A protein assembly mediates Xist localization and gene silencing

Nuclear compartments play diverse roles in regulating gene expression, yet the molecular forces and components driving compartment formation remain largely unclear(1). The long non-coding RNA Xist establishes 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 paradigm for understanding how RNA-mediated recruitment of diffusible proteins induces a functional compartment. Interestingly, the properties of the inactive X (Xi)-compartment change over time because upon initial Xist spreading and transcriptional shutoff a state is reached where gene silencing remains stable even if Xist is turned off(8). Here, we show that the Xist RNA-binding-proteins (RBPs) PTBP1(9), MATR3(10), TDP43(11), and CELF1(12) assemble on the multivalent E-repeat-element of Xist(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 anchoring of Xist to the Xi-territory and can be sustained in the absence of Xist. Notably, these E-repeat-binding RBPs become essential coincident with transition to the Xist-independent XCI-phase(8), indicating that the condensate seeded by the E-repeat underlies the developmental switch from Xist-dependence to Xist-independence. Taken together, our data reveal that Xist forms the Xi-compartment by seeding a heteromeric condensate consisting of ubiquitous RBPs and uncover an unanticipated mechanism for heritable gene silencing.