SUMMARY Once protein-coding, the X-inactivation center (Xic) is now dominated by large noncoding RNAs (ncRNA). X-chromosome inactivation (XCI) equalizes gene expression between mammalian males and females by inactivating one X in female cells. XCI requires Xist, a ncRNA that coats the X and recruits Polycomb proteins. How Xist is controlled remains unclear but likely involves negative and positive regulators. For the active X, the antisense Tsix RNA is an established Xist repressor. For the inactive X, here we identify Xic-encoded Jpx as an Xist activator. Jpx is developmentally regulated and accumulates during XCI. Deleting Jpx blocks XCI and is female-lethal. Posttranscriptional Jpx knockdown recapitulates the knockout, while supplying Jpx in trans rescues lethality. Thus, Jpx is trans-acting and functions as ncRNA. Furthermore, ΔJpx is rescued by truncating Tsix, indicating an antagonistic relationship between the ncRNAs. We conclude that Xist is controlled by two RNA-based switches – Tsix for Xa, and Jpx for Xi.

Summary In mammals, dosage compensation between XX and XY individuals occurs through X chromosome inactivation (XCI). The noncoding Xist RNA is expressed and initiates XCI only when more than one X chromosome is present. Current models invoke a dependency on the X-to-autosome ratio (X:A), but molecular factors remain poorly defined. Here, we demonstrate that molecular titration between an X-encoded RNA and an autosomally encoded protein dictates Xist induction. In pre-XCI cells, CTCF protein represses Xist transcription. At the onset of XCI, Jpx RNA is upregulated, binds CTCF, and extricates CTCF from one Xist allele. We demonstrate that CTCF is an RNA-binding protein and is titrated away from the Xist promoter by Jpx RNA. Thus, Jpx activates Xist by evicting CTCF. The functional antagonism via molecular titration reveals a role for long noncoding RNA in epigenetic regulation.