The mammal has evolved an epigenetic mechanism to silence one of two X chromosomes in the XX female to equalize gene dosages with the XY male. Once established, the inactivated X chromosome (Xi) is extremely stable and is maintained through the lifetime of the female mammal. The principal regulator, Xist, is a long noncoding RNA that orchestrates the silencing process along the Xi. Xist is believed to operate as a scaffold to recruit and spread repressive complexes, such as Polycomb Repressive Complex 2, along the X chromosome. The identities of crucial interacting factors, however, have remained largely unknown.

Although the Xi’s epigenetic stability is a necessary homeostatic property, an ability to unlock this epigenetic state is of great current interest. The X chromosome is home to nearly 1000 genes, at least 50 of which have been implicated in X-linked diseases, such as Rett syndrome and fragile X syndrome. The Xi is therefore a reservoir of functional genes that could be tapped to replace expression of a disease allele on the active X (Xa). A major gap in current understanding is the lack of a comprehensive Xist interactome. Progress toward a full interactome would advance knowledge of epigenetic regulation by long noncoding RNA and potentially inform treatment of X-linked diseases.

We have developed an RNA-centric proteomic method called iDRiP (identification of direct RNA-interacting proteins).

Using iDRiP, we identified 80 to 200 proteins in the Xist interactome. The interactors fall into several functional categories, including cohesins, condensins, topoisomerases, RNA helicases, chromatin remodelers, histone modifiers, DNA methyltransferases, nucleoskeletal factors, and nuclear matrix proteins. Targeted inhibition demonstrates that Xi silencing can be destabilized by disrupting multiple components of the interactome, consistent with the idea that these factors synergistically repress Xi transcription. Triple-drug treatments lead to a net increase of Xi expression and up-regulation of ~100 to 200 Xi genes. We then carry out a focused study of X-linked cohesin sites. Chromatin immunoprecipitation sequencing analysis demonstrates three types of cohesin sites on the X chromosome: Xi-specific sites, Xa-specific sites, and biallelic sites. We find that the Xa-specific binding sites represent a default state. Ablating Xist results in restoration of Xa-specific sites on the Xi. These findings demonstrate that, while Xist attracts repressive complexes to the Xi, it actively repels chromosomal architectural factors such as the cohesins from the Xi. Finally, we examine how Xist and the repulsion of cohesins affect Xi chromosome structure. In wild-type cells, the Xa is characterized by ~112 topologically associated domains (TADs) and the Xi by two megadomains. Intriguingly, loss of Xist and restoration of cohesin binding result in a reversion of the Xi to an Xa-like chromosome conformation. Hi-C analysis shows that TADs return to the Xi in a manner correlated with the reappearance of cohesins and with a transcriptionally permissive state.

Our study unveils many layers of Xi repression and demonstrates a central role for RNA in the topological organization of mammalian chromosomes. Our study also supports a model in which Xist RNA simultaneously acts as (i) a scaffold for the recruitment of repressive complexes to establish and maintain the inactive state and (ii) a repulsion mechanism to extrude architectural factors such as cohesins to avoid acquisition of a transcription-favorable chromatin conformation. Finally, our findings indicate that the stability of the Xi can be perturbed by targeted …