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.

  • Article
  • Published:

The A-repeat links ASF/SF2-dependent Xist RNA processing with random choice during X inactivation

Nature Structural & Molecular Biology volume 17, pages 948–954 (2010)Cite this article

Subjects

Abstract

One X chromosome, selected at random, is silenced in each female mammalian cell. Xist encodes a noncoding RNA that influences the probability that the cis-linked X chromosome will be silenced. We found that the A-repeat, a highly conserved element within Xist, is required for the accumulation of spliced Xist RNA. In addition, the A-repeat is necessary for X-inactivation to occur randomly. In combination, our data suggest that normal Xist RNA processing is important in the regulation of random X-inactivation. We propose that modulation of Xist RNA processing may be part of the stochastic process that determines which X chromosome will be inactivated.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: XΔAX cells undergo primary nonrandom X-inactivation.
Figure 2: XΔAX cells do not show allelic enrichment for histone modifications.
Figure 3: Reduced levels of correctly spliced Xist transcripts from the ΔA mutant chromosome.
Figure 4: The A-repeat binds ASF/SF2.
Figure 5: Binding of ASF/SF2 to the A-repeat depends on ASF/SF2 consensus sequences and not predicted secondary structure.
Figure 6: An RNA-based model for randomness and mutual exclusivity.

Similar content being viewed by others

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. Martin, G.R. et al. X-chromosome inactivation during differentiation of female teratocarcinoma stem cells in vitro. Nature 271, 329–333 (1978).

    Article  CAS  Google Scholar 

  3. Avner, P. & Heard, E. X-chromosome inactivation: counting, choice and initiation. Nat. Rev. Genet. 2, 59–67 (2001).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Gribnau, J., Luikenhuis, S., Hochedlinger, K., Monkhorst, K. & Jaenisch, R. X chromosome choice occurs independently of asynchronous replication timing. J. Cell Biol. 168, 365–373 (2005).

    Article  CAS  Google Scholar 

  6. 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 

  7. Luikenhuis, S., Wutz, A. & Jaenisch, R. Antisense transcription through the Xist locus mediates Tsix function in embryonic stem cells. Mol. Cell. Biol. 21, 8512–8520 (2001).

    Article  CAS  Google Scholar 

  8. Wutz, A. & Jaenisch, R. A shift from reversible to irreversible X inactivation is triggered during ES cell differentiation. Mol. Cell 5, 695–705 (2000).

    Article  CAS  Google Scholar 

  9. 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 

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

    Article  CAS  Google Scholar 

  11. 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 

  12. Hoki, Y. et al. A proximal conserved repeat in the Xist gene is essential as a genomic element for X-inactivation in mouse. Development 136, 139–146 (2009).

    Article  CAS  Google Scholar 

  13. Cattanach, B.M. & Rasberry, C. Identification of the Mus castaneus Xce allele. Mouse Genome 92, 114–115 (1994).

    Google Scholar 

  14. Panning, B., Dausman, J. & Jaenisch, R. X chromosome inactivation is mediated by Xist RNA stabilization. Cell 90, 907–916 (1997).

    Article  CAS  Google Scholar 

  15. Mlynarczyk-Evans, S. et al. X chromosomes alternate between two states prior to random X-inactivation. PLoS Biol. 4, e159 (2006).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. 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 

  18. Norris, D.P. et al. Evidence that random and imprinted Xist expression is controlled by preemptive methylation. Cell 77, 41–51 (1994).

    Article  CAS  Google Scholar 

  19. 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 

  20. 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 

  21. Perocchi, F., Xu, Z., Clauder-Munster, S. & Steinmetz, L.M. Antisense artifacts in transcriptome microarray experiments are resolved by actinomycin D. Nucleic Acids Res. 35, e128 (2007).

    Article  Google Scholar 

  22. Lin, S., Xiao, R., Sun, P., Xu, X. & Fu, X.D. Dephosphorylation-dependent sorting of SR splicing factors during mRNP maturation. Mol. Cell 20, 413–425 (2005).

    Article  CAS  Google Scholar 

  23. Fazzio, T. & Panning, B. Condensin complexes regulate mitotic progression and interphase chromatin structure in embryonic stem cells. J. Cell Biol. 188, 491–503 (2010).

    Article  CAS  Google Scholar 

  24. Mathews, D.H., Sabina, J., Zuker, M. & Turner, D.H. Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J. Mol. Biol. 288, 911–940 (1999).

    Article  CAS  Google Scholar 

  25. Zuker, M. & Jacobson, A.B. “Well-determined” regions in RNA secondary structure prediction: analysis of small subunit ribosomal RNA. Nucleic Acids Res. 23, 2791–2798 (1995).

    Article  CAS  Google Scholar 

  26. Cartegni, L., Wang, J., Zhu, Z., Zhang, M.Q. & Krainer, A.R. ESEfinder: A web resource to identify exonic splicing enhancers. Nucleic Acids Res. 31, 3568–3571 (2003).

    Article  CAS  Google Scholar 

  27. Zhao, J., Sun, B.K., Erwin, J.A., Song, J.J. & Lee, J.T. Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome. Science 322, 750–756 (2008).

    Article  CAS  Google Scholar 

  28. Maenner, S. et al. 2-D structure of the A region of Xist RNA and its implication for PRC2 association. PLoS Biol. 8, e1000276 (2010).

    Article  Google Scholar 

  29. 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 

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

    Article  CAS  Google Scholar 

  31. Csankovszki, G., Panning, B., Bates, B., Pehrson, J.R. & Jaenisch, R. Conditional deletion of Xist disrupts histone macroH2A localization but not maintenance of X inactivation. Nat. Genet. 22, 323–324 (1999).

    Article  CAS  Google Scholar 

  32. Heard, E. et al. Methylation of histone H3 at Lys-9 is an early mark on the X chromosome during X inactivation. Cell 107, 727–738 (2001).

    Article  CAS  Google Scholar 

  33. Rougeulle, C. et al. Differential histone H3 Lys-9 and Lys-27 methylation profiles on the X chromosome. Mol. Cell. Biol. 24, 5475–5484 (2004).

    Article  CAS  Google Scholar 

  34. Cartegni, L., Chew, S.L. & Krainer, A.R. Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat. Rev. Genet. 3, 285–298 (2002).

    Article  CAS  Google Scholar 

  35. Herzing, L.B., Romer, J.T., Horn, J.M. & Ashworth, A. Xist has properties of the X-chromosome inactivation centre. Nature [see comments] 386, 272–275 (1997).

    Article  CAS  Google Scholar 

  36. Lee, J.T., Lu, N. & Han, Y. Genetic analysis of the mouse X inactivation center defines an 80-kb multifunction domain. Proc. Natl. Acad. Sci. USA 96, 3836–3841 (1999).

    Article  CAS  Google Scholar 

  37. Monkhorst, K., Jonkers, I., Rentmeester, E., Grosveld, F. & Gribnau, J. X inactivation counting and choice is a stochastic process: evidence for involvement of an X-linked activator. Cell 132, 410–421 (2008).

    Article  CAS  Google Scholar 

  38. Nesterova, T.B. et al. Skewing X chromosome choice by modulating sense transcription across the Xist locus. Genes Dev. 17, 2177–2190 (2003).

    Article  CAS  Google Scholar 

  39. Nesterova, T.B. et al. Dicer regulates Xist promoter methylation in ES cells indirectly through transcriptional control of Dnmt3a. Epigenetics Chromatin 1, 2 (2008).

    Article  Google Scholar 

  40. Sado, T., Hoki, Y. & Sasaki, H. Tsix defective in splicing is competent to establish Xist silencing. Development 133, 4925–4931 (2006).

    Article  CAS  Google Scholar 

  41. Shibata, S. & Lee, J.T. Characterization and quantitation of differential Tsix transcripts: implications for Tsix function. Hum. Mol. Genet. 12, 125–136 (2003).

    Article  CAS  Google Scholar 

  42. Wutz, A. & Gribnau, J. X inactivation Xplained. Curr. Opin. Genet. Dev. 17, 387–393 (2007).

    Article  CAS  Google Scholar 

  43. 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 

  44. Blewitt, M.E. et al. SmcHD1, containing a structural-maintenance-of-chromosomes hinge domain, has a critical role in X inactivation. Nat. Genet. 40, 663–669 (2008).

    Article  CAS  Google Scholar 

  45. Fazzio, T.G., Huff, J.T. & Panning, B. An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity. Cell 134, 162–174 (2008).

    Article  CAS  Google Scholar 

  46. Cohen, H.R. & Panning, B. XIST RNA exhibits nuclear retention and exhibits reduced association with the export factor TAP/NXF1. Chromosoma 116, 373–383 (2007).

    Article  CAS  Google Scholar 

  47. Plath, K. et al. Role of histone H3 lysine 27 methylation in X inactivation. Science 300, 131–135 (2003).

    Article  CAS  Google Scholar 

  48. Huang, Y., Yario, T.A. & Steitz, J.A. A molecular link between SR protein dephosphorylation and mRNA export. Proc. Natl. Acad. Sci. USA 101, 9666–9670 (2004).

    Article  CAS  Google Scholar 

  49. Nusinow, D.A. et al. The histone domain of macroH2A1 contains several dispersed elements that are each sufficient to direct enrichment on the inactive X chromosome. J. Mol. Biol. 371, 11–18 (2007).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank K. Worringer, J. Huff, T. Fazzio, M.K. Alexander, L. Spector, P. O'Farrell and I. Listerman for critical reading of the manuscript, X.-D. Fu and S. Lin (University of California, San Diego) for tet-repressible ASF/SF2 cells, N. Krogan for mass spectrometry and A. Krainer (Cold Spring Harbor) for ASF/SF2 antibodies. This work was funded in part by US National Institutes of Health grant R01 GM088506.

Author information

Author notes

  1. Morgan E Royce-Tolland and Angela A Andersen: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, University of California, San Francisco, California, USA

    Morgan E Royce-Tolland, Hannah R Koyfman, Dale J Talbot & Barbara Panning

  2. Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah, USA

    Angela A Andersen

  3. The Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, UK

    Anton Wutz

  4. Queensland Institute of Medical Research, Brisbane, Australia

    Ian D Tonks & Graham F Kay

Authors
  1. Morgan E Royce-Tolland
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Angela A Andersen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hannah R Koyfman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dale J Talbot
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Anton Wutz
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ian D Tonks
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Graham F Kay
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Barbara Panning
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.E.R.-T., A.A.A., H.R.K., D.J.T. and B.P. designed and performed experiments and wrote the manuscript, and A.W., I.D.T. and G.F.K. provided cell lines.

Corresponding author

Correspondence to Barbara Panning.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1-5 and Supplementary Table 1 (PDF 1421 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Royce-Tolland, M., Andersen, A., Koyfman, H. et al. The A-repeat links ASF/SF2-dependent Xist RNA processing with random choice during X inactivation. Nat Struct Mol Biol 17, 948–954 (2010). https://doi.org/10.1038/nsmb.1877

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1877

This article is cited by

Search

Advanced 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