H3K27 tri-methylation spreads from the hotspot into Xist in the absence of Tsix transcription. (A) Map of Xic (see Figure 1A). (B) ChIP analysis of H3K27 trimethylation in male Tsix-truncated embryonic stem (ES) cells (Ma2L cell line, red line) and in the corresponding revertant (Ma1L, black line). In the Ma2L cell line, Tsix transcription has been truncated through the insertion of a loxP-flanked transcriptional STOP signal downstream of the Tsix promoter (represented in the graph by the STOP symbol). The Ma1L revertant was obtained from Ma2L after deletion of the STOP signal. (C) Similar analysis in male wild-type (CK35 cell line, black line) and mutated (ΔPas34, red line) ES cells in which Tsix transcription is drastically reduced. The ΔPas34 cell line was generated by deleting DXPas34, an enhancer of Tsix located near the Tsix major promoter. Dotted lines in the graph indicate the location of the 1.2-kb deletion carried by ΔPas34 ES cells.