Chromosoma

, 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

Research Article

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.

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 (RXi = 0.7253, upper panel) demonstrates co-localization between ATRX protein and the inactive X chromosome. The co-localization coefficient for the active X chromosome (RXa = 0.3115, arrowhead) implies lack of co-localization. Similarily, the (R) value for ubiquitinated H2A (green) in trophoblast stem cells during interphase (RXi = 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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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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