Suppression of liquid-liquid phase separation by 1,6-hexanediol partially compromises the 3D genome organization in living cells

Abstract

Liquid-liquid phase separation (LLPS) contributes to the spatial and functional segregation of molecular processes. 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 modulation on higher-order chromatin organization in living cells. We found that 1,6-HD treatment caused the enlargement of nucleosome nanodomains 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, but increased further mixing of the chromatin compartments and resulted in more compact repressed chromatin than in untreated cells. 1,6-HD treatment also weakened enhancer-promoter interactions 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.

Time-dependent effect of 1,6-hexanediol on biomolecular condensates and 3D chromatin organization

“…S1A). These conditions seem to work well in previous studies, probably owing to the fact that some studies only focused on dissolving specific type of condensate they are interested in without detecting cell variability, or used tumor-derived cell lines (e.g., Hela [ 49 ], K562 [ 50 ]) which show activation of anti-apoptotic pathways [ 52 ]. Despite cell variability, cell size also matters.…”

“…Two recent studies treating cells with high concentration (5% and 10%, respectively) and long time (15 min and 20 min, respectively) of 1,6-HD have reported different results in 3D reorganization [ 49 , 50 ]. One observed moderate 3D reorganization upon treatment [ 49 ] whereas the other observed a global 3D reorganization that short-range interactions decreased and long-range interactions increased [ 50 ], which is consistent with our observation in cells upon 1.5%, long-term (10 min and 30 min) 1,6-HD treatment and opposite to our results in proper condition.…”

“…Two recent studies treating cells with high concentration (5% and 10%, respectively) and long time (15 min and 20 min, respectively) of 1,6-HD have reported different results in 3D reorganization [ 49 , 50 ]. One observed moderate 3D reorganization upon treatment [ 49 ] whereas the other observed a global 3D reorganization that short-range interactions decreased and long-range interactions increased [ 50 ], which is consistent with our observation in cells upon 1.5%, long-term (10 min and 30 min) 1,6-HD treatment and opposite to our results in proper condition. The opposite effect of different conditions of 1,6-HD treatment might occur because high concentration (5% or 10%) of 1,6-HD treatment caused chromatin “freeze [ 13 ],” chromatin hyper-condensation, nucleus shrinkage, and abnormal protein aggregates in live cells, while low condition and short-time treatment does not have those side effects (Additional file 1 : Fig.…”

Inhibition of HSF1 and SAFB Granule Formation Enhances Apoptosis Induced by Heat Stress

“…The report by Ulianov et al showed that 5% 1,6-HD, which is structurally similar to 2,5-HD, partially compromises 3D genome organization [ 58 ]. Further, 5% and 10% 1,6-HD cause chromatin hyper-condensation, whereas 2.5% 1,6-HD causes nucleosome clustering slightly similar to that in untreated cells [ 59 ].…”

A modified protocol of Capture-C allows affordable and flexible high-resolution promoter interactome analysis

“…These datasets were processed identically to promoter Capture-C reads (except usage of nofill: 0 setting in HiCUP). We used currently unpublished data on topologically associated domain (TAD) boundaries positions in HeLa cells 35 to analyse localization of detected Capture-C interactions relative to TAD boundaries. To assess the expected number of inter-TAD loops we performed simulations, in which we randomly assigned orientation (forward or backward relative to the reference genome) to the set of intra-chromosomal interactions, which we had described (overall 5107 such interactions).…”