Mechanisms of X-Chromosome Counting and Pairing in Mouse Embryonic Stem Cells

Sex chromosome gene dosage is balanced between male and female mammals early during development by transcriptionally silencing an entire X-chromosome in female cells. This X-chromosome inactivation (XCI) process begins by correctly counting X-chromosome number and choosing silent and active fates, determined by expression of the silencing Xist RNA from only one X. How the cell designates this asymmetric Xist expression remains unclear. The studies in this thesis dissect X-counting and choice mechanisms and provide key contributions to the field.;In the first study, we revisit the one- or two-factor X-counting and choice models. Observational differences regarding Xist induction in males have led to divergent models that require either a single 'blocking factor' to prevent XCI or both a blocking factor and a second 'inactivating factor' to initiate silencing. We investigated different methods of cell differentiation as a potential cause for these Xist discrepancies, and found that differentiation method---specifically retinoic acid use---affected Xist expression independently of the XCI program. Given that Xist expression alone was unable to completely silence X-linked genes, this implicated that an additional inactivating factor is required to trigger full silencing. Our findings therefore support the two-factor counting model.;In the second study, we tested the hypothesis that X-counting and choice depend upon somatic X-X pairing, which occurs just prior to asymmetric Xist upregulation. How the Xs communicate at the molecular level remains ambiguous. Using in vivo and in vitro protein/RNA-binding assays as well as shRNA knockdown technology, we found novel and essential roles for RNA and cohesins in pairing and XCI progression. Moreover, we observed skewed binding of factors on one X that correlates with the timing of pairing. Taken together, our findings suggest a two-step pairing model in which RNA first attracts the Xs together, and cohesins cohere X-X pairs long enough to distribute factors specifically onto one X. Thus, pairing couples the X-counting and choice steps by determining X-chromosome content through physical homolog interactions and allowing for the mutually exclusive exchange of blocking and inactivating factors to assign disparate transcriptional fates.