Project Summary / Abstract The goal of this proposal is to define the role of the long non-coding RNA (lncRNA) XACT (X-active coating transcript). XACT is a primate-specific and X-chromosome-linked lncRNA expressed exclusively in pluripotent stem cells. In naïve human embryonic stem cells (hESCs), which model the pluripotent state of the pre-implantation embryo, the XACT RNA forms two clouds in female and one cloud in male cells over the respective X-chromosome territories. At this stage of development, female and male cells express X-linked genes at comparable levels despite both female X-chromosomes being transcriptionally active. This gene dosage compensation mechanism, known as X-chromosome dampening (XCD), is unique to human development. Upon implantation, female pluripotent cells switch to complete silencing of one X-chromosome via X-chromosome inactivation (XCI), a process mediated by the well-studied chromatin-associated lncRNA XIST. In primed hESCs, which capture the pluripotent state of the post-implantation embryo, XACT expression is silenced on the inactive X-chromosome (Xi) of female cells, but is retained on the active X-chromosome in female and male cells. Considering that many lncRNAs function as transcriptional regulators, we hypothesize that XACT modulates the naïve and primed pluripotent states in early human development, potentially by regulating X- chromosome dosage compensation. Intriguingly, preliminary findings in our lab have shown that XIST also controls X-chromosome dampening in naïve pluripotent cells, but does not induce complete silencing in this developmental state. One possible function of XACT may be to counteract the silencing capacity of XIST in naïve pluripotent cells, in which XCD is characterized by concurrent expression of XACT and XIST from the X- chromosome. We posit that XACT antagonizes the activity of XIST during XCD through competitive chromatin association, thus preventing premature XCI prior to implantation. To unveil the role of XACT in pluripotent states and in the regulation of X-chromosome dosage compensation, we will: 1) delete XACT in naïve and primed hESCs and analyze the effect on cell states and transcriptomes; and 2) define its molecular mechanism of action as a chromatin-associated lncRNA. Taken together, these approaches will elucidate the contribution of XACT to the control of pluripotent states and its role in X-chromosome gene dosage regulation. By exploring the link between XACT function and the modulation of XIST in early human development, we may uncover a novel, primate-specific strategy for gene dosage compensation and define fundamental features of human pluripotency.