Dosage compensation defects due to roX RNA loss are rescued by recalibration of X/autosome stoichiometry.

Metazoa evolved regulatory networks to balance sex chromosome expression. In Drosophila, males have a single gene-rich X chromosome, whereas females have two. Balanced X/autosome expression is essential for viability, and in male flies is achieved by activation of genes on the X through the male-specific-lethal (MSL) dosage compensation complex (DCC). This ribonucleoprotein assembly contains long non-coding roX RNAs. To dissect the functional requirements of roX in a cell-based system, we deleted the roX2 gene in male S2 cells and selected two independent lines lacking detectable roX RNA. In the absence of roX, the remaining MSL protein complex was unable to associate with known or newly identified binding sites and thus failed to activate transcription. Surprisingly, the X/autosome expression ratio appeared nevertheless compensated. Cytogenetic and genomic analyses revealed that both roX-deficient cell populations had acquired additional X chromosomes. We propose that uncompensated X expression compromises fitness and gives a selective growth advantage to cells that rebalance their genomes through chromosome mis-segregation. Remarkably, ectopic expression of roX2 reversed this selection, restored DCC binding, and normalized the karyotype. These findings illustrate that X chromosome dosage compensation is critical for viability even in cultured cells, and provide a striking example of rapid evolution under stringent selection.