Mutational Analysis of Xist and the X-inactivation Center

In placental mammals, dosage compensation of the sex chromosomes isachieved through inactivation of one X chromosome in female cells. This Xchromosome inactivation (XCI) requires tight developmental regulation to ensure all butone X chromosome is silenced.At the center of this process is Xist, a long non-coding RNA. Upon differentiationof a female cell, Xist spreads in cis to coat and silence the inactive X chromosome.While upregulation of Xist, has been shown to be sufficient for X inactivation to occur,no one has thoroughly investigated whether Xist is necessary for the establishment of Xchromosome inactivation. In this thesis I provide evidence that Xist is not required fordosage compensation of the X chromosome during epiblast-like cell differentiation. Thisresult suggests Xist-independent silencing mechanisms for this essential process maybe in place.A 1-2 Mb region of the X chromosome, termed the X-inactivation center (Xic) isnecessary in two copies for XCI to occur, indicating it is necessary for cells to count thenumber of X chromosomes present. I delete one copy of the putative 2 Mb Xic in maleand female mouse embryonic stem cells and present evidence that this deletion is notwell tolerated, suggesting that this region requires finer resolution mapping to identifythe minimal element required for counting.Finally, I finish with a review which elaborates on studies that enlighten ourunderstanding of activators and repressors that control XCI. Our findings challengeexisting dogmas in the field and provide the foundation for future work focused onuncovering the molecular mechanisms behind Xist-independent silencing of the Xchromosome.