Evidence that homologous X-chromosome pairing requires transcription and Ctcf protein

X-chromosome inactivation (XCI) ensures the equality of X-chromosome dosages in male and female mammals by silencing one X in the female(1). To achieve the mutually exclusive designation of active X (Xa) and inactive X ( Xi), the process necessitates that two Xs communicate in trans through homologous pairing(2,3). Pairing depends on a 15-kb region within the genes Tsix and Xite(2). Here, we dissect molecular requirements and find that pairing can be recapitulated by 1- to 2-kb subfragments of Tsix or Xite with little sequence similarity. However, a common denominator among them is the presence of the protein Ctcf, a chromatin insulator(4-7) that we find to be essential for pairing. By contrast, the Ctcf-interacting partner, Yy1 (ref. 8), is not required. Pairing also depends on transcription. Transcriptional inhibition prevents new pair formation but does not perturb existing pairs. The kinetics suggest a pairing half-life of < 1 h. We propose that pairing requires Ctcf binding and co-transcriptional activity of Tsix and Xite.