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Fig. 6. Bisulfite genomic sequencing of a Pst3 tissue-specific differentially methylated region (TDM): sequence of cloned PCR products (10 clones each) amplified from bisulfite-treated DNA. Open circles indicate unmethylated CpGs (Thy), and filled circles indicate methylated CpGs (Cyt). The sequenced region contains 26 CpGs in 131 bp (see Fig. 4). The results are similar to those obtained by direct sequencing of the PCR product (Fig. 4); the testis is unmethylated, whereas muscle and liver are densely methylated.

Fig. 7. Restriction landmark genomic scanning (RLGS) analyses of tissues from different mice indicate a consistent, tissue-specific RLGS profile.

Fig. 8. Full-page version of Fig. 2. Restriction landmark genomic scanning (RLGS)-inferred tissue methylation profiles for 150 tissue-specific differentially methylated regions (TDMs) identified by RLGS and virtual RLGS. Black squares indicate the RLGS spot was absent (methylated), purple squares indicate reduced intensity (partial methylation), and white squares indicate full diploid intensity (unmethylated). ND, the spot intensity could not be determined. Note that 64 of 150 loci were unmethylated in only one tissue and that 43 were unmethylated in testis only. Table 4 provides a complete listing of the TDMs and their locations in the genome.

Fig. 9. Bisulfite genomic sequence analysis of a Pvu2 tissue-specific differentially methylated region (TDM). The unmodified genomic sequence within Pvu2, provided for reference, is shown above the trace. The locations of the CpG dinucleotides are highlighted. In the trace sequence from liver, the Cyt residues in the CpGs are retained as Cyt residues, indicating that they are methylated, whereas in the testis, they are converted to Thy residues, indicating that they are unmethylated. Because the PCR product was sequenced directly without being cloned, the trace represents an approximation of the average sequence at each position. Note that non-CpG Cyt residues in both sequences are converted to Thy residues, indicating complete conversion of unmethylated Cyt. A diagrammatic representation of the bisulfite sequencing results is also shown. Note that the TDM is within the third exon of the monocarboxylate transporter-like gene and is not within a region defined as a CpG island.

Fig. 10. Full-page version of Fig. 4. Bisulfite genomic sequencing of TDM Pst3. (a) Sequence traces obtained from the PCR products from bisulfite-treated DNA by using primers that would amplify a portion of the Pst3 locus. Because the PCR products were not cloned, the trace represents an approximation of the "average" methylation status at each CpG residue. The normal sequence and bisulfite sequence are shown. CpG sites are highlighted. (b) Diagrammatic representation of PstIII within the 5′ promoter region of Ddx4 (DEAD-box protein 4) on chromosome 13. The CpG island includes the 5′ promoter, exon 1, and a portion of the first intron. The arrow indicates the direction of transcription. The methylation status of the CpGs (black circles, fully methylated; blue circles, partial methylation; white circles, unmethylated) within a 450-bp region of Pst3 is shown along with the location of the restriction landmark NotI site. Note the dense hypermethylation in liver, kidney, colon, muscle, and brain and the hypomethylation in testis. Fig. 6 shows the bisulfite sequence of a portion of the Pst3 region that was determined from cloned PCR products. The results are in general agreement, although the cloned sequences exhibit considerable heterogeneity in methylation.