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Role of the region 3′ to Xist exon 6 in the counting process of X-chromosome inactivation

Nature Genetics volume 19, pages 249–253 (1998)Cite this article

Abstract

During early embryogenesis of female mammals, one of the two X chromosomes is randomly chosen to be inactivated in each cell1, leading to the transcriptional silencing of thousands of genes on this chromosome. This random X-inactivation process also occurs during in vitro differentiation of female embryonic stem (ES) cells2,3,4. A locus on the X chromosome, the X inactivation centre (Xic) is initially 'counted', given that at least two copies of Xic must be present per diploid genome in order for inactivation to occur2. The counting process ensures that one X chromosome remains active in diploid cells. In the mouse, the essential functions of Xic can be assured by a 450-kb region5,6 containing the Xist gene. Xist maps within Xic (refs 7, 8, 9, 10) and is necessary in cis for inactivation11,12. The Xist transcript is a 15-kb RNA which is confined within the nucleus and coats the inactive X chromosome13. In order to characterize functional elements within Xic and the Xist gene, we created a 65-kb cre/loxP deletion extending 3′ to Xist exon 6. In undifferentiated ES cells, Xist expression from the deleted X chromosome was markedly reduced. In differentiated XX ES cells containing one deleted X chromosome, the X inactivation process still occurred but was never initiated from the unmutated X chromosome. In differentiated ES cells that were essentially XO, the mutated Xic was capable of initiating X inactivation, even in the absence of another Xic. These results demonstrate a role for the region 3′ to Xist exon 6 in the counting process and suggest that counting is mediated by a repressive mechanism which prevents inactivation of a single X chromosome in diploid cells.

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Figure 1: Strategy for deleting a 65-kb region 3′ to Xist exon 6.
Figure 2: A 65-kb deletion targeted 3′ to Xist exon 6 on the bPa X chromosome.
Figure 3: In ES cells, Xist expression from the del-bPa X chromosome is highly reduced; in differentiated ES cells, silencing occurs at the del-bPa X chromosome.
Figure 4: Initiation of inactivation of the del-bPa X chromosome is insensitive to X-chromosome counting.

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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. Rastan, S. & Robertson, E.J. X-chromosome deletions in embryo-derived (EK) cell lines associated with lack of X-chromosome inactivation. J. Embryol. exp. Morph. 90, 379–388 (1985).

    CAS  Google Scholar 

  3. Sheardown, S.A. et al. Stabilization of Xist RNA mediates initiation of X chromosome inactivation . Cell 91, 99–107 (1997).

    Article  CAS  Google Scholar 

  4. Panning, B., Dausman, J. & Jaenisch, R. X chromosome inactivation is mediated by Xist RNA stabilization. Cell 90, 907–16 ( 1997).

    Article  CAS  Google Scholar 

  5. Lee, J.T., Strauss, W.M., Dausman, J.A. & Jaenisch, R. A 450 kb transgene displays properties of the mammalian X-inactivation center. Cell 86, 83–94 ( 1996).

    Article  CAS  Google Scholar 

  6. Herzing, L.B.K., Romer, J.T., Horn, J.M. & Ashworth, A. Xist has properties of the X-chromosome inactivation centre. Nature 386, 272–75 (1997).

    Article  CAS  Google Scholar 

  7. Borsani, B. et al. Characterization of a murine gene expressed from the inactive X chromosome . Nature 351, 325–329 (1991).

    Article  CAS  Google Scholar 

  8. 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 

  9. Brockdorff, N. et al. Conservation of position and exclusive expression of mouse Xist from the inactive X chromosome. Nature 351, 329 –331 (1991).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Penny, G.D., Kay, G.F., Sheardown, S.A., Rastan, S. & Brockdorff, N. The Xist gene is required in cis for X chromosome inactivation. Nature 379, 131 –137 (1996).

    Article  CAS  Google Scholar 

  12. Marahrens, Y., Panning, B., Dausman, J., Strauss, W. & Jaenisch, R. Xist-deficient mice are defective in dosage compensation but not spermatogenesis. Genes Dev. 11, 156–166 (1997).

    Article  CAS  Google Scholar 

  13. Clemson, C.M., Mc Neil, J.A., Willard, H.F. & Lawrence, J.B. XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure. J. Cell Biol. 132, 259–275 ( 1996).

    Article  CAS  Google Scholar 

  14. Ramirez-Solis, R., Liu, P. & Bradley, A. Chromosome engineering in mice. Nature 378, 720–724 (1995).

    Article  CAS  Google Scholar 

  15. Simmler, M.C. et al. A 94kb genomic sequence 3′ to the murine Xist gene reveals an AT rich region containing a new testis specific gene Tsx. Hum. Mol. Genet. 5, 1713–1726 (1996).

    Article  CAS  Google Scholar 

  16. Panning, B. & Jaenisch, R. DNA hypomethylation can activate Xist expression and silence X-linked genes. Genes Dev. 10, 1991–2002 (1996).

    Article  CAS  Google Scholar 

  17. Simmler, M.C. et al. Localization and expression analysis of a novel conserved brain expressed transcript, Brx/BRX, lying within the Xic/XIC candidate region. Mamm. Genome 8, 760– 766 (1997).

    Article  CAS  Google Scholar 

  18. Marahrens, Y., Loring, J. & Jaenisch, R. Role of Xist gene in X chromosome choosing. Cell 92, 657–664 (1998).

    Article  CAS  Google Scholar 

  19. Rastan, S. Non-random X-chromosome inactivation in mouse X-autosome translocation embryos - location of the inactivation centre. J. Embryo. exp. Morph. 78, 1– 22 (1983).

    CAS  Google Scholar 

  20. Plenge, M.R. et al. A promoter mutation in the Xist gene in two unrelated families with skewed X-chromosome inactivation. Nature Genet. 17, 353–356 (1997).

    Article  CAS  Google Scholar 

  21. Cattanach, B.M., Perez, J.N. & Pollard, C.E. Controlling elements in the mouse X-chromosome. Genet. Res. 15, 189–195 (1970).

    Google Scholar 

  22. Cattanach, B.M. Control of chromosome inactivation. Annu. Rev. Genet. 75, 1–18 (1975).

    Article  Google Scholar 

  23. Rastan, S. Primary non-random X-inactivation caused by controlling elements in the mouse demonstrated at the cellular level. Genet. Res. 40, 139– 147 (1982).

    Article  CAS  Google Scholar 

  24. Courtier, B., Heard, E., Arnaud, D. & Avner, P. Xce haplotypes show modified methylation in a region of the active X chromosome lying 3′ to Xist. Proc. Natl Acad. Sci. USA 92, 3531–3535 (1995).

    Article  CAS  Google Scholar 

  25. Heard, E. et al. Physical mapping and YAC contig analysis of the region surrounding Xist on the mouse X chromosome. Genomics 15, 559–569 (1993).

    Article  CAS  Google Scholar 

  26. Kuehn, M., Bradley, A., Robertson, E.J. & Evans, M. A potential animal model for Lesh-Nyhan syndrome through introduction of HPRT mutations into mice. Nature 326, 295–298 (1987).

    Article  CAS  Google Scholar 

  27. Kay, G.F. et al. Expression of Xist during mouse development suggests a role in initiation of X chromosome inactivation. Cell 72, 171 –182 (1993).

    Article  CAS  Google Scholar 

  28. Robertson, E.J. Teratocarcinomas and embryonic stem cells, a Practical approach (IRL, Oxford, 1987).

    Google Scholar 

  29. Lawrence, J.B., Singer, R.H. & Marselle, L.M. Highly localized tracks of specific transcripts within interphase nuclei visualized by in situ hybridization. Cell 57, 493–502 ( 1989).

    Article  CAS  Google Scholar 

  30. Rougeulle, C., Colleaux, L., Dujon, B. & Avner, P. Generation and characterization of an ordered lambda clone array for the 460kb region surrounding the murine Xist sequence. Mamm. Genome 5, 416– 423 (1994).

    Article  CAS  Google Scholar 

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Acknowledgements

Thanks are due to D. Arnaud for generating the HP3.10 ES cell line and for technical help with the 6-TG selection and to E. Heard and F. Mongelard for establishing RNA FISH in the laboratory. F. Huetz and K. Rajewsky provided helpful advice on homologous recombination, and D. Cunningham, E. Heard, B. Robert and M. Wiles fruitful discussions. We are grateful to A. Bird, A. Bradley, S. O'Gorman, J.T. Lee and C. Vourc'h for the gifts of plasmids pBam7.6, pPGKpuro.bpA, pOG231, pXist3K, and p68. We thank F. Huetz and A. Stafford for critical reading of the manuscript. This work was supported by grants to P. A. from the Association Francaise contre la Myopathie (AFM), and the Association pour la Recherche contre le Cancer (ARC).

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  1. Génétique Moléculaire Murine, Institut Pasteur, Paris, 75015, 25 rue du Docteur Roux, France

    Philippe Clerc & Philip Avner

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Correspondence to Philippe Clerc.

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Clerc, P., Avner, P. Role of the region 3′ to Xist exon 6 in the counting process of X-chromosome inactivation. Nat Genet 19, 249–253 (1998). https://doi.org/10.1038/924

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