X chromosome inactivation (XCI) is fundamental to mammalian XX female development, ensuring balanced X-linked gene dosage with XY males by randomly silencing one of the two X chromosomes during pre-implantation development. XCI is initiated by monoallelic upregulation of the non-coding RNA Xist, which is expressed from and coats the future inactive X chromosome (Xi) in cis, coordinating downstream silencing events. This process is still incompletely understood.
We performed a targeted genetic screen in differentiating female mouse embryonic stem cells (mESCs), which recapitulate the XCI observed during development in vitro, and revealed E1A-binding protein p400 as a potential activator of XCI initiation. Encoded by Ep400, this chromatin remodeler mainly catalyses histone variant H2A.Z and H3.3 deposition, but also promotes histone acetylation through the TIP60-p400 complex. p400 has not previously been identified in the XCI context.
Using female mESCs with X-linked fluorescent markers (Xmas), we investigated the impact of p400 on different XCI stages. Depleting Ep400 early in differentiation by RNAi consistently reduced Xist expression and delayed XCI onset, confirming its activating effect on XCI initiation. Conversely, Ep400 depletion in post-XCI cells did not consistently affect Xist expression or XCI, suggesting a role in establishing, but not maintaining, silencing.
We are now working to determine the precise role of p400 in XCI, including its contribution to the repressive chromatin landscape of the inactive X.