A comprehensive Xist interactome reveals cohesin repulsion and an RNA-directed chromosome conformation

摘要: INTRODUCTION The mammal has evolved an epigenetic mechanism to silence one of two X chromosomes in the XX female equalize gene dosages with XY male. Once established, inactivated chromosome (Xi) is extremely stable and maintained through lifetime mammal. principal regulator, Xist, a long noncoding RNA that orchestrates silencing process along Xi. Xist believed operate as scaffold recruit spread repressive complexes, such Polycomb Repressive Complex 2, chromosome. identities crucial interacting factors, however, have remained largely unknown. RATIONALE Although Xi’s stability necessary homeostatic property, ability unlock this state great current interest. home nearly 1000 genes, at least 50 which been implicated X-linked diseases, Rett syndrome fragile syndrome. Xi therefore reservoir functional genes could be tapped replace expression disease allele on active (Xa). A major gap understanding lack comprehensive interactome. Progress toward full interactome would advance knowledge regulation by potentially inform treatment diseases. RESULTS We developed RNA-centric proteomic method called iDRiP (identification direct RNA-interacting proteins). Using iDRiP, we identified 80 200 proteins interactors fall into several categories, including cohesins, condensins, topoisomerases, helicases, chromatin remodelers, histone modifiers, DNA methyltransferases, nucleoskeletal nuclear matrix proteins. Targeted inhibition demonstrates can destabilized disrupting multiple components interactome, consistent idea these factors synergistically repress transcription. Triple-drug treatments lead net increase up-regulation ~100 genes. then carry out focused study cohesin sites. Chromatin immunoprecipitation sequencing analysis three types sites chromosome: Xi-specific sites, Xa-specific biallelic find binding represent default state. Ablating results restoration These findings demonstrate that, while attracts complexes Xi, it actively repels chromosomal architectural cohesins from Finally, examine how repulsion affect structure. In wild-type cells, Xa characterized ~112 topologically associated domains (TADs) megadomains. Intriguingly, loss result reversion Xa-like conformation. Hi-C shows TADs return manner correlated reappearance transcriptionally permissive CONCLUSION Our unveils many layers repression central role for topological organization mammalian chromosomes. also supports model simultaneously acts (i) recruitment establish maintain inactive (ii) extrude avoid acquisition transcription-favorable our indicate perturbed targeted