X-chromosome inactivation (XCI) is essential for female (XX) mammalian development, equalising X-linked gene dosage with XY males by randomly silencing one X chromosome during pre-implantation development. XCI is triggered by monoallelic upregulation of the long non-coding RNA Xist, which coats the future inactive X (Xi) in cis and coordinates downstream silencing machineries. Although Xist is a well-defined initiator, the epigenetic mechanisms behind its activation remain incompletely understood.
Using an in vitro differentiation model of female murine embryonic stem cells (mESCs) that faithfully recapitulates the progression of XCI, we identified the E1A-binding protein p400 (p400/Ep400) as a positive regulator of XCI onset.
RNAi-mediated Ep400 depletion during mESC differentiation led to reduced Xist expression and a delayed appearance of Xist-coated Xi domain, indicating that p400 potentially acts upstream of Xist activation. In contrast, Ep400 depletion in post-XCI cells had minimal impacts on Xist level or maintenance of silencing, suggesting a stage-specific mechanism.
p400 functions as the essential scaffolding subunit of the TIP60–p400 complex, which couples ATP-dependent chromatin remodelling (including H2A.Z/H3.3 deposition) with histone acetylation (notably of H2A.Z and H4). Disrupting Ep400 is therefore likely to impair both the integrity and catalytic output of the entire complex. To dissect which activities are necessary for Xist activation, we are now perturbing additional TIP60–p400 components to separate the contributions of different catalytic activities. Meanwhile, we are inducing Ep400 knockout in differentiating female mESCs to study potential redundancy and dosage-dependent effects on Xist activity.
Together, our current data support a model in which TIP60–p400 activity promotes Xist upregulation and timely XCI initiation. This work provides an entry point to delineate the dynamic epigenetic mechanisms that precede stable Xi silencing.