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 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.

Molecular insights into the effect of alkanediols on FUS liquid-liquid phase separation

“…Inspired by recent studies on the effect of hexanediol in the organization of chromatin(Shi et al , 2021; Liu et al , 2021; Ulianov et al , 2021) and RNA granules(Fuller et al ,2020) as well as by reports on the action of chemotherapeutics on nucleolar proteins and ribosomal RNA (rRNA) synthesis(Sutton & DeRose, 2021), we studied the capacity of condensate-modifying agents to inhibit phase separation of FUS in the presence of RNA as a model for the types of protein-RNA interactions that contribute to biomolecular condensates formed in cells. As previously(Burke et al , 2015; Monahan et al , 2017), we imaged FUS full-length after the addition of TEV protease to cleave the solubilizing MBP-tag ( Figure 2A ).…”

Subcellular Localization of Seed-Expressed LEA_4 Proteins Reveals Liquid-Liquid Phase Separation for LEA9 and for LEA48 Homo- and LEA42-LEA48 Heterodimers

“…The disorder content analysis of LEA42 and LEA48 using MFDp2 [ 40 ] predicted both proteins to be 100% disordered, suggesting the involvement of LLPS in complex formation. To assess whether LLPS was involved in the formation of these droplet-like structures 1,6-hexanediol, a chemical known to diffuse LLPS structures [ 58 ] was applied. Infiltrated leave samples were treated with 10% 1,6-hexanediol for 1 h followed by 10 min of vacuum-infiltration.…”

Chromatin Conformation in Development and Disease

“…Phase separation could explain several confusing observations, like how transcriptional activation occurs without direct physical contact between enhancers and promoters through eRNAs ( Cai et al, 2020 ), or multi-enhancer and multi-promoter contacts ( Li G. et al, 2012 ; Jin et al, 2013 ), or simultaneous regulation of more than one gene by a single enhancer ( Fukaya et al, 2016 ). In parallel, recent data suggest that forces other than the ones derived from LLPS could also stabilize transcription factories ( Ulianov et al, 2021 ).…”