Xmas ESC: A new female embryonic stem cell system that reveals the BAF complex as a key regulator of the establishment of X chromosome inactivation

Abstract

Although female pluripotency significantly differs to male, complications with in vitro culture of female embryonic stem cells (ESC) have severely limited the use and study of these cells. We report a replenishable female ESC system, Xmas, that has enabled us to optimise a protocol for preserving the XX karyotype. Our protocol also improves male ESC fitness. We utilised our Xmas ESC system to screen for regulators of the female-specific process of X chromosome inactivation, revealing chromatin remodellers Smarcc1 and Smarca4 as key regulators of establishment of X inactivation. The remodellers create a nucleosome depleted region at gene promotors on the inactive X during exit from pluripotency, without which gene silencing fails. Our female ESC system provides a tractable model for XX ESC culture that will expedite study of female pluripotency and has enabled us to discover new features of the female-specific process of X inactivation.

New Insights into X-Chromosome Reactivation during Reprogramming to Pluripotency

“…Several in vitro systems have been used to induce and study XCR. These include cell fusion [ 145 , 146 , 147 , 148 ], and nuclear transfer to mouse or frog oocytes [ 52 , 70 , 134 ] (reviewed in [ 22 , 135 , 149 ]) and somatic cell reprogramming to induced pluripotent stem cells (iPSCs) [ 23 , 25 , 42 , 48 , 49 , 150 ].…”

“…In the initiation phase of XCR, reprogramming induces reacquisition of PRC2 enrichment on the Xi [ 25 ]. The progression phase induces reactivation of endogenous pluripotency genes [ 23 , 25 , 42 , 48 , 49 ] and include, Tsix reactivation [ 23 , 25 , 42 ], Xist silencing [ 23 , 25 , 42 ], loss of PRC2, H3K27me3 and macroH2A enrichment on the Xi [ 23 , 25 , 42 ], as well as gain of histone acetylation and active RNA Polymerase II [ 25 , 42 , 48 ], DNA demethylation [ 25 , 42 , 51 ], and transcriptional reactivation [ 23 , 25 , 42 , 48 , 49 , 150 , 154 ]. In the completion phase, stable and heritable gene silencing is reversed and the X chromosome reacquires the ability to undergo random XCI upon differentiation [ 42 ].…”

“…A key remaining question is: which additional chromatin regulators are involved in XCR and whether active or passive mechanisms are used? Candidate factors of outstanding interest for remodeling of chromatin during XCR are Smarrc1 and Smarca4 , which were recently identified in a screen for factors involved in XCI [ 150 ].…”