Abstract
The spatial organisation of eukaryotic genomes is critical for coordinating DNA-dependent processes such as transcription and replication. Cohesin contributes to this organisation by extruding chromatin loops; however, how cohesin achieves processive translocation along nucleosome-dense chromatin in vivo remains poorly understood. Here, we identify the histone chaperone FACT as a regulator of cohesin dynamics in human cells. We show that the FACT subunit SUPT16H colocalises and interacts with cohesin on chromatin. Depletion of FACT impairs cohesin translocation from its loading sites, leading to reduced chromatin looping and decreased boundary strength of topologically associating domains (TADs), as revealed by Hi-C. In addition, FACT depletion results in the accumulation of cohesin within gene bodies that are not engaged in loop formation, which is associated with reduced transcription. Importantly, degradation of cohesin in FACT-depleted cells restores transcriptional output, indicating that cohesin mislocalisation underlies this effect. Together, these findings demonstrate that FACT facilitates cohesin translocation along chromatin, promoting efficient loop extrusion while preventing its aberrant accumulation within transcribed regions. This work provides mechanistic insight into how chromatin structure modulates cohesin function to coordinate genome organisation with transcription in mammalian cells.