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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 female1. 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 pairing2,3. Pairing depends on a 15-kb region within the genes Tsix and Xite2. 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 insulator4,5,6,7 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.

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Figure 1: Growth and differentiation characteristics of new transgenic cell lines.
Figure 2: Induction of ectopic X-A pairing, disruption of endogenous X-X pairing and compromised Xist upregulation.
Figure 3: Ctcf protein is essential for pairing at Tsix.
Figure 4: New Ctcf sites in Xite.
Figure 5: Ctcf protein is also required for pairing at Xite.
Figure 6: Analysis of transcription requirements.


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We thank all members of the Lee laboratory for stimulating discussion and feedback throughout this work. S.S.S. is partially funded by a doctoral fellowship from the Gulbenkian Institute–Portugal (Fundacao para a Ciencia e a Tecnologia SFRH/BD/9614/2002). This work was supported by an US National Institutes of Health grant (RO1-GM58839) to J.T.L., who is an investigator of the Howard Hughes Medical Institute.

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N.X., M.E.D. and J.T.L. designed this study; N.X. created and analyzed transgenes and performed pairing, RNA FISH, immunofluorescence and transcription analyses; M.E.D. performed knockdowns, western blot, ChIP, EMSA, bioinformatics and qRT-PCR analyses; S.S. contributed to pairing analyses; J.T.L. wrote the paper and supervised the research.

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Correspondence to Jeannie T Lee.

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Xu, N., Donohoe, M., Silva, S. et al. Evidence that homologous X-chromosome pairing requires transcription and Ctcf protein. Nat Genet 39, 1390–1396 (2007).

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