Abstract The development and reproduction of female mammals is dependent on dosage compensation from sex chromosomes. Indeed, female mammals silence one of their two X chromosomes (X) in a process called X inactivation (XCI), a paradigm for epigenetic gene silencing. In mice, XCI occurs early during female embryogenesis in two waves. In embryos at the 2-4 cell stage, an early imprinted form of XCI (iXCI) silences exclusively the paternal X (Xp). Around implantation, however, female epiblast cells that give rise to somatic embryonic tissues undergo a major epigenetic switch: These cells reactivate the silenced Xp (XCR) and undergo another, random form of XCI (rXCI), silencing the paternal or maternal X with equal probability. Thus, tissues in the adult female mouse generally display a random XCI pattern. Xist RNA is crucial for X- silencing both during iXCI and rXCI and paints the inactive X from which it is expressed as a cloud, triggering downstream repressive chromatin modifications and X-silencing. We discovered that in mice the X-linked ubiquitin ligase RLIM is crucial for Xist functioning and maintenance of Xist clouds during iXCI and that lack of Rlim disrupts embryo implantation due to trophoblast failure. However, Rlim is dispensable for rXCI in epiblast cells of peri- and post- implantation embryos. Thus, the reasons of how X dosage compensation defects inhibit trophoblast functions as well as the pathways that regulate Xist during rXCI are unknown, and regulatory pathways triggering the developmental switch from iXCI to rXCI in epiblasts remain enigmatic. The research proposed in this grant is based on our published and preliminary work and will use mouse genetics combined with a newly generated female ESC model that permits investigations of XCI in the absence of Rlim. In particular, we will explore the effects of iXCI failure on trophoblast stem cell fates (Aim 1), examine novel mechanisms of rXCI regulation in female ESCs (Aim 2) and unravel in vivo mechanisms of XCR in female peri-implantation embryos (Aim 3). The proposed research will identify novel mechanisms of trophoblast stem cell maintenance, underlying regulation of Xist both during rXCI and iXCI and illuminate mechanisms underlying XCR. The combined results will allow designing the first comprehensive model of a iXCI/XCR/rXCI epigenetic switch that occurs in epiblast cells of developing female mice. Expected results will have transformative impact in the fields of early mouse development, stem cell biology, female reproduction and sex-specific epigenetic regulation.