, Volume 118, Issue 2, pp 209–222 | Cite as

ATRX marks the inactive X chromosome (Xi) in somatic cells and during imprinted X chromosome inactivation in trophoblast stem cells

  • Claudia Baumann
  • Rabindranath De La FuenteEmail author
Research Article


Mammalian X chromosome inactivation (XCI) is an essential mechanism to compensate for dosage imbalances between male and female embryos. Although the molecular pathways are not fully understood, heterochromatinization of the Xi requires the coordinate recruitment of multiple epigenetic marks. Using fluorescence in situ hybridization analysis combined with immunocytochemistry, we demonstrate that the chromatin remodeling protein ATRX decorates the chromatids of a single, late replicating X chromosome in female somatic cells and co-localizes with the bona fide marker of the Xi, macroH2A1.2. Chromatin immunoprecipitation using somatic, embryonic stem (ES) cells and trophoblast stem (TS) cells as model for random and imprinted XCI, respectively, revealed that, in somatic and TS cells, ATRX exhibits a specific association with sequences located within the previously described H3K9me2-hotspot, a region 5′ to the X inactive-specific transcript (Xist) locus. While no ATRX-Xi interaction was detectable in undifferentiated ES cells, an enrichment of ATRX was observed after 8 days of differentiation, indicating that ATRX associates with the Xi following the onset of random XCI, consistent with a potential role in maintenance of XCI. These results have important implications regarding a previously described escape from imprinted XCI in ATRX-deficient mice as well as cases of skewed XCI in patients with ATRX syndrome.


Chromosome Inactivation Embryonic Stem Cell Differentiation Extraembryonic Tissue Bold Arrow Trophoblast Giant Cell 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We would like to thank Drs. J. Rossant and N. Brockdorff for kindly providing the TS cell line and the PGK12.1 ES cell line, respectively. We are grateful to Drs. D. Higgs, D. Garrick and J. Pehrson for generous gifts of antibodies and to Drs. M. M. Viveiros and F. Yang for helpful discussions and comments during manuscript preparation. This research was supported by a grant from the National Institute of Child Health and Human Development (NICHD) National Institutes of Health (HD042740) to R. De La Fuente.

Supplementary material

412_2008_189_MOESM1_ESM.jpg (633 kb)
Supplemental Figure S1. a Chromatin immunoprecipitation (ChIP) analysis of X chromosome-specific sequences in male MEFs using specific antibodies against ATRX, H3K4me2 (upstate) and H3K9me3 (abcam) with an anti-IgG antibody against as control. No significant enrichment of DNA sequences encoding the constitutive H3K9me2-hotspot were detected in samples immunoprecipitated with anti-ATRX antibodies compared to the IgG control. Error bars represent the STD of three independent experiments, and different superscripts indicate significant differences (P < 0.05). A representative gel image is shown. b Summary of ChIP analysis data from undifferentiated TS cells, differentiating ES cells and female MEF’s. Individual data points represent IgG-corrected immunoprecipitation values expressed as percentages of the input value. STD indicates variability between experimental replicates (DOC 632 KB)
412_2008_189_MOESM2_ESM.jpg (2.1 mb)
Supplemental Figure S2. a Trophoblast stem cell colonies are characterized by an epithelial sheet-like morphology; here growing on a feeder layer of male MEFs (magnification ×100, Image Pro Plus, Nikon Eclipse TE300 imaging system). The multipotent state of TS cells is marked by a transient accumulation of the histone modification ubH2A at the inactive X chromosome (green, arrow). b Nonbiased co-localization analysis using CoLocalizer Pro software to calculate the overlap coefficient according to Manders (R), which is specifically designed to determine the degree of overlap between signals obtained from differing fluorescent channels (Zinchuk and Zinchuk 2008). Values indicating true co-localization range from 0.6 to 1.0, while values between 0 and 0.6 indicate absence of co-localization. The (R) coefficient indicated for the territory of the inactive X chromosome in an interphase granulosa cell (R Xi = 0.7253, upper panel) demonstrates co-localization between ATRX protein and the inactive X chromosome. The co-localization coefficient for the active X chromosome (R Xa = 0.3115, arrowhead) implies lack of co-localization. Similarily, the (R) value for ubiquitinated H2A (green) in trophoblast stem cells during interphase (R Xi = 0.8642, lower panel) reveals co-localization with ATRX protein at the Xi. Scale bars = 10 μm. c About 10% (n = 164) of metaphase spreads from embryonic stem cells present ATRX localization (red) to the chromatids (bold arrow) in all autosomes and X chromosomes (green) as well as a lack of staining at pericentromeric domains (thin arrow, inset). d Timely onset of differentiation in ES cell cultures was monitored by indirect immunofluorescence using an antibody against ubH2A (green) with signals predominantly detectable at inactive X chromosomes in cultures differentiated for 4 days. No significant accumulation at the Xi was observed at earlier or later time points (d0 and d8), which is in accordance with previous reports demonstrating the transient nature of associations between ubH2A and the Xi in ES cells during early differentiation stages (de Napoles et al. 2004; Fang et al. 2004) (DOC 2.14 MB)


  1. Abbondanzo S, Gadi I, Stewart C (1993) Derivation of embryonic stem cell lines. In Methods Enzymology, vol. 225. Academic Press, San Diego, pp 803-823Google Scholar
  2. Bannister AJ, Zegerman P, Partridge JF, Miska EA, Thomas JO, Allshire RC, Kouzarides T (2001) Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature 410:120–124PubMedCrossRefGoogle Scholar
  3. Baumann C, Schmidtmann A, Muegge K, De La Fuente R (2008) Association of ATRX with pericentric heterochromatin and the Y chromosome of neonatal mouse spermatogonia. BMC Mol Biol 9:29Google Scholar
  4. Berube NG, Smeenk CA, Picketts DJ (2000) Cell cycle-dependent phosphorylation of the ATRX protein correlates with changes in nuclear matrix and chromatin association. Hum Mol Genet 9:539–547PubMedCrossRefGoogle Scholar
  5. Boggs BA, Cheung P, Heard E, Spector DL, Chinault AC, Allis CD (2002) Differentially methylated forms of histone H3 show unique association patterns with inactive human X chromosomes. Nat Genet 30:73–76PubMedCrossRefGoogle Scholar
  6. Brockdorff N (2002) X-chromosome inactivation: closing in on proteins that bind Xist RNA. Trends in Genetics 18:352–358PubMedCrossRefGoogle Scholar
  7. Brown C, Willard H (1994) The human X-inactivation centre is not required for maintenance of X-chromosome inactivation. Nature 368:154–156PubMedCrossRefGoogle Scholar
  8. Cardoso C, Timsit S, Villard L, Khrestchatisky M, Fontes M, Colleaux L (1998) Specific interaction between the XNP/ATR-X gene product and the SET domain of the human EZH2 protein. Hum Mol Genet 7:679–684PubMedCrossRefGoogle Scholar
  9. Chadwick BP, Willard HF (2003) Chromatin of the Barr body: histone and non-histone proteins associated with or excluded from the inactive X chromosome. Hum Mol Genet 12:2167–2178PubMedCrossRefGoogle Scholar
  10. Chaumeil J, Okamoto I, Guggiari M, Heard E (2002) Integrated kinetics of X chromosome inactivation in differentiating embryonic stem cells. Cytogenet Genome Res 99:75–84PubMedCrossRefGoogle Scholar
  11. Chow J, Brown C (2003) Forming facultative heterochromatin: silencing of an X chromosome in mammalian females. Cell Mol Life Sci 60:2586–2603PubMedCrossRefGoogle Scholar
  12. Clemson C, McNeil J, Willard H, Lawrence J (1996) XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure. J Cell Biol 132:259–275PubMedCrossRefGoogle Scholar
  13. Costanzi C, Pehrson JR (1998) Histone macroH2A1 is concentrated in the inactive X chromosome of female mammals. Nature 393:599–601PubMedCrossRefGoogle Scholar
  14. Costanzi C, Stein P, Worrad DM, Schultz RM, Pehrson JR (2000) Histone macroH2A1 is concentrated in the inactive X chromosome of female preimplantation mouse embryos. Development 127:2283–2289PubMedGoogle Scholar
  15. Csankovszki G, Panning B, Bates B, Pehrson J, Jaenisch R (1999) Conditional deletion of Xist disrupts histone macroH2A localization but not maintenance of X inactivation. Nat Genet 22:323–324PubMedCrossRefGoogle Scholar
  16. Csankovszki G, Nagy A, Jaenisch R (2001) Synergism of Xist RNA, DNA methylation, and histone hypoacetylation in maintaining X chromosome inactivation. J Cell Biol 153:773–784PubMedCrossRefGoogle Scholar
  17. Czermin B, Melfi R, McCabe D, Seitz V, Imhof A, Pirrotta V (2002) Drosophila enhancer of Zeste/ESC complexes have a histone H3 methyltransferase activity that marks chromosomal Polycomb sites. Cell 111:185–196PubMedCrossRefGoogle Scholar
  18. De La Fuente R, Viveiros M, Wigglesworth K, Jj E (2004) ATRX, a Member of the SNF2 Family of Helicase /ATPases, is required for chromosome alignment and meiotic spindle organization in metaphase II stage mouse oocytes. Developmental Biology 272:1–14PubMedCrossRefGoogle Scholar
  19. de Napoles M, Mermoud JE, Wakao R, Tang YA, Endo HM, Appanah R, Nesterova TB, Silva J, Otte AP, Vidal M et al (2004) Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X inactivation. Dev Cell 7:663–676PubMedCrossRefGoogle Scholar
  20. Drouin R, Lemieux N, Richer C (1990) Analysis of DNA replication during S-phase by means of dynamic chromosome banding at high resolution. Chromosoma 99:273–280PubMedCrossRefGoogle Scholar
  21. Fang J, Chen T, Chadwick BP, Li E, Zhang Y (2004) Ring1b-mediated H2A ubiquitination associates with inactive X chromosomes and is involved in initiation of X inactivation. J Biol Chem 279:52812–52815PubMedCrossRefGoogle Scholar
  22. Fischle W, Wang Y, Jacobs SA, Kim Y, Allis CD, Khorasanizadeh S (2003) Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 by Polycomb and HP1 chromodomains. Genes Dev 17:1870–1881PubMedCrossRefGoogle Scholar
  23. Garrick D, Sharpe JA, Arkell R, Dobbie L, Smith AJH, Wood WG, Higgs DR, Gibbons RJ (2006) Loss of atrx affects trophoblast development and the pattern of x-inactivation in extraembryonic tissues. PLoS Genetics 2:438–450CrossRefGoogle Scholar
  24. Gartler SM, Varadarajan KR, Luo P, Norwood TH, Canfield TK, Hansen RS (2006) Abnormal X: autosome ratio, but normal X chromosome inactivation in human triploid cultures. BMC Genet 7Google Scholar
  25. Gibbons RJ, Picketts DJ, Villard L, Higgs DR (1995) Mutations in a putative global transcriptional regulator cause X-linked mental retardation with alpha-thalassemia (ATR-X syndrome). Cell 80:837–845PubMedCrossRefGoogle Scholar
  26. Gibbons RJ, Bachoo S, Picketts DJ, Aftimos S, Asenbauer B, Bergoffen J, Berry SA, Dahl N, Fryer A, Keppler K et al (1997) Mutations in the transcriptional regulator ATRX establish the functional significance of a PHD-like domain. Nat Genet 17:146–148PubMedCrossRefGoogle Scholar
  27. Gibbons RJ, McDowell TL, Raman S, O’Rourke DM, Garrick D, Ayyub H, Higgs DR (2000) Mutations in ATRX, encoding a SWI/SNF-like protein, cause diverse changes in the pattern of DNA methylation. Nat Genet 24:368–371PubMedCrossRefGoogle Scholar
  28. Gilbert DM (2002) Replication timing and transcriptional control: beyond cause and effect. Curr Opin Cell Biol 14:377PubMedCrossRefGoogle Scholar
  29. Gilbert SL, Sharp PA (1999) Promoter-specific hypoacetylation of X-inactivated genes. PNAS 96:13825–13830PubMedCrossRefGoogle Scholar
  30. Gilbert C, Muldal S, Lajtha L, Rowley J (1962) Time-sequence of human chromosome duplication. Nature 195:869–875PubMedCrossRefGoogle Scholar
  31. Heard E, Disteche CM (2006) Dosage compensation in mammals: fine-tuning the expression of the X chromosome. Genes Dev 20:1848–1867PubMedCrossRefGoogle Scholar
  32. Heard E, Rougeulle C, Arnaud D, Avner P, Allis CD, Spector DL (2001) Methylation of Histone H3 at Lys-9 Is an Early Mark on the X Chromosome during X Inactivation. Cell 107:727–738PubMedCrossRefGoogle Scholar
  33. Hemberger M (2007) Epigenetic landscape required for placental development. Cell Mol Life Sci 64:2422–2436PubMedCrossRefGoogle Scholar
  34. Huynh KD, Lee JT (2005) X-chromosome inactivation: a hypothesis linking ontogeny and phylogeny. Nat Rev Genet 6:410–408PubMedCrossRefGoogle Scholar
  35. Kalantry S, Magnuson T (2006) The Polycomb group protein EED is dispensable for the initiation of random X-chromosome inactivation. PLoS Genet 2:e66PubMedCrossRefGoogle Scholar
  36. Kalantry S, Mills KC, Yee D, Otte AP, Panning B, Magnuson T (2006) The Polycomb group protein Eed protects the inactive X-chromosome from differentiation-induced reactivation. Nat Cell Biol 8:195–202PubMedCrossRefGoogle Scholar
  37. Kieran R, Seah C, Moulin J, Isaac C, Dick F, Bérubé NG (2008) Loss of ATRX leads to chromosome cohesion and congression defects. J Cell Biol 180:315–324CrossRefGoogle Scholar
  38. Kohlmaier A, Savarese F, Lachner M, Martens J, Jenuwein T, Wutz A (2004) A chromosomal memory triggered by Xist regulates histone methylation in X inactivation. PLoS Biol 2:E171PubMedCrossRefGoogle Scholar
  39. Kourmouli N, Sun Y-M, van der Sar S, Singh PB, Brown JP (2005) Epigenetic regulation of mammalian pericentric heterochromatin in vivo by HP1. Biochem Biophys Res Commun 337:901–907PubMedGoogle Scholar
  40. Lachner M, O’Carroll D, Rea S, Mechtler K, Jenuwein T (2001) Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 410:116–120PubMedCrossRefGoogle Scholar
  41. Le Douarin B, Nielsen A, Garnier J, Ichinose H, Jeanmougin F, Losson R, Chambon P (1996) A possible involvement of TIF1a and TIF1b in the epigenetic control of transcription by nuclear receptors. EMBO J 15:6701–6715PubMedGoogle Scholar
  42. Lyon MF (1961) Gene action in the X-chromosome of the mouse (Mus musculus L.). Nature 190:372–373PubMedCrossRefGoogle Scholar
  43. Mak W, Baxter J, Silva J, Newall AE, Otte AP, Brockdorff N (2002) Mitotically stable association of polycomb group proteins eed and enx1 with the inactive x chromosome in trophoblast stem cells. Curr Biol 12:1016–1020PubMedCrossRefGoogle Scholar
  44. Mak W, Nesterova T, de Napoles M, Appanah R, Yamanaka S, Otte A, Brockdorff N (2004) Reactivation of the paternal X chromosome in early mouse embryos. Science 303:666–669PubMedCrossRefGoogle Scholar
  45. Masui O, Heard E (2006) RNA and protein actors in X-chromosome inactivation. Cold Spring Harbor Symp Quant Biol 71:419–428PubMedCrossRefGoogle Scholar
  46. McDowell TL, Gibbons RJ, Sutherland H, O’Rourke DM, Bickmore WA, Pombo A, Turley H, Gatter K, Picketts DJ, Buckle VJ et al (1999) Localization of a putative transcriptional regulator (ATRX) at pericentromeric heterochromatin and the short arms of acrocentric chromosomes. Proc Natl Acad Sci USA 96:13983–13988PubMedCrossRefGoogle Scholar
  47. Mermoud JE, Costanzi C, Pehrson JR, Brockdorff N (1999) Histone macroH2A1.2 relocates to the inactive X chromosome after initiation and propagation of X-inactivation. J Cell Biol 147:1399–1408PubMedCrossRefGoogle Scholar
  48. Mermoud JE, Popova B, Peters AH, Jenuwein T, Brockdorff N (2002) Histone H3 lysine 9 methylation occurs rapidly at the onset of random X chromosome inactivation. Curr Biol 12:247–251PubMedCrossRefGoogle Scholar
  49. Ng K, Pullirsch D, Leeb M, Wutz A (2007) Xist and the order of silencing. EMBO Rep 8:34–39PubMedCrossRefGoogle Scholar
  50. Nusinow DA, Hernández-Muñoz I, Fazzio TG, Shah GM, Kraus WL, Panning B (2007) Poly(ADP-ribose) polymerase 1 is inhibited by a histone H2A variant, MacroH2A, and contributes to silencing of the inactive X chromosome. J Biol Chem 282:12851–12859PubMedCrossRefGoogle Scholar
  51. Okamoto I, Otte AP, Allis CD, Reinberg D, Heard E (2004) Epigenetic dynamics of imprinted X inactivation during early mouse development. Science 303:644–649PubMedCrossRefGoogle Scholar
  52. Okamoto I, Arnaud D, Le Baccon P, Otte AP, Disteche CM, Avner P, Heard E (2005) Evidence for de novo imprinted X-chromosome inactivation independent of meiotic inactivation in mice. Nature 438:369–373PubMedCrossRefGoogle Scholar
  53. Penny GD, Kay GF, Sheardown SA, Rastan S, Brockdorff N (1996) Requirement for Xist in X chromosome inactivation. Nature 379:131–137PubMedCrossRefGoogle Scholar
  54. Peters AH, Mermoud JE, O’Carroll D, Pagani M, Schweizer D, Brockdorff N, Jenuwein T (2002) Histone H3 lysine 9 methylation is an epigenetic imprint of facultative heterochromatin. Nat Genet 30:77–80PubMedCrossRefGoogle Scholar
  55. Plath K, Fang J, Mlynarczyk-Evans SK, Cao R, Worringer KA, Wang H, de la Cruz CC, Otte AP, Panning B, Zhang Y (2003) Role of Histone H3 lysine 27 methylation in X inactivation. Science 300:313–135CrossRefGoogle Scholar
  56. Plath K, Talbot D, Hamer KM, Otte AP, Yang TP, Jaenisch R, Panning B (2004) Developmentally regulated alterations in Polycomb repressive complex 1 proteins on the inactive X chromosome. J Cell Biol 167:1025–1035PubMedCrossRefGoogle Scholar
  57. Rougeulle C, Chaumeil J, Sarma K, Allis CD, Reinberg D, Avner P, Heard E (2004) Differential histone H3 Lys-9 and Lys-27 methylation profiles on the X chromosome. Mol Cell Biol 24:5475–5484PubMedCrossRefGoogle Scholar
  58. Silva J, Mak W, Zvetkova I, Appanah R, Nesterova TB, Webster Z, Peters AH, Jenuwein T, Otte AP, Brockdorff N (2003) Establishment of histone h3 methylation on the inactive X chromosome requires transient recruitment of Eed-Enx1 polycomb group complexes. Dev Cell 4:481–495PubMedCrossRefGoogle Scholar
  59. Sugawara O, Takagi N, Sasaki M (1985) Correlation between X-chromosome inactivation and cell differentiation in female preimplantation mouse embryos. Cytogenet Cell Genet 39:210–219PubMedCrossRefGoogle Scholar
  60. Takagi N, Sasaki M (1975) Preferential inactivation of the paternally derived X chromosome in the extraembryonic membranes of the mouse. Nature 256:640–642PubMedCrossRefGoogle Scholar
  61. Takagi N, Sugawara O, Sasaki M (1982) Regional and temporal changes in the pattern of X-chromosome replication during the early post-implantation development of the female mouse. Chromosoma 85:275–286PubMedCrossRefGoogle Scholar
  62. Tanaka S, Kunath T, Hadjantonakis A, Nagy A, Rossant J (1998) Promotion of trophoblast stem cell proliferation by FGF4. Science 282:2072–2075PubMedCrossRefGoogle Scholar
  63. Wang J, Mager J, Chen Y, Schneider E, Cross JC, Nagy A, Magnuson T (2001) Imprinted X inactivation maintained by a mouse polycomb group gene. Nat Genet 28:371–375PubMedCrossRefGoogle Scholar
  64. Wutz A (2007) Xist function: bridging chromatin and stem cells. Trends Genet 23:457–464PubMedCrossRefGoogle Scholar
  65. Wutz A, Jaenisch R (2000) A shift from reversible to irreversible X inactivation is triggered during ES cell differentiation. Mol Cell Biol 5:695–705Google Scholar
  66. Zhang J, Xu F, Hashimshony T, Keshet I, Cedar H (2002) Establishment of transcriptional competence in early and late S phase. Nature 420:198–202PubMedCrossRefGoogle Scholar
  67. Zinchuk V, Zinchuk O (2008) Quantitative colocalization analysis of confocal fluorescence microscopy images. Curr Protoc Cell Biol Chapter 4Google Scholar
  68. Zvetkova I, Apedaile A, Ramsahoye B, Mermoud JE, Crompton LA, John R, Feil R, Brockdorff N (2005) Global hypomethylation of the genome in XX embryonic stem cells. Nat Genet 37:1274–1279PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  1. 1.Female Germ Cell Biology Group, Department of Clinical Studies, Center for Animal Transgenesis and Germ Cell Research, School of Veterinary MedicineUniversity of PennsylvaniaKennett SquareUSA

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