CEBPA phase separation links transcriptional activity and 3D chromatin hubs

Cell identity is orchestrated through an interplay between transcription factor (TF) action and genome architecture. The mechanisms used by TFs to shape three-dimensional (3D) genome organization remain incompletely understood. Here we present evidence that the lineage-instructive TF CEBPA drives extensive chromatin compartment switching and promotes the formation of long-range chromatin hubs during induced B cell to macrophage transdifferentiation. A large intrinsically disordered region (IDR) enables CEBPA to undergo in vitro phase separation and to co-condense with transcriptional partners, which is at least partially mediated by aromatic residues. Furthermore, CEBPA forms visible nuclear condensates in transdifferentiating B cells that co-localize with co-activator condensates and recover rapidly upon photobleaching. Finally, native CEBPA-expressing cell types such as primary granulocyte-macrophage progenitors (GMPs), liver cells and trophectoderm cells also reveal nuclear CEBPA condensates and long-range 3D chromatin hubs at CEBPA-bound regions. These findings support a model in which CEBPA acts as a 3D genome structural organizer and suggest that this effect is mediated at least in part by its phase-separation capacity. Overall design: HiC datasets were generated from BLaER cells knocked out for a CEBPA binding site in the FGGY locus before and after 24h of CEBPA induction (n=2)