A biophysical basis for the spreading behavior and limited diffusion of Xist [RNA-Seq]
Here we reveal a biophysical basis for the spreading behavior of Xist RNA on the inactive X-chromosome (Xi). Xist and HNRNPK together drive a liquid-liquid phase separation (LLPS) that encapsulates the Xi. HNRNPK and Xist exert mutual pulling forces that lead to RNA internalization into the condensate. While HNRNPK is sufficient for condensate formation, Xist induces a further phase transition into a "soft" droplet by altering elasticity, adhesiveness, and wetting properties of the condensate in vitro. Once phase transition occurs, other Xist-interacting factors are internalized and concentrated within the condensate. We attribute LLPS to HNRNPK's RGG and Xist's Repeat B (RepB) motifs. Mutating these motifs causes Xist diffusion, disrupts Polycomb recruitment, and precludes the required mixing of Xi chromatin for establishing the Xi superstructure. Thus, phase transitions driven by RepB and HNRNPK create a membrane-less subnuclear compartment for the localization and limited diffusion of Xist ribonucleoprotein complexes on the Xi. Overall design: RNA-seq on WT  and HNRNPK 6A mutant of day 0 and differenated day 7 mESCs