Pluripotency factor binding and Tsix expression act synergistically to repress Xist in undifferentiated embryonic stem cells

Here we employ a transgene strategy to test the role of the intron 1 element and Tsix in repressing Xist in ES cells. We find that deletion of the intron 1 element causes a small increase in Xist expression and that simultaneous deletion of the antisense regulator Tsix enhances this effect.We conclude that Tsix and pluripotency factors act synergistically to repress Xist in undifferentiated embryonic stem cells. Double mutants do not exhibit maximal levels of Xist expression, indicating that other pathways also play a role.In female mammals a developmentally regulated process, X inactivation, ensures silencing of a single X chromosome, balancing levels of X-linked genes relative to males [1]. X inactivation is mediated by the cis-acting non-coding RNA Xist that is transcribed from and coats the inactive X chromosome (Xi) elect [2]. Coating by Xist RNA triggers epigenetic modifications that silence transcription and establish a heritable heterochromatic state [3].X inactivation in the mouse occurs in two waves; imprinted X inactivation of the paternal X chromosome (Xp) that is initiated in two to four cell embryos and maintained in all cells until the blastocyst stage, and random X inactivation, initiated in the postimplantation epiblast. Embryo precursors in the inner cell mass (ICM) of the blastocyst reactivate Xp, reversing imprinted X inactivation and setting the ground state for the onset of random X inactivation [4,5]. XX embryonic stem (ES) cells, which are derived from the ICM, mirror this ground state, retaining two active X chromosomes [6,7]. In contrast extraembryonic trophectoderm and primitive endoderm lineages and cell lines derived thereof retain the imprinted X inactivation pattern through embryogenesis [8-11].X chromosome reactivation also occurs in XX primordial germ cells during migration towards the genital ridges [12-14], and similarly during experimental reprogramming of XX somatic cells, either by cloning, cell fusion with pluripotent cells or