UCLA

X-active coating transcript (XACT) is a primate-specific long non-coding RNA (lncRNA) first detected in human embryonic stem cells (hESCs) and encoded on the X-chromosome. XACT is expressed in both the na�ve and primed pluripotent states, which represent the distinct cellular identities of pre- and post-implantation epiblast cells, respectively. Its expression patterns and colocalization with the X-chromosome implicate this lncRNA in X-linked gene dosage regulation in early human development; however, XACT’s specific role and mechanism of action in human embryogenesis remain unclear. Here, we show that in primed human hESCs that have undergone X-chromosome inactivation (XCI) and no longer express the XCI master regulator XIST, XACT does not appreciably impact X-linked gene expression. We establish that XACT is associated with chromatin, and the loss of its genomic locus as well as transcript expression affects the distribution of mRNAs between chromatin and the cytoplasm. Additionally, primed hESCs missing XACT are attenuated in their potential to differentiate into primordial germ cell-like cells, suggesting that XACT may not only mark, but function to preserve the pluripotent state. Overall, these results indicate a possible role of XACT outside X-linked gene dosage compensation. In contrast to XCI in primed hESCs, in the pre-implantation blastocyst and in na�ve hESCs, X-linked genes are transcriptionally downregulated (but not silenced) from both female X chromosomes to equilibrate gene dosage with male cells. The ensuing X-chromosome “dampening” (XCD) is unique to humans and coincides with the coating of both X chromosomes by XIST and XACT. We show that XIST is required for XCD, yet displays a lower accumulation and different distribution on the dampened compared to the inactive X chromosome. Surprisingly, XIST was also found to accumulate near and regulate the expression of autosomal genes involved in development. Together these results provide insight into the XIST-mediated mechanism of XCD, and invite potential for validating the putative relationship between XIST and XACT in regulating human X-linked gene dosage compensation in pre-implantation development.