EVOLUTION OF X CHROMOSOME INACTIVATION ESCAPE IN MAMMALS Andrea Jane Slavney Ph. D. Cornell University 2018 In eutherian mammals, dosage compensation between XX females and XY males occurs in part through X chromosome inactivation (XCI) of one X homolog per cell in females. XCI was previously assumed to be complete across the X-specific regions of X chromosomes, but recently genes outside the pseudoautosomal regions (PARs) have been observed to exhibit low but significant expression from the inactive X in several species. These genes are termed XCI “escapers”. XCI escape introduces gene expression variation between females and males and inflates variation among females. However, it remains unclear whether XCI escape is generally a “bug” in the XCI system – that is, merely a consequence of inefficient XCI – or a “feature” required for normal biological function. By comparing XCI profiles across mammals, we can gain insight into the evolutionary history of XC and XCI escape, with the hope that this will improve our understanding of their mechanisms and functions. Here, I describe three analyses motivated by these goals. First, we expanded upon earlier findings to show that human XCI escapers exhibit a greater degree of strong purifying selection, as well as higher and broader gene expression than inactivated genes in both sexes. These results suggest a role for gene expression patterns in determining XCI status after divergence from the Y chromosome. Next, we generated a novel XCI profile for the domestic dog from single cell RNA-seq from two female F1 crossbreed dogs. Cell-level gene expression data was used to classify X genes as showing either monoallelic or bialleleic expression in multiple cells. Using this method, we identified 45 putative XCI escapers and 98 putative X-inactivated genes. Lastly, we performed a comparative analysis of XCI profiles across human, mouse, dog, and opossum 1:1 X orthologs. This analysis revealed that XCI escape is highly lineage-specific. Despite this, XCI escapers in these species overlap highly in their Gene Ontology biological process annotations. XCI escapers also appear to be under greater evolutionary constraint than inactivated genes within most species, though the magnitude varies. Overall, our findings suggest that the forces driving XCI escape evolution vary extensively across genes within and between species.