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

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Abstract

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.

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References

  • Abbondanzo S, Gadi I, Stewart C (1993) Derivation of embryonic stem cell lines. In Methods Enzymology, vol. 225. Academic Press, San Diego, pp 803-823

  • 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 410:120–124

    Article  PubMed  CAS  Google Scholar 

  • 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 9:29

    Google Scholar 

  • 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 9:539–547

    Article  PubMed  CAS  Google Scholar 

  • 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 30:73–76

    Article  PubMed  CAS  Google Scholar 

  • Brockdorff N (2002) X-chromosome inactivation: closing in on proteins that bind Xist RNA. Trends in Genetics 18:352–358

    Article  PubMed  CAS  Google Scholar 

  • Brown C, Willard H (1994) The human X-inactivation centre is not required for maintenance of X-chromosome inactivation. Nature 368:154–156

    Article  PubMed  CAS  Google Scholar 

  • 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 7:679–684

    Article  PubMed  CAS  Google Scholar 

  • 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 12:2167–2178

    Article  PubMed  CAS  Google Scholar 

  • Chaumeil J, Okamoto I, Guggiari M, Heard E (2002) Integrated kinetics of X chromosome inactivation in differentiating embryonic stem cells. Cytogenet Genome Res 99:75–84

    Article  PubMed  CAS  Google Scholar 

  • Chow J, Brown C (2003) Forming facultative heterochromatin: silencing of an X chromosome in mammalian females. Cell Mol Life Sci 60:2586–2603

    Article  PubMed  CAS  Google Scholar 

  • 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 132:259–275

    Article  PubMed  CAS  Google Scholar 

  • Costanzi C, Pehrson JR (1998) Histone macroH2A1 is concentrated in the inactive X chromosome of female mammals. Nature 393:599–601

    Article  PubMed  CAS  Google Scholar 

  • 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 127:2283–2289

    PubMed  CAS  Google Scholar 

  • 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 22:323–324

    Article  PubMed  CAS  Google Scholar 

  • Csankovszki G, Nagy A, Jaenisch R (2001) Synergism of Xist RNA, DNA methylation, and histone hypoacetylation in maintaining X chromosome inactivation. J Cell Biol 153:773–784

    Article  PubMed  CAS  Google Scholar 

  • 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 111:185–196

    Article  PubMed  CAS  Google Scholar 

  • 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 272:1–14

    Article  PubMed  Google Scholar 

  • 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 7:663–676

    Article  PubMed  Google Scholar 

  • Drouin R, Lemieux N, Richer C (1990) Analysis of DNA replication during S-phase by means of dynamic chromosome banding at high resolution. Chromosoma 99:273–280

    Article  PubMed  CAS  Google Scholar 

  • 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 279:52812–52815

    Article  PubMed  CAS  Google Scholar 

  • 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 17:1870–1881

    Article  PubMed  CAS  Google Scholar 

  • 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 2:438–450

    Article  CAS  Google Scholar 

  • 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 7

  • 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 80:837–845

    Article  PubMed  CAS  Google Scholar 

  • 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 17:146–148

    Article  PubMed  CAS  Google Scholar 

  • 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 24:368–371

    Article  PubMed  CAS  Google Scholar 

  • Gilbert DM (2002) Replication timing and transcriptional control: beyond cause and effect. Curr Opin Cell Biol 14:377

    Article  PubMed  CAS  Google Scholar 

  • Gilbert SL, Sharp PA (1999) Promoter-specific hypoacetylation of X-inactivated genes. PNAS 96:13825–13830

    Article  PubMed  CAS  Google Scholar 

  • Gilbert C, Muldal S, Lajtha L, Rowley J (1962) Time-sequence of human chromosome duplication. Nature 195:869–875

    Article  PubMed  CAS  Google Scholar 

  • Heard E, Disteche CM (2006) Dosage compensation in mammals: fine-tuning the expression of the X chromosome. Genes Dev 20:1848–1867

    Article  PubMed  CAS  Google Scholar 

  • 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 107:727–738

    Article  PubMed  CAS  Google Scholar 

  • Hemberger M (2007) Epigenetic landscape required for placental development. Cell Mol Life Sci 64:2422–2436

    Article  PubMed  CAS  Google Scholar 

  • Huynh KD, Lee JT (2005) X-chromosome inactivation: a hypothesis linking ontogeny and phylogeny. Nat Rev Genet 6:410–408

    Article  PubMed  CAS  Google Scholar 

  • Kalantry S, Magnuson T (2006) The Polycomb group protein EED is dispensable for the initiation of random X-chromosome inactivation. PLoS Genet 2:e66

    Article  PubMed  Google Scholar 

  • 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 8:195–202

    Article  PubMed  CAS  Google Scholar 

  • 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 180:315–324

    Article  Google Scholar 

  • 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:E171

    Article  PubMed  Google Scholar 

  • 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 337:901–907

    PubMed  CAS  Google Scholar 

  • 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 410:116–120

    Article  PubMed  CAS  Google Scholar 

  • 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 15:6701–6715

    PubMed  Google Scholar 

  • Lyon MF (1961) Gene action in the X-chromosome of the mouse (Mus musculus L.). Nature 190:372–373

    Article  PubMed  CAS  Google Scholar 

  • 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 12:1016–1020

    Article  PubMed  CAS  Google Scholar 

  • 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 303:666–669

    Article  PubMed  CAS  Google Scholar 

  • Masui O, Heard E (2006) RNA and protein actors in X-chromosome inactivation. Cold Spring Harbor Symp Quant Biol 71:419–428

    Article  PubMed  CAS  Google Scholar 

  • 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 96:13983–13988

    Article  PubMed  CAS  Google Scholar 

  • 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 147:1399–1408

    Article  PubMed  CAS  Google Scholar 

  • 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 12:247–251

    Article  PubMed  CAS  Google Scholar 

  • Ng K, Pullirsch D, Leeb M, Wutz A (2007) Xist and the order of silencing. EMBO Rep 8:34–39

    Article  PubMed  CAS  Google Scholar 

  • 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 282:12851–12859

    Article  PubMed  CAS  Google Scholar 

  • Okamoto I, Otte AP, Allis CD, Reinberg D, Heard E (2004) Epigenetic dynamics of imprinted X inactivation during early mouse development. Science 303:644–649

    Article  PubMed  CAS  Google Scholar 

  • 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 438:369–373

    Article  PubMed  CAS  Google Scholar 

  • Penny GD, Kay GF, Sheardown SA, Rastan S, Brockdorff N (1996) Requirement for Xist in X chromosome inactivation. Nature 379:131–137

    Article  PubMed  CAS  Google Scholar 

  • 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 30:77–80

    Article  PubMed  CAS  Google Scholar 

  • 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 300:313–135

    Article  Google Scholar 

  • 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 167:1025–1035

    Article  PubMed  CAS  Google Scholar 

  • 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 24:5475–5484

    Article  PubMed  CAS  Google Scholar 

  • 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 4:481–495

    Article  PubMed  CAS  Google Scholar 

  • Sugawara O, Takagi N, Sasaki M (1985) Correlation between X-chromosome inactivation and cell differentiation in female preimplantation mouse embryos. Cytogenet Cell Genet 39:210–219

    Article  PubMed  CAS  Google Scholar 

  • Takagi N, Sasaki M (1975) Preferential inactivation of the paternally derived X chromosome in the extraembryonic membranes of the mouse. Nature 256:640–642

    Article  PubMed  CAS  Google Scholar 

  • 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 85:275–286

    Article  PubMed  CAS  Google Scholar 

  • Tanaka S, Kunath T, Hadjantonakis A, Nagy A, Rossant J (1998) Promotion of trophoblast stem cell proliferation by FGF4. Science 282:2072–2075

    Article  PubMed  CAS  Google Scholar 

  • 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 28:371–375

    Article  PubMed  CAS  Google Scholar 

  • Wutz A (2007) Xist function: bridging chromatin and stem cells. Trends Genet 23:457–464

    Article  PubMed  CAS  Google Scholar 

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

    CAS  Google Scholar 

  • Zhang J, Xu F, Hashimshony T, Keshet I, Cedar H (2002) Establishment of transcriptional competence in early and late S phase. Nature 420:198–202

    Article  PubMed  CAS  Google Scholar 

  • Zinchuk V, Zinchuk O (2008) Quantitative colocalization analysis of confocal fluorescence microscopy images. Curr Protoc Cell Biol Chapter 4

  • 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 37:1274–1279

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

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.

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  1. Female Germ Cell Biology Group, Department of Clinical Studies, Center for Animal Transgenesis and Germ Cell Research, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center, 382 West Street Road, Kennett Square, PA, 19348, USA

    Claudia Baumann & Rabindranath De La Fuente

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  1. Claudia Baumann
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Correspondence to Rabindranath De La Fuente.

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Communicated by: G. Almouzni

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412_2008_189_MOESM1_ESM.jpg

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

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)

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Baumann, C., De La Fuente, R. ATRX marks the inactive X chromosome (Xi) in somatic cells and during imprinted X chromosome inactivation in trophoblast stem cells. Chromosoma 118, 209–222 (2009). https://doi.org/10.1007/s00412-008-0189-x

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