Osmotic Stress Response Reveals Distinct Genome Architectures in Drosophila and Humans

The 3D genome organization is mediated by chromatin architectural elements such as insulator binding proteins and cohesin and serves as a crucial epigenetic regulatory mechanism. In Drosophila, architectural proteins translocate from chromatin to the nucleoplasm in response to hyperosmotic stress forming condensates known as insulator bodies, which exhibit liquid-liquid phase separation (LLPS) features. We harnessed the osmotic stress response to gain insights into the differences in genome organization principles between human and fly genomes across several scales. We found that under identical osmotic stress conditions, CTCF also translocates to the nucleoplasm in human cells but does not form condensates. Using genome-wide chromosome conformation capture (Hi-C), we found that in both human and Drosophila, osmotic stress induces a loss of genome structure, characterized by the loss of compartments and the weakening of TAD boundary strength throughout the genome, as well as a significant increase in cross-compartment interactions in the long-range. In both Drosophila and human genomes, looping interactions are also lost during stress. The genome organization in human cells almost fully recovers after 1 hour in normal media post osmotic stress, regaining full compartment structure, TAD boundary strength and loop formation. Recovery of Drosophila cells after 1 hour in normal media, however, is incomplete as genomes fail to regain normal compartment structure and TAD boundary strength remains significantly reduced. Interestingly, loops are re-formed at this time point. Analysis of these differences revealed that the Drosophila genome does not replicate the canonical A/B compartment organization found in vertebrates. In Drosophila, A compartments engage in A-to-A long-range interactions in a manner similar to that found in the human genome. However, long range B-B interactions are rarely observed. Interestingly, A compartments are specifically enriched in ?H2Av and Su(Hw) insulator proteins, suggesting that the LLPS properties of these proteins may be required for compartment formation. Overall design: Drosophila S2 cells were sub-cultured for 3 days at 25°C. Human A375 cells were sub-cultured for 3 days prior to experiment. Osmotic stress was induced in the cells by quickly replacing the conditioned media with 250 mM NaCl (from a 5M stock) prepared with complete Schneider's Drosophila media or DMEM media (for A375 cells) while the control cells were kept in the conditioned media. The cells were kept in the increased NaCl for 1 hour, then for Recovery conditions, cells were returned to original media for 1 hour. Hi-C experiments were performed on Control, Stress, and Recovery conditions.