Strong Constraint on Human Genes Escaping X-Inactivation Is Modulated by their Expression Level and Breadth in Both Sexes

Abstract

In eutherian mammals, X-linked gene expression is normalized between XX females and XY males through the process of X chromosome inactivation (XCI). XCI results in silencing of transcription from one ChrX homolog per female cell. However, approximately 25% of human ChrX genes escape XCI to some extent and exhibit biallelic expression in females. The evolutionary basis of this phenomenon is not entirely clear, but high sequence conservation of XCI escapers suggests that purifying selection may directly or indirectly drive XCI escape at these loci. One hypothesis is that this signal results from contributions to developmental and physiological sex differences, but presently there is limited evidence supporting this model in humans. Another potential driver of this signal is selection for high and/or broad gene expression in both sexes, which are strong predictors of reduced nucleotide substitution rates in mammalian genes. Here, we compared purifying selection and gene expression patterns of human XCI escapers with those of X-inactivated genes in both sexes. When we accounted for the functional status of each ChrX gene's Y-linked homolog (or "gametolog"), we observed that XCI escapers exhibit greater degrees of purifying selection in the human lineage than X-inactivated genes, as well as higher and broader gene expression than X-inactivated genes across tissues in both sexes. These results highlight a significant role for gene expression in both sexes in driving purifying selection on XCI escapers, and emphasize these genes' potential importance in human disease.

Fig. 1.

XCI escapers show larger fractions of sites under strong purifying selection (S) and smaller fractions of neutral sites (N) than X-inactivated genes with the same Y gametolog status. XCI escaper-to-X-inactivated ratios of the values of N and S are presented, with standard deviations based on 1,000 bootstrap samples of genes in each XCI/Y category (error bars denote ±1 standard deviation). The numbers (n) of XCI escaper and X-inactivated genes in each Y gametolog category are shown below the category names at the top of the plot. The whole ChrX category includes genes from all other categories, as well as 18 genes that did not have a Y gametolog status assignment (12 XCI escapers and 6 X-inactivated genes). The no Y category is the union of the pseudogenized and lost Y categories.

Fig. 2.

(a) XCI category average gene expression by Y gametolog status, tissue, and sex. Scatterplots show the XCI/Y category averages of gene expression values for each sex in each GTEx tissue. Shaded areas show the standard deviation of the average across genes. In both plots, individual expression values were filtered by tissue-specific cutoffs (see Methods) before being incorporated into gene averages. (b) Global mean expression for each gene by XCI status, Y gametolog status, and sex. Each point in these one-dimensional scatterplots shows the global mean expression value of a single gene in female (left) and male (right) samples. The white diamonds indicate the mean expression value of the genes in that XCI/Y category. Areas of darker color indicate overlap of two or more points. In both plots, individual expression values were filtered by tissue-specific cutoffs (see Methods) before being incorporated into gene averages.

Fig. 3.

XCI escapers are more broadly expressed than X-inactivated genes with the same Y gametolog status in both sexes: Points show τ statistic values for all genes in each XCI/Y category for female (left) and male (right) samples. White diamonds show the mean of τ across all genes in that XCI/Y category. Areas of darker color indicate overlap of two or more points. Values of τ range from 0 to 1, with higher values corresponding to greater tissue specificity.