Altered Cohesin Dynamics During Cellular Differentiation

Abstract

The cohesin complex plays essential roles in chromosome organization and gene regulation, yet how cohesin dynamics are controlled during cell-state transitions remains poorly understood. Here, we examined how cohesin regulation is remodeled during the differentiation of mouse embryonic stem cells (mESCs) into the cardiomyocyte lineage using an in vitro differentiation system. We found that core cohesin subunits remain broadly stable at the protein level. In contrast, the levels of cohesin regulators, including the cohesin removal protein WAPL and the cohesin stabilizing protein ESCO1, decline sharply despite modest transcript-level changes. The cohesion maintenance factor Sororin was also reduced. To better understand the net effect of these changes on cohesin dynamics, we use live-cell FRAP of RAD21, which revealed increased cohesin mobility in differentiated cells without a change in recovery kinetics, consistent with reduced stable chromatin engagement or redistribution into a chromatin-unbound nuclear pool. To test functional consequences, we generated homozygous degron alleles for Wapl and Esco1 and induced acute degradation using a dTAG-based system. Loss of WAPL altered cell-cycle dynamics in stem cells and produced a characteristic "vermicelli" chromosome phenotype, consistent with abnormally high and lethal cohesin retention on chromatin. Surprisingly, depletion of ESCO1 had no clear impact on viability and cell cycle progression. Notably, despite loss of detectable WAPL protein in the differentiated cell population, we find that WAPL remains functionally required to maintain a viable interphase chromosome organization. Together, these findings identify cohesin regulators, rather than cohesin abundance, as central drivers of changes in cohesin dynamics during differentiation. They further show that even very low levels of WAPL continue to provide critical structural plasticity of chromosomes following cell cycle exit and lineage commitment.