Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

Abstract

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

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.

References

  1. Lyon, M.F. Gene action in the X-chromosome of the mouse (Mus musculus L.). Nature 190, 372–373 (1961).

    Article  CAS  Google Scholar 

  2. Xu, N., Tsai, C.L. & Lee, J.T. Transient homologous chromosome pairing marks the onset of X inactivation. Science 311, 1149–1152 (2006).

    Article  CAS  Google Scholar 

  3. Bacher, C.P. et al. Transient colocalization of X-inactivation centres accompanies the initiation of X inactivation. Nat. Cell Biol. 8, 293–299 (2006).

    Article  CAS  Google Scholar 

  4. Chao, W., Huynh, K.D., Spencer, R.J., Davidow, L.S. & Lee, J.T. CTCF, a candidate trans-acting factor for X-inactivation choice. Science 295, 345–347 (2002).

    Article  CAS  Google Scholar 

  5. Bell, A. & Felsenfeld, G. Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene. Nature 405, 482–485 (2000).

    Article  CAS  Google Scholar 

  6. Hark, A.T. et al. CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus. Nature 405, 486–489 (2000).

    Article  CAS  Google Scholar 

  7. Kanduri, C. et al. Functional association of CTCF with the insulator upstream of the H19 gene is parent of origin-specific and methylation-sensitive. Curr. Biol. 10, 853–856 (2000).

    Article  CAS  Google Scholar 

  8. Donohoe, M.E., Zhang, L., Xu, N., Shi, Y. & Lee, J.T. Identification of a CTCF co-factor, YY1, for the X-chromosome binary switch. Mol. Cell 25, 43–56 (2007).

    Article  CAS  Google Scholar 

  9. Ogawa, Y. & Lee, J.T. Xite, X-inactivation intergenic transcription elements that regulate the probability of choice. Mol. Cell 11, 731–743 (2003).

    Article  CAS  Google Scholar 

  10. Lee, J.T., Davidow, L.S. & Warshawsky, D. Tsix, a gene antisense to Xist at the X-inactivation centre. Nat. Genet. 21, 400–404 (1999).

    Article  CAS  Google Scholar 

  11. Lee, J.T. & Lu, N. Targeted mutagenesis of Tsix leads to nonrandom X inactivation. Cell 99, 47–57 (1999).

    Article  CAS  Google Scholar 

  12. Sado, T., Wang, Z., Sasaki, H. & Li, E. Regulation of imprinted X-chromosome inactivation in mice by Tsix. Development 128, 1275–1286 (2001).

    CAS  PubMed  Google Scholar 

  13. Brockdorff, N. et al. The product of the mouse Xist gene is a 15 kb inactive X-specific transcript containing no conserved ORF and located in the nucleus. Cell 71, 515–526 (1992).

    Article  CAS  Google Scholar 

  14. Brown, C.J. et al. The human XIST gene: analysis of a 17 kb inactive X-specific RNA that contains conserved repeats and is highly localized within the nucleus. Cell 71, 527–542 (1992).

    Article  CAS  Google Scholar 

  15. Wutz, A., Rasmussen, T.P. & Jaenisch, R. Chromosomal silencing and localization are mediated by different domains of Xist RNA. Nat. Genet. 30, 167–174 (2002).

    Article  CAS  Google Scholar 

  16. Lee, J.T. Regulation of X-chromosome counting by Tsix and Xite sequences. Science 309, 768–771 (2005).

    Article  CAS  Google Scholar 

  17. Vigneau, S., Augui, S., Navarro, P., Avner, P. & Clerc, P. An essential role for the DXPas34 tandem repeat and Tsix transcription in the counting process of X chromosome inactivation. Proc. Natl. Acad. Sci. USA 103, 7390–7395 (2006).

    Article  CAS  Google Scholar 

  18. Lee, J.T. Homozygous Tsix mutant mice reveal a sex-ratio distortion and revert to random X-inactivation. Nat. Genet. 32, 195–200 (2002).

    Article  CAS  Google Scholar 

  19. Navarro, P., Pichard, S., Ciaudo, C., Avner, P. & Rougeulle, C. Tsix transcription across the Xist gene alters chromatin conformation without affecting Xist transcription: implications for X-chromosome inactivation. Genes Dev. 19, 1474–1484 (2005).

    Article  CAS  Google Scholar 

  20. Sado, T., Hoki, Y. & Sasaki, H. Tsix silences Xist through modification of chromatin structure. Dev. Cell 9, 159–165 (2005).

    Article  CAS  Google Scholar 

  21. Sun, B.K., Deaton, A.M. & Lee, J.T. A transient heterochromatic state in Xist preempts X inactivation choice without RNA stabilization. Mol. Cell 21, 617–628 (2006).

    Article  CAS  Google Scholar 

  22. Cohen, D.E. et al. The Dxpas34 repeat regulates random and imprinted X-inactivation. Dev. Cell 12, 57–71 (2007).

    Article  CAS  Google Scholar 

  23. Stavropoulos, N., Rowntree, R.K. & Lee, J.T. Identification of developmentally specific enhancers for Tsix in the regulation of X chromosome inactivation. Mol. Cell. Biol. 25, 2757–2769 (2005).

    Article  CAS  Google Scholar 

  24. Kurukuti, S. et al. CTCF binding at the H19 imprinting control region mediates maternally inherited higher-order chromatin conformation to restrict enhancer access to Igf2. Proc. Natl. Acad. Sci. USA 103, 10684–10689 (2006).

    Article  CAS  Google Scholar 

  25. Ling, J.Q. et al. CTCF mediates interchromosomal colocalization between Igf2/H19 and Wsb1/Nf1. Science 312, 269–272 (2006).

    Article  CAS  Google Scholar 

  26. Syken, J., De-Medina, T. & Munger, K. TID1, a human homolog of the Drosophila tumor suppressor l(2)tid, encodes two mitochondrial modulators of apoptosis with opposing functions. Proc. Natl. Acad. Sci. USA 96, 8499–8504 (1999).

    Article  CAS  Google Scholar 

  27. Kim, T.H. et al. Analysis of the vertebrate insulator protein CTCF-binding sites in the human genome. Cell 128, 1231–1245 (2007).

    Article  CAS  Google Scholar 

  28. Workman, C.T. et al. enoLOGOS: a versatile web tool for energy normalized sequence logos. Nucleic Acids Res. 33, W389–92 (2005).

    Article  CAS  Google Scholar 

  29. Spilianakis, C.G., Lalioti, M.D., Town, T., Lee, G.R. & Flavell, R.A. Interchromosomal associations between alternatively expressed loci. Nature 435, 637–645 (2005).

    Article  CAS  Google Scholar 

  30. Nichols, J. et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95, 379–391 (1998).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Contributions

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.

Corresponding author

Correspondence to Jeannie T Lee.

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1, Supplementary Tables 1 and 2 (PDF 466 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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). https://doi.org/10.1038/ng.2007.5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ng.2007.5

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing