Epigenetic gene silencing facilitates cell-type specific transcriptional signatures and is therefore fundamental to shaping cell identity in both development and disease. The silencing process is necessarily complex, multilayered and not fully understood despite significant research effort. X chromosome inactivation (XCI) is the mammalian compensation mechanism that ensures equal gene dosage between XX females and XY males, resulting in near complete silencing of one female X chromosome. XCI is therefore a powerful system in which to study epigenetic silencing across hundreds of loci in parallel.
In vitro, female embryonic stem cells (ESCs), like the blastocyst cells from which they derive, have activity from both X chromosomes. Upon differentiation ESCs undergo XCI creating an active (Xa) and an inactive (Xi) X chromosome in an apparently random process. Female ESCs therefore present as an enticing system in which to study XCI, however complications with in vitro maintenance of these cells have severely limited their use. To create a tractable female ESC line, we have tagged each X chromosome with different fluorescent reporters, to monitor activity from each X rapidly and accurately by FACs, allowing us to perform the first screens for regulators of XCI in native female cells. We reveal the chromatin remodellers Smarcc1 and Smarca4, members of the BAF complex, as key regulators of establishment of X inactivation. These remodellers create a nucleosome depleted region at gene promotors on the inactive X during exit from pluripotency, without which gene silencing fails. Chromatin relaxation may therefore be a required initial step in the process of establishing epigenetic gene silencing.