GSE138543
Suppression of liquid-liquid phase separation by 1,6-hexanediol partially compromises the 3D genome organization in living cells
Liquid-liquid phase separation (LLPS) contributes to the spatial and functional segregation of molecular processes within the cell nucleus. However, the role played by LLPS in chromatin folding in living cells remains unclear. Here, using stochastic optical reconstruction microscopy (STORM) and Hi-C techniques, we studied the effects of 1,6-hexanediol (1,6-HD)- mediated LLPS disruption/modulation on higher-order chromatin organization in living cells. We found that 1,6-HD treatment caused the enlargement of nucleosome clutches and their more uniform distribution in the nuclear space. At a megabase-scale, chromatin underwent moderate but irreversible perturbations that resulted in the partial mixing of A and B compartments. The removal of 1,6-HD from the culture medium did not allow chromatin to acquire initial configurations, and resulted in more compact repressed chromatin than in untreated cells. 1,6-HD treatment also weakened enhancer-promoter interactions and TAD insulation but did not considerably affect CTCF-dependent loops. Our results suggest that 1,6-HD-sensitive LLPS plays a limited role in chromatin spatial organization by constraining its folding patterns and facilitating compartmentalization at different levels. Overall design: This submission contains the following experiments on untreated (Control and Control_T), 1,6-hexanediol treated (Hex5), Tween-20 treated (Tween), 1,6-hexanediol treated + recovery (Recovery) and Tween-20 treated + recovery (Recovery_T) HeLa cells: 2 biological replicates of Hi-C in Control, Control_T, Hex5, Tween, Recovery and Recovery_T cells, 2 biological replicates of CTCF ChIP-seq in Control, Hex5 and Recovery cells.