FACT depletion demonstrates a role for nucleosome organization in TAD formation

Mammalian genomes are organized into distinct chromatin structures, which include small-scale nucleosome arrays and large-scale topologically associating domains (TADs). The mechanistic interplay between chromatin structures across scales is poorly understood. Here, we investigate how changes in nucleosome organization impact TAD structure by studying the role of the histone chaperone facilitates chromatin transcription (FACT) in 3D genome organization. We show that FACT depletion perturbs TADs, causing decreased insulation and weaker CTCF loops. These changes in TAD structure cannot be attributed to changes in chromatin occupancy of CTCF or cohesin and occur specifically in transcribed regions of the genome, where we observe perturbed nucleosome organization in the absence of FACT. FACT depletion therefore allows us to separate the role of nucleosome organization and CTCF binding and to demonstrate that the organization of nucleosomes at TAD boundaries contributes to TAD formation.