X-chromosome inactivation (XCI) in mammals compensates for the imbalance in X chromosome number between females (XX) and males (XY). How the number of X chromosomes is counted by the cell is a long-standing problem. A recent study has implicated an X-encoded long non-coding RNA (lncRNA) in X-chromosome counting that titrates an autosomal transcription factor away from the X chromosome to inactivate it.

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Mammalian XCI is known to occur when the X to autosome ratio (X/A) is ≥1, leading to the expression of inactive X specific transcript (Xist), which is an lncRNA that silences the X chromosome. However, a full understanding of how the cell calculates the X/A ratio through X-encoded numerators and autosomally encoded denominators has been elusive.

A recent study has implicated an X-encoded long non-coding RNA in X-chromosome counting

Sun et al. postulated that the X-encoded lncRNA Jpx, which is known to activate the transcription of Xist, is a good candidate for an X-encoded numerator. In support of this hypothesis, transgene experiments in mouse embryonic stem cells (ESCs) showed that Xist displays a dosage-dependent response to Jpx expression, so Xist expression is switched on only when the levels of Jpx are high enough.

The authors then chose to investigate CTCF as a potential autosomal regulator of XCI initiation — that is, as an autosome-encoded denominator of the X/A ratio — as putative binding sites for the CTCF transcription factor had previously been identified in the Xist promoter. They used chromatin immunoprecipitation (ChIP) to show that CTCF binds to the promoter site P2 in ESCs prior to XCI. However, on differentiation in female cells, CTCF binding at this site is depleted in the future inactive X chromosome. Furthermore, CTCF transgene studies indicated that CTCF was a direct Xist repressor.

Sun et al. were then able to show a link between Jpx and CTCF: Jpx overexpression relieved the effects of CTCF overexpression. Furthermore, they showed direct binding of Jpx to CTCF in vivo. Finally, ChIP confirmed that when CTCF was overexpressed, the binding of CTCF to P2 was increased, and Xist upregulation was compromised. However, when Jpx alone was overexpressed, CTCF binding to Xist was reduced approximately by half.

The authors' data point towards a model in which, in order to initiate XCI, Jpx is activated and titrates repressive CTCF away from the Xist promoter through competitive binding; this is the first time that such a function has been reported for an lncRNA. The allele-specific nature of these interactions suggests that further regulatory factors are yet to be found.