Fig. 3
Xist RNA domain on the maternal inactive X in I-XCI revertant clones is associated with and accumulation of H3K27me3 at the majority of expressed X-linked genes. a Left, scatterplot of H3K27me3 percentages along the body of expressed X-linked genes in female F3 (y-axis) relative to male F2 (x-axis) TS cells. Each dot represents a single gene and its respective percentage of H3K27me3 in male and female TS cells. k-means clustering was applied, which led to the identification of three classes of genes. Genes subject to I-XCI (red dots) are concentrated in the upper left quadrant consistently, with them being depleted in H3K27me3 in male cells and enriched in H3K27me3 in female cells as previously established [20]. Genes escaping from I-XCI in TS cells (blue dots) are significantly depleted in H3K27me3 in both male and female cells. In agreement with H3K27me3 marking preferentially the silent state, very few expressed genes are enriched in H3K27me3 in both male and female cells ([0] genes; indicated by maroon dots). These [0] genes may have resulted from the fact that gene expression data [29] and our ChIP-chip experiment data were obtained from different TS cell lines. Middle, scatterplot of H3K27me3 percentages along the body of expressed X-linked genes in the I-XCI revertant clone 1#A (y-axis) relative to female F3 (x-axis) TS cells. Genes marked with red triangles “H3K27me3-low in 1#A” represent genes that have not regain H3K27me3 on the inactive X after the reversal of I-XCI patterns. Right, scatterplot of H3K27me3 percentages along the body of expressed X-linked genes in the I-XCI revertant clone 1#A (y-axis) relative to male F2 (x-axis) TS cells. “H3K27me3-low in 1#A” genes show a percentage of H3K27me3 that is not significantly different from the low levels of H3K27me3 on active allele in male TS cells by Kolmogorov–Smirnov test. The H3K27me3 profiles of Ndufa1, Lamp2 and Atp7a analysed in panel c and d and of Cox7b and Hmgn5 analysed in Additional file 3C are shown. b Representative examples of H3K27me3 distribution along expressed genes in male, female and I-XCI clone 1#A TS cells. University of California Santa Clara (UCSC) Genome Browser screenshots (mm8 build) are shown. IP immunoprecipitated DNA; IN input DNA. c Representative images of immuno-RNA-FISH for H3K27me3 (yellow) and Hprt1 (green) combined with the indicated X-linked gene (red) in F3 parental cells (upper panels) or in the indicated XCI revertant clones (lower panels). The chromosomal position of the genes analysed is depicted on the diagram on the left. In F3 cells, active alleles of Cybb, Ndufa1, Lamp2 and Atp7a are located away from the H3K27me3 coated inactive X indicating that these genes are expressed from the active X. In revertant clones 1#B and 1#F, active alleles of all four genes are located at the vicinity of the Hprt1 signal marking the XP and away from the H3K27me3 accumulation which indicates that these genes are transcribed from the active X. Moreover, Cybb and Ndufa1 are also transcribed, in these clones, from the inactive X as illustrated by a faint signal at the periphery of H3K27me3 accumulation (arrowheads). Maximal projection after deconvolution. Scale bar 5 μm. d Cumulative histograms of the percentages of nuclei with the depicted expression pattern in F3 TS cells and in nuclei of 4 independent XCI revertant clones (analysis performed at passage 8 or later after clone picking). An Hprt1 signal (green pinpoint) identifies the XP, while an H3K27me3 domain marks the inactive X (yellow domain). In XCI revertant clone 1#A, X-linked genes Lamp2 and Atp7a that show high levels of H3K27me3 as assessed by ChIP-on-chip show a preferential expression from the reactivated XP. In contrast, X-linked genes Cybb and Ndufa1 displaying low levels H3K27me3 by ChIP-on-chip show significant bi-allelic expression compared to F3 cells (χ 2 test p value <0.05). No significant difference is observed amongst XCI revertant clones (χ 2 test p value >0.05). See Additional file 10 for probe position. n > 150