Figure 1

Progressive gain of DNA methylation over the human retinoblastoma promoter. A, Scheme of the transgene reporter carrying the core Rb promoter. The reporter gene (black rectangle) corresponds to the GFP gene, and locations of the relevant transcription factor binding sites including that of CTCF are shown. The vertical lanes correspond to the CpG dinucleotides. Locations of primers for PCR amplification of the bisulfite modified genome are shown as horizontal arrows. The probe used for Southern blots to validate the integrity of the transgenes and to determine copy number is also shown. B, DNA methylation status of a HeLa cell line in which the transgene shown in A is integrated as a single-copy. The individual and global percentages of methylation are shown at 130 and 170 days of continuous cell culture (left panel). As shown in a previous study [8], at 100 days in culture there is no expression of the transgene, but here we clearly observe a progressive increase in DNA methylation over the Rb promoter. At the center, the flow cytometry profile of the clone is shown at the same days, where 10,000 cells were analyzed. The intensity of fluorescence was plotted; M1, corresponds to the non-fluorescent cell population, and M2, the fluorescent detectable part of the graph. The plot summarizing the percentage of DNA methylation is shown (right panel).