SEQUENOM mass spectrometry analysis of Xist CpG island DNA methylation in Xist mutant XY embryonic stem cell lines. (A) Schematic representation of the Xist promoter region and 5'end of exon 1 (CpG regions 1 and 2). The P1 and P2 start sites and the direction of transcription are indicated by arrows. The grey shaded box shows the position of the 5'repeats. Individual CpG sites are represented by small circles above the schematic; grey circles indicate the sites that were analysed. Polymerase chain reaction fragments A, C and D incorporate sites A1–15, C1–22 and D1–10 (see Methods). The graphs show the percentage of methylation of specific Xist CpG sites in wild-type (wt) XY and XX embryonic stem (ES) and somatic cells (B) and in Δ5' (C), SPA (D) and Δhs (E) Xist mutants [12, 13] and in pAA2Δ1.7 and pSS1Δ2.7 (F) Tsix mutants . The wt 129/1 XY ES cell line is included as a reference control on each graph. The insert shows the type and position of the mutation. X inactivation skewing phenotypes for each mutation are indicated alongside. The dots are joined by lines where consecutive sites were analysed. CpG sites numbered in grey below the graphs indicate that the data points are not available due to low or high fragment mass or due to duplication or overlay of two or more fragments. The average data for two or three CpG sites (for example, A7/8/9) is shown in cases when the sites reside close to each other and could not be resolved as separate fragments. Note the direct correlation between hypomethylation of the Xist promoter region in mutant ES cells and primary (1°) non-random X inactivation in vivo.