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Modelling human escape from X-chromosome inactivation in mouse Peeters, Samantha

Abstract

A long-standing question concerning X-chromosome inactivation has been how some genes avoid the otherwise stable chromosome-wide heterochromatinization of the inactive X. As 20% or more of human X-linked genes escape from inactivation, such genes are important contributors to sex differences in gene expression, and identifying the mechanism by which these exceptions occur will inform our understanding of X-inactivation and broader questions of epigenetic regulation. While bioinformatic studies have generated a list of candidate features, the nature of the elements or definitive evidence that any one particular element is necessary or sufficient for a gene to escape, is still elusive and requires experimental validation. Mouse models offer a well-characterized and readily manipulated system in which to study X-inactivation and escape, but have far fewer genes and gene clusters that escape than humans. Given these differences, it was unclear whether the mechanism of escape gene regulation is conserved between species, and thus, this thesis addresses conservation of the escape process and the potential to model human escape gene regulation using mouse systems. Bacterial artificial chromosomes carrying genes known to escape from X-inactivation in humans were targeted to the Hprt locus and studied on the inactive X in mice. They were examined for escape by expression and inactivation-associated DNA methylation of promoter CpG islands. Expression from the inactive X and corresponding low promoter DNA methylation of human gene RPS4X demonstrated that the mouse system is capable of recognizing human elements. Furthermore, the escape status of the transgene remained stable between developmental time points, tissues, and individual females. A second human escape gene, KDM5C, was targeted to the Hprt locus and was surprisingly subject to inactivation, suggesting that its mechanism of escape was not conserved or that the critical elements for escape were not contained in the transgene. To further interrogate the escape elements involved in both human genes analyzed, as well as additional constructs of interest, a docking site at Hprt was generated in a female mouse embryonic stem cell line. Overall, this thesis contributed to the development of approaches to examine human escape from inactivation, and characterization of two human escape regions.

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