biostudies-literatureDing MNICHD NIH HHSNational Institute of Child Health and Human Development978-997.e25https://www.ebi.ac.uk/biostudies/studies/S-EPMC11863002biostudies-literatureUnknown188(4)Xist RNA initiates X inactivation as it spreads in cis across the chromosome. Here, we reveal a biophysical basis for its cis-limited diffusion. Xist RNA and HNRNPK together drive a liquid-liquid phase separation (LLPS) that encapsulates the chromosome. HNRNPK droplets pull on Xist and internalize the RNA. Once internalized, Xist induces a further phase transition and "softens" the HNRNPK droplet. Xist alters the condensate's deformability, adhesiveness, and wetting properties in vitro. Other Xist-interacting proteins are internalized and entrapped within the droplet, resulting in a concentration of Xist and protein partners 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,CellA biophysical basis for the spreading behavior and limited diffusion of Xist.PMC11863002R01 HD097665Jiang JLee JTWang DLappala ARivera CLi PChen HWeissbein UKesner BGrimm NBDing MLou JfalseA biophysical basis for the spreading behavior and limited diffusion of Xist.Xist RNA initiates X inactivation as it spreads in cis across the chromosome. Here, we reveal a biophysical basis for its cis-limited diffusion. Xist RNA and HNRNPK together drive a liquid-liquid phase separation (LLPS) that encapsulates the chromosome. HNRNPK droplets pull on Xist and internalize the RNA. Once internalized, Xist induces a further phase transition and "softens" the HNRNPK droplet. Xist alters the condensate's deformability, adhesiveness, and wetting properties in vitro. Other Xist-interacting proteins are internalized and entrapped within the droplet, resulting in a concentration of Xist and protein partners 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,2025-01-01T00:00:00Z2025 Feb2026-07-16T09:16:34.952Z2026-07-09T10:46:49.885ZS-EPMC118630023982418310.1016/j.cell.2024.12.004