Dosage Compensation in the Mouse Balances Up-Regulation and Silencing of X-Linked Genes

Abstract

Dosage compensation in mammals involves silencing of one X chromosome in XX females and requires expression, in cis, of Xist RNA. The X to be inactivated is randomly chosen in cells of the inner cell mass (ICM) at the blastocyst stage of development. Embryonic stem (ES) cells derived from the ICM of female mice have two active X chromosomes, one of which is inactivated as the cells differentiate in culture, providing a powerful model system to study the dynamics of X inactivation. Using microarrays to assay expression of X-linked genes in undifferentiated female and male mouse ES cells, we detect global up-regulation of expression (1.4- to 1.6-fold) from the active X chromosomes, relative to autosomes. We show a similar up-regulation in ICM from male blastocysts grown in culture. In male ES cells, up-regulation reaches 2-fold after 2–3 weeks of differentiation, thereby balancing expression between the single X and the diploid autosomes. We show that silencing of X-linked genes in female ES cells occurs on a gene-by-gene basis throughout differentiation, with some genes inactivating early, others late, and some escaping altogether. Surprisingly, by allele-specific analysis in hybrid ES cells, we also identified a subgroup of genes that are silenced in undifferentiated cells. We propose that X-linked genes are silenced in female ES cells by spreading of Xist RNA through the X chromosome territory as the cells differentiate, with silencing times for individual genes dependent on their proximity to the Xist locus. In organisms such as fruit flies and humans, major chromosomal differences exist between the sexes: females have two large, gene-rich X chromosomes, and males have one X and one small, gene-poor Y. Various strategies have evolved to balance X-linked gene expression between the single X and the autosomes, and between the sexes (a phenomenon called dosage compensation). In Drosophila melanogaster, expression from the male X is up-regulated approximately 2-fold, thereby balancing both X-to-autosome and female-to-male expression. In contrast, mammals silence one of the two female Xs in a process requiring the untranslated RNA product of the Xist gene. This balances female-to-male expression but leaves both sexes with only one functional X chromosome. Using mouse embryonic stem cells and microarray expression analysis, we found that dosage compensation in mice is more complex than previously thought, with X-linked genes up-regulated in both male and female cells so as to balance X-to-autosome expression. As differentiation proceeds, female cells show progressive loss of expression from one of the two initially active Xs. Surprisingly, silencing occurs on a gene-by-gene basis over 2–3 week of differentiation; some genes escape altogether, whereas a subgroup of genes, often adjacent to the Xist locus, is silenced even in undifferentiated cells. We propose that female X-linked genes are silenced by progressive spreading of Xist RNA through the X chromosome territory as differentiation proceeds.