ATRX marks the inactive X chromosome (Xi) in somatic cells and during imprinted X chromosome inactivation in trophoblast stem cells
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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.
KeywordsChromosome Inactivation Embryonic Stem Cell Differentiation Extraembryonic Tissue Bold Arrow Trophoblast Giant Cell
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.
- 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
- 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
- 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
- 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
- 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
- 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
- Zinchuk V, Zinchuk O (2008) Quantitative colocalization analysis of confocal fluorescence microscopy images. Curr Protoc Cell Biol Chapter 4Google Scholar