FIGURE 4.

5-Aza-CdR treatment led to E-box demethylation and occupation by c-Myc in the promoters of the TfR1, Alas2, and Fech genes. A, bisulfite sequencing of the methylation status of the E-boxes in TfR1 promoters (+285 bp and −6 kb E-boxes) before and after 5-aza-CdR treatment. B, ChIP assay of the E-box in the CpG island of the TfR1 promoter (+285 bp). *, p < 0.01 compared with the control MEL cells. C, ChIP assay of E-boxes outside the CpG islands of the TfR1, Alas2, and Fech genes. *, p < 0.01 compared with the control MEL cells. A specific antibody to c-Myc was used for the ChIP assays of the TfR1, Fech, and Alas2 promoters, followed by sequence-specific PCR analysis of specific binding sites of c-Myc. D, transcriptional activities of the constructs containing different E-boxes of TfR1 promoters (+285, −6 kb, or −8 kb E-boxes). Luciferase activities of the constructs containing different fragments of TfR1 promoter, including a 700-bp fragment (−183 to +550), 1700-bp fragment (−1230 to +550), 700-bp fragment plus −6 kb E-box (−6267 to −6061), and 700-bp fragment plus −8 kb E-box (−8689 to −8421) were measured under co-transfection with the expression vectors of c-Myc, Max, or both. All of these E-boxes in these promoter fragments were in demethylated status. Luciferase activities of the constructs containing methylated 700- and 1700-bp fragments were also measured. Firefly luciferase activities were normalized by Renilla luciferase. Error bars, S.E.