H3K27me3 accumulates on the inactive X chromosome in female XEN cells. (A) Heatmap of single-cell, steady-state RNA levels for the ten most stringent epigenetic discriminants for trophoblast stem (TS) cells and extraendoderm stem (XEN) cells (P < 5 × 10−4 by F-test). The complete data set is available in Additional file 2B. R, Regulator preferentially associated with transcription repression; D, Regulator associated with a dual function; it can be involved in transcription repression or activation. n = 72 female XEN cells (GHP7/9). (B) Representative image of immunofluorescence followed by fluorescence in situ hybridisation on RNA for H3K27me3 (green) and Xist (red) on female XEN cells (GHP7/3 cell line). Similar results were obtained with the GHP7/9 cell line (data not shown). Quantification of fluorescence intensities for Xist and H3K27me3 across the inactive X domain show that the two domains do not strictly overlap. Maximal projections after deconvolution are shown. Scale bar = 5 μm. (C) Pie charts showing the percentage of nuclei exhibiting an accumulation of Xist RNA only (red), coaccumulation of Xist RNA and H3K27me3 (yellow) and accumulation of H3K27me3 only (green) in female XEN cells (GHP7/3 cell line). (D) Boxplots showing the distribution of volumes occupied by Xist RNA and H3K27me3 on the inactive X in XEN cells (GHP7/3 cell line). The two distributions are significantly different. P < 0.05 by Kolmogorov–Smirnov test (n > 50). Vertical bars below and above the box-plots show the minimal and maximal values in the cell population respectively.