The mammalian blastocyst forms several days after one of the smallest cells - the sperm - fertilizes one of the largest cells -the egg. Depending on sex chromosome contribution from the sperm, either a female (XX genotype) or a male (XY genotype) embryo develops. To compensate for the X chromosome genetic imbalance between males and females, female cells transcriptionally silence one of their two X chromosomes. This phenomenon of X-chromosome inactivation (XCI) occurs in the blastocyst inner cell mass (ICM) cells – which form the embryo – as it implants to the uterine wall. The transition of the naï¿½ve pluripotent ICM cells with two active X chromosomes to primed pluripotency with an active and an inactive X chromosome (Xa and Xi, respectively) is mediated by the lncRNA Xist. While both mouse and human cells arising after implantation have an Xist-expressing Xi and an Xist-negative Xa, the X-chromosome state in the naï¿½ve pre-implantation development is rather different in the two species. In mice, Xist is not expressed in the ICM cells with two active X chromosomes since Xist expression invariably leads to XCI. Yet, human pre-implantation blastocysts, including cells of the ICM, express XIST from active X chromosomes. We demonstrate that the presence of an XIST-expressing Xa, which is unique to human pre-implantation development, is a robust marker of human naï¿½ve pluripotency. We utilize this marker to identify a culture condition that, for the first time, allows detailed molecular studies of X-chromosome regulation of human pre-implantation development using cultured human pluripotent stem cells (hPSCs). We demonstrate that naï¿½ve hPSCs, despite having two active X chromosomes, compensate expression of X-chromosome genes via chromosome- wide transcriptional dampening and mediate XIST-mediated XCI upon differentiation to a somatic lineage. Lastly, we determine that naï¿½ve culture conditions obliterate the epigenetic abnormalities of the Xi characteristic to conventional hPSCs of developmentally advanced – primed – pluripotent state.
Once established, the Xi is kept transcriptionally silent for the life of the cell and its progeny, namely due to the many epigenetic layers forming the facultative heterochromatin of the Xi. In addition to covalent modifications of histone proteins, CpG islands of X-chromosome genes are methylated at cytosine residues to keep this silent state. To uncover the mechanisms which work in synergy with DNA methylation to maintain the inactive state of the Xi, we performed an RNAi and small chemical screen using engineered mouse cells with a reporter gene on the silent X chromosome. Our screen identified a synergistic combination of two FDA-approved chemicals that together lead to increased DNA demethylation not only of the Xi, but genome-wide, and works in synergy to reduce viability of leukemic cells. Thus, understanding XCI at the molecular level can be used to optimize the epigenetic activity of drug combinations.