The inactive X chromosome is still epigenetically distinguishable from its active counterpart. (A) Representative examples of immunofluorescence show the status of H4ac (white) depletion/enrichment on X chromosomes (X-paint DNA FISH, red) on metaphase spreads from normal (WI-38) and breast cancer cell lines (ZR-75-1, SK-BR-3, and MDA-MB-436). On the right, MDA-MB-436 cells carry six X-chromosome fragments with a “2-by-2” homology, as assessed by the presence or absence of the NXT2 (white) or XIC loci (green), and line scans show H4ac enrichment variation between these X-fragments and the neighboring autosomal regions. As expected, one X chromosome (Xi) lacks H4ac staining in normal WI-38 cells (and HMEC, not shown). ZR-75-1 and SK-BR-3 cell lines harbor a reduced H4ac staining on one and two X chromosomes, respectively, in agreement with the number of XIST-coated X chromosomes shown in Figure 1A. In MDA-MB-436 cells, homologous X-chromosome fragments (two containing the XIC locus, two containing the NXT2 locus, and two with none of them) display opposite H4ac staining, suggesting that there is still one inactive and one active X chromosome linked to those loci, although fragmented. (B) Representative examples of immunofluorescence show the status of H3K4me2 (white) depletion/enrichment on X chromosomes (X-paint DNA FISH, red) on metaphase spreads from normal and breast cancer cell lines. On the right, line scans show H3K4me2 enrichment variation between the six X-fragments (for details, see A) and the neighboring autosomal regions in MDA-MB-436 cells. In each tumoral cell line, H3K4me2 depletion patterns follow the H4ac profiles found in A. (C) Representative examples of immunofluorescence show the status of H3K27me3 (white) enrichment on X chromosomes (X-paint DNA FISH, red) in metaphase spreads from breast cancer cell lines. ZR-75-1 and SK-BR-3 cell lines harbor an accumulation of H3K27me3 on one and two X chromosomes, respectively, in agreement with the number of XIST-coated X chromosomes shown in A. In MDA-MB-436 cells, H3K27me3 staining was only enriched on the X-chromosome fragment, where the XIC region lies. Indeed, RNA/DNA FISH analysis showed that this X fragment corresponds to the one coated by XIST RNA in interphase cells, which is not the case for the other fragments (Supplemental Fig. S3F). In SK-BR-3 and MDA-MB-436 cell lines, H3K27me3 spreads into the autosomal fragments translocated to the XIC-containing fragment. (D) Schematic view of H4ac, H3K4me2, and H3K27me3 patterns on X-chromosomes in the three tumor cell lines.
