X-chromosome inactivation (XCI) is the mechanism of dosage compensation in mammals whereby one X-chromosome in every female cell is transcriptionally silenced during early embryonic development to ensure equivalent dosage of X-chromosome-linked genes between males and females. The process of XCI is closely coupled to exit from the pluripotent state. Pluripotency factors inhibit the process of XCI, and conversely the presence of two active X-chromosomes in female cells delays the exit from the pluripotent state. Female mouse embryonic stem cells, that have two active X-chromosomes, have been previously reported to display a more n ̈aive state of pluripotency including higher expression of pluripotency factors, slower kinetics of downregulation of pluripotency factors upon induction of differentiation. Additionally, they show decreased expression of MAPK and GSK3 target genes, but elevated levels of MAPK pathway intermediates such pMEK. However, the molecular mechanisms underlying the observed differences between the mESCs based on differential X-dosage are not well understood. In my doctoral project, I combined systematic perturbation experiments with mathematical modeling to interrogate the signaling network that regulates XCI and pluripotency in mESCs and to find differences in this network based on the differences in X-chromosome dosage. A female mESC line and its subclone that has lost one X-chromosome were used as a model system. I quantified the response of cells with one (XO) or two X chromosomes (XX) to a variety of inhibitors and growth factors. I then built models of the signaling networks in XX and XO cells through a semi-quantitative modeling approach based on modular response analysis (MRA). MRA-based modeling resulted in the discovery of novel links in the network and quantitative comparison of the model parameters using profile likelihood led to identification of the links underlying X-dosage-based differences. A novel negative feedback was identified in the PI3K-AKT pathway through GSK3. Moreover, it was found that the presence of a single active X makes mESCs more sensitive to the differentiation-promoting Activin A signal and leads to a stronger RAF1-mediated negative feedback in the FGF-triggered MAPK pathway. The differential response to these differentiation-promoting pathways can explain the impaired differentiation propensity of female mESCs.
