovery patterns of these structures were distinct between the Drosophila and human genomes suggesting differences in the overall mode of genome restructuring between these organisms. In Drosophila, the gypsy insulator is one of the best characterized architectural elements and contains binding sites for the Suppressor of Hairy-wing [Su(Hw)] protein. Our previous analysis suggests an interaction between Su(Hw) and the phosphorylated histone variant H2Av (γH2Av). Interestingly, mutation of H2Av triggers an immune and metabolic response characterized by melanotic mass and large lipid droplet (LD) formation in fat bodies. We explored the mechanism behind these immuno-metabolic features. We found that in contrast to the null H2Av mutant phenotype, H2Av over-expression and mutants of other Gypsy interactors produce smaller LDs. Mechanistically, we discovered that the increased lipid droplets are linked to H2Av phosphorylation, down-regulation of adipogenic genes, and association of differentially expressed genes (DEGs) with genome organization architectural proteins. Our results suggest that Drosophila insulator proteins likely regulate metabolic genes through 3D genome organization.