X-chromosome inactivation (XCI) is a process that equalizes X-linked gene dosage in female organisms. Incomplete silencing is linked to severe phenotypes such as dysplasia and Rett syndrome while failure in XCI is lethal. While the genetic basis for XCI is well known, the mechanistic understanding on how the silencing network is organized is still missing. In this study we aimed to investigate protein complexes dynamics upon XCI in a time resolved manner with the goal of better understanding the systems level interplay between epigenetic modifiers during chromosome-wide silencing.
Isolated nuclei from ES cells undergoing X-inactivation at different times, were lysed under mild conditions, fractionated on a size exclusion column, and measured in DIA mode. We implemented a hybrid machine learning framework to infer novel complexes from co-elution features. Following the discovery of aninteraction between Polycomb Repressive Complex 2 (PRC2) and Spen, a protein genetically linked to XCI, we proceeded to investigate the effect of Spen deletion on PRC2 activity and recruitment.
We characterized > 400 differentially regulated nuclear protein complexes of which 120 are either submodules with distinct coelution features or entirely novel. Of these putative complexes, we selected one formed by PRC2 and Spen. We validated this interaction by performing endogenous immunoprecipitation of Spen. We further probed the role of Spen in PRC2 recruitment and we were able to show that ablation of Spen reduced PRC2 recruitment. Finally, Spen deletion abrogated XCI thereby pointing towards a pivotal role of this protein for epigenetic silencing.
We profiled nuclear protein complex dynamics in ES cells undergoing epigenetic silencing. Besides systems level insights into changes in nuclear proteome organization we identified Spen as a novel PRC2 associated protein which controls chromatin-wide epigenetic remodelling via regulation of PRC2 recruitment.