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. 2003 Dec;23(23):8429–8439. doi: 10.1128/MCB.23.23.8429-8439.2003

FIG. 5.

FIG. 5.

Expression and methylation status of GATA downstream target genes in cultured colon cancer and GC cells and normal tissues. (A) RT-PCR analyses of the expression of each gene in the same colon cancer and GC cell lines and normal tissues as given for GATA analyses in Fig. 1A. Also, to the far right, is shown an analysis of expression, carried out as described above, of the TFF genes and inhibinα in the HCT116 colon cancer wild-type (WT) and DNMT1−/− plus DNMT3b−/− cells (DKO) shown in Fig. 1C. Lanes: M, mock treatment of cells; A, treatment with DAC; NC, normal colon; PBL, normal lymphocytes; H2O, no RNA added. (B) Methylation status of the inhibinα gene. A schematic of the 5′ region of the gene is shown above in which the rectangle depicts the first exon, and the blackened area denotes the coding region within this exon. The black triangles represent positions of consensus GATA-binding sites, and the vertical lines each represent a CpG site. The large arrow (BS) denotes a region of bisulfite sequencing for the CpG poor region previously thought to be the only promoter region (see the text), and the smaller arrows (MSP) represent the positions of primers used for the MSP analysis in all of the cancer cell lines of the newly defined CpG island discussed in the text and shown in the lower part of panel B. Lanes: U, unmethylated alleles; M, methylated alleles; PBL, normal lymphocytes; NC, normal colon; H2O, no DNA added. (C) Methylation status of Dab2. A schematic of Dab2 is shown above in which a 5′ untranslated exon 1 (open box) is located upstream from exon 2 which contains the ATG for start of the coding region (black area within the square for exon 2). Arching line, mRNA splicing which joins exon 1 to exon 2; black triangles, positions of GATA-binding sites; vertical lines, CpG sites and the island around exon 1; arrows at the bottom (MSP), position of MSP primers used to analyze the methylation status of the CpG island as shown in the panel below. The lower part of panel C shows examples of MSP results for the methylation status of the Dab2 5′ CpG island. Lanes: U, unmethylated alleles; M, methylated alleles; PBL, normal lymphocytes; NC, normal colon; C1C and C2C, colon cancers.