Fig. 4.

ATRX is a stable marker of the inactive X chromosome throughout TS cell differentiation. a Analysis of ATRX immunolocalization patterns (red) and subsequent DNA-FISH analysis (green)of the X chromosomes revealed a stable association of the protein with the Xi (arrow, insets) throughout TS cell differentiation as depicted for day 4 of differentiation. The active X chromosome remains indistinguishable from the autosomes (arrowhead, insets). Scale bars represent 10 μm. b Quantification of ATRX associations with the Xi in TS cell metaphase spreads. Data indicate the mean percentage of diploid metaphase spreads with ATRX association at a single X chromosome at different days (d0–d6) following spontaneous differentiation. Error bars represent the STD of three independent experiments, and different superscripts indicate significant differences (p< 0.05). c ChIP assays were performed on trophoblast stem cells using specific antibodies against ATRX, H3K27_me3, and H3K9_me2. Precipitated genomic DNA was amplified, and a significant enrichment of DNA fragments encoding the constitutive H3K9_me2-hotspot located 5′ of the Xist locus for ATRX and the histone modifications compared to the controls (IgG and TFIIB) was detected. A representative gel image is shown below the graph. Error bars represent the STD of three independent experiments, and different superscripts indicate significant differences (p<0.05)