Analysis of Xist promoter DNA methylation in Xist mutants. (A) Schematic representation spanning Xist and the immediate upstream gene, Enox, including pS12x and pS19x. Xist and Enox TSS and the direction of transcription are indicated by arrows. The restriction methylation-sensitive enzymes used in the analysis are shown underneath the schematic. The grey bar shows the position of the probe used for Southern blot hybridisation. The three targeted Xist mutants Δ5', SPA  and XT67E1  are shown. The dotted red line shows the deletions in Δ5' and XT67E1 mutants and the lilac box below the schematic represents an insertion of a floxed PGKneo cassette. The small yellow box shows the position of the SPA insertion. (B) MSRE analysis of the Xist promoter in wt (129/1) and two mutant (Δ5'+neo and SPA+neo) XY ES cell lines. The different sizes of parental EcoRI fragments in Xist mutants are due to deleted/inserted sequences. The increased intensity of digested fragments in mutant samples indicates partial hypomethylation. (C) Quantitation of the degree of hypomethylation of MluI, HaeII and SacII sites in wt and mutant cell lines. (D) MSRE analysis of the Xist promoter in wt XY (129/1), wt XX (Pgk12.1) and mutant (XT67E1) XX ES cell lines. The blue arrow indicates the methylated wt PGK fragment and the red arrow to the larger mutant XT67E1 fragment. Note the complete loss of DNA methylation in the Xist upstream region on the XT67E1 mutant allele. (E) Strand-specific RT-PCR analysis of the Xist 5'region in wt Pgk12.1 and mutant XT67E1 XX ES cell lines. The position of the primers for amplicon 4 (amp 4), amplicon 51 (amp 51), amplicon 51mut (amp51mut) and the direction of sense (s, green) and antisense (as, red) transcripts are shown on the schematic above. Note the expression of ectopic sense transcript in XT67E1, attributable to the mutant allele.