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

Hong Lin,Vibhor Gupta,Bryan M Turner,Jeannie T Lee,Matthew D Vermilyea,Francesco Falciani,Laura P O'Neill,Wolf Reik

doi:10.1371/journal.pbio.0050326

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.

Highlights

IntroductionSex determination mechanisms have evolved in a way that has generated chromosomal differences between the sexes
In many higher eukaryotes, sex determination mechanisms have evolved in a way that has generated chromosomal differences between the sexes
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

Summary

Introduction

Sex determination mechanisms have evolved in a way that has generated chromosomal differences between the sexes. X inactivation alone exacerbates the X:autosome imbalance, leaving both sexes functionally monosomic for Xlinked genes This problem was highlighted many years ago, and a balancing, 2-fold up-regulation of genes from the single, active X was proposed as a possible solution [2,8]. Comparisons of the mean, overall expression levels of X-linked and autosomal genes in various cell and tissue types, usually from mixtures of male and female, gives an X:autosome expression ratio of approximately 1 [10,11,12] Given that both XY male and XX female cells have only a single, transcriptionally active X, and two copies of each autosome, without up-regulation of X-linked genes the mean ratio should be closer to 0.5. The results provide evidence, albeit circumstantial, for a balancing up-regulation of expression from the active X

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Conclusion