Pyrosequencing Analysis of the Methylation Status of the CpG Island in the Retinoic Acid Receptor-β₂ (RAR-β₂) Gene Promoter.

CpG islands are associated with the 5′-ends of most housekeeping genes and many regulated genes. We have hypothesized that the methylation status of CpG islands in the promoter region of all-trans retinoic acid (ATRA) target genes such as retinoic acid receptor-β₂ (RAR-β₂) may be related to ATRA resistance and relapse of acute promyelocytic leukemia (APL). In the present study, we developed a highly quantitative method to assess the degree of DNA methylation at specific sites using Pyrosequencing^TM technology (Biotage, Uppsala, Sweden). This method is more quantitative than methylation-specific PCR, and is as accurate as but simpler and more robust than combined bisulfite restriction analysis (COBRA) or direct sequencing of plasmid clones of PCR products. We used this method to study 14 CpG dinucleotides in the CpG island of the RAR-β₂ promoter. In reconstruction experiments in which 100% methylated and 100% unmethylated DNAs were admixed in different proportions (100:0; 80:20, 60:40, etc), a straightline graph was obtained over the entire range from 0 – 100% for each of the 14 CpG dinucleotides (r² > 0.98). The results were highly reproducible and the variation between the results obtained from repetitive pyrosequencing of the same DNA was very low (S.D.<2%). Also the standard deviation between measurements of different PCR-amplified, bisulfite-converted DNAs prepared in separate experiments was <5%. We then used this method to measure the methylation level of the CpG island of the RAR-β₂ promoter in several leukemia cell lines. Of 3 APL cell lines, the two with PML-RARα mutations, i.e., UF-1 and AP-1060, had higher overall methylation, compared to the NB4 cell line with non-mutant PML-RARα (mean ± SD = 52 ± 25% and 55 ± 21%, versus 43 ± 20%; p = 0.04 and 0.08, respectively; SD calculated from the variation across the 14 CpG dinucleotides for each source). Two myeloid leukemia cell lines with predominantly erythroid lineage characteristics, K562 and TF-1, had much lower levels of RAR-β₂ methylation (2.6 ± 0.9% and 8.9 ± 3.2%, respectively). In the AP-1060 culture system, recently developed in our lab, there was little difference in methylation status between the patient bone marrow source and an intermediate, non-immortalized cell strain AP-1060S (27 ± 13% vs. 31 ± 25%). Further, there was no difference between lower and higher passage generations of AP-1060S (31 ± 25% vs. 30 ± 26%), which had markedly different replicative potential, indicating that replicative senescence at higher AP-1060 passages was not associated with altered methylation of the RAR-β₂ gene promoter. However, the established, immortalized AP-1060 cell line had significantly greater methylation (52 ± 25%) than either the bone marrow source or AP-1060S (p <0.0001 and p = 0.0002, respectively), consistent with published reports of increased promoter methylation of cell lines. In conclusion, pyrosequencing is a high throughput method with great quantitative strength, and can be used for accurate and consistent analysis of methylation status in large numbers of samples.