PROJECT SUMMARY The overall goal of this proposal is to study the regulation of X-chromosome dosage compensation by long- noncoding (lnc) RNAs in human embryonic stem cells (hESCs) to fundamentally advance the understanding of early human development, the regulation of pluripotency states, and mechanisms of gene regulation by lncRNAs. X-chromosome dosage compensation is an essential process that equalizes X-linked gene expression between females and males and is initiated early in development. In mammals, it is mediated by X-chromosome inactivation (XCI), the transcriptional silencing of genes on one of the two X-chromosomes in females. Mechanistic studies of XCI have been carried out in mice and established that the lncRNA Xist, encoded on the X chromosome, spreads along the X chromosome to mediate XCI. Although XCI occurs in both mouse and human post-implantation development, the regulation of X-chromosome dosage in pre-implantation embryos has evolved differently between the two species. In pre-implantation development, mouse embryos undergo an imprinted form of XCI, while human embryos lack imprinted XCI and instead regulate gene expression by dampening transcription on both X-chromosomes. X-chromosome dampening (XCD) is different from XCI as it occurs on both X chromosomes in contrast to the complete transcriptional silencing of one X. Additional remarkable differences between human and mouse include the unprecedented expression and accumulation of XIST on the active (dampened) X-chromosomes and the expression of the primate- and pluripotency-specific lncRNA XACT from the X in human pre-implantation embryos. Thus, X-linked gene dosage in humans is regulated first by XCD and upon implantation by XCI, and remarkably, XIST expression is uncoupled from silencing when XCD takes place. A mechanistic understanding of XCD, lack of silencing by XIST in pre- implantation embryos, XIST's transition to mediating XCI, and the role of the lncRNA XACT is lacking, and it is not known if XIST has any role in human pre-implantation cells or XCD. We discovered that naïve hESCs recapitulate in vitro many of the unique features of human X-chromosome dosage compensation, including XCD, expression of XIST on active X-chromosomes, XACT expression, and initiation of XCI upon differentiation. Here, we will take advantage of naïve hESCs to investigate how XIST expression does not induce silencing early in development and later acquires the ability to silence, to reveal regulatory mechanisms of human XCD and XCI, and if XACT controls XIST functions. We have the following Specific Aims: 1) We will define the function of XIST in naïve hESCs, its requirement for XCD, and mechanisms of XCD. 2) We will define chromatin targets and protein interactors of XIST to understand its differential silencing ability in naïve and primed hESCs. 3) We will characterize the function and localization of the lncRNA XACT. Understanding the mechanisms of human X- chromosome dosage compensati...