Courtois, A.; Menchero, S.; Ogushi, S.; Varsally, W.; Wood, S.; Decarpentrie, F.; Yoshimi, R.; Shiraishi, A.; Inoue, K.; Abe, T.; VandeBerg, J. L.; Snell, D. M.; Kiyonari, H.; Turner, J. M. A.

X-chromosome inactivation balances X-gene dosage between the sexes in eutherians and marsupials. The lncRNA Xist mediates X-inactivation in eutherians but is absent from marsupials. An evolutionarily unrelated lncRNA, RSX, has been identified in marsupials, but its role in X-inactivation is unresolved because genome editing in these mammals has only recently become feasible. Using CRISPR-Cas9 in the opossum, we show that RSX is required for the initiation of X-inactivation in marsupial embryos. RSX deletion later, in differentiated cells, causes modest X-gene de-repression, with repressive chromatin marks H3K27me3 and H3K9me3 retained. H2AK119ub is not enriched on the opossum inactive X, revealing divergent Polycomb-mediated regulation in therians. Our findings reveal stage-specific roles of RSX in marsupial X-inactivation and identify conserved and divergent features of X-dosage compensation in mammals.