Living with two X chromosomes: of mice and women : studies on the initiation mechanisms of X chromosome inactivation in stem cells and mouse models, and the role of RNF12 herein

__Abstract__ In this thesis work, we have investigated mechanisms involved in regulation of the initiation of X chromosome inactivation (XCI). Starting point was our earlier hypothesis that the initiation of XCI is a stochastic process, controlled in trans by autosomally-encoded XCI-inhibitors and X-encoded XCI-activators. The XCI-inhibitors prevent initiation of XCI in undifferentiated cells or early embryos. Upon differentiation of these cells during development, XCI is initiated in females only, by the activity of X-linked XCI-activators, which reach a higher concentration in female cells compared to male cells, due to the location of the encoding genes on the X chromosome. As described in Chapter 2, we found that the E3 ubiquitin ligase RNF12 acts as an important X-encoded activator of X chromosome inactivation. When extra copies of Rnf12 are transgenically expressed in male ES cells, the encoded higher level of RNF12 is able to induce XCI on the single X chromosome, whereas such over-expression in female ES cells leads to ectopic XCI on both X chromosomes in a significant portion of the cells. Rnf12 becomes up-regulated in female ES cells during the developmental time window when XCI is normally occurring, and genetic ablation of one copy of Rnf12 in female ES cells results in a significant delay in the XCI process. Chapter 4 discusses this discovery in the context of the stochastic model for XCI initiation, controlled by XCI-activators and XCI-inhibitors, and further provides evidence that all features of XCI initiation can indeed be explained by this model. A novel BAC targeting strategy described in Chapter 3 enabled efficient, fast and reliable genetic modifications of mouse ES cells, and this strategy was used throughout this thesis work. Among others, it allowed us to generate the Rnf12 homozygous knockout ES cells, described in Chapter 5. Using these cells, we provide evidence that RNF12 is essential for XCI to occur, and mediates its effect mainly throu