HNRNPU facilitates chromatin anchoring of Xist RNA independently of cis localisation.

X chromosome inactivation (XCI) is orchestrated by the non-coding RNA Xist which localises in cis over the length of the X chromosome, recruiting factors that confer transcriptional silencing of underlying genes. The RNA-binding protein HNRNPU, which interacts with Xist RNA, plays a role in regulating X chromosome localisation, as evidenced by nuclear dispersal of Xist transcripts in HNRNPU loss-of-function experiments. In this study, we develop an interspecific XX mouse embryonic stem cell model with an inducible Xist promoter together with dTAG degron-tagged HNRNPU, enabling precise analysis of XCI parameters following acute depletion of HNRNPU. Using this system, we show that HNRNPU depletion at the onset of XCI results in reduced chromatin anchoring and increased steady-state levels of Xist RNA, likely underlying the observed nucleoplasmic dispersal. However, Xist RNA continues to localise to preferred association sites on the X chromosome and moreover is able to induce X-linked gene silencing, albeit with reduced efficiency. We discuss our findings in the context of models for Xist RNA localisation in cis and the role of HNRNPU.