A protein assembly mediates Xist localization and gene silencing

Nuclear compartments have diverse roles in regulating gene expression, yet the molecular forces and components that drive compartment formation remain largely unclear(1). The long non-coding RNAXistestablishes an intra-chromosomal compartment by localizing at a high concentration in a territory spatially close to its transcription locus(2)and binding diverse proteins(3-5)to achieve X-chromosome inactivation (XCI)(6,7). The XCI process therefore serves as a paradigm for understanding how RNA-mediated recruitment of various proteins induces a functional compartment. The properties of the inactive X (Xi)-compartment are known to change over time, because after initialXistspreading and transcriptional shutoff a state is reached in which gene silencing remains stable even ifXistis turned off(8). Here we show that theXistRNA-binding proteins PTBP1(9), MATR3(10), TDP-43(11)and CELF1(12)assemble on the multivalent E-repeat element ofXist(7)and, via self-aggregation and heterotypic protein-protein interactions, form a condensate(1)in the Xi. This condensate is required for gene silencing and for the anchoring ofXistto the Xi territory, and can be sustained in the absence ofXist. Notably, these E-repeat-binding proteins become essential coincident with transition to theXist-independent XCI phase(8), indicating that the condensate seeded by the E-repeat underlies the developmental switch fromXist-dependence toXist-independence. Taken together, our data show thatXistforms the Xi compartment by seeding a heteromeric condensate that consists of ubiquitous RNA-binding proteins, revealing an unanticipated mechanism for heritable gene silencing. A protein condensate formed by multivalent interactions between the long non-coding RNA Xist and specific RNA-binding proteins drives the compartmentalization required to perpetuate gene silencing on the inactive X chromosome.