QAMA RT‐PCR analysis of the correlation of OCT4 expression with CpG methylation status at specific sites in the OCT4 promoter

Abstract

Promoter DNA demethylation at CpG sites is associated with expression for many genes. Current approaches for measuring this phenomenon are labor-intensive, insensitive and/or limited to a subset of CpG sites. A novel protocol known as QAMA (Quantitative Analysis of Methylated Alleles) was recently described by Zeschnigk et al. (Nucleic Acids Research 32:e125, 2004). QAMA involves RT-PCR using a single set of primers, combined with a pair of probes that distinguish methylated from unmethylated target sequences. We have adapted and simplified this technique in order to assay the methylation status of 3 individual CpG sites in the promoter region of the murine pluripotency-associated gene OCT4 (Pou5f1). The proportion of DNA molecules methylated at each site was determined for embryonic stem cells (ESCs; high OCT4 expression) and adult liver (no OCT4 expression). These 3 targeted CpG sites had been previously reported to vary widely in the extent to which demethylation correlated with expression. Results from the simplified QAMA assay confirmed the findings and extended the resolution of previous assays. In a second experiment, ESCs were allowed to aggregate into embryoid bodies and initiate differentiation for 3 days, during which OCT4 expression declines. QAMA comparison of OCT4 promoter regions in DNA isolated from ESCs or 3-day embryoid bodies revealed little change in methylation status. This suggests that a decrease in OCT4 promoter activation precedes CpG methylation at these 3 sites. We are currently testing other genes for promoter methylation changes associated with expression changes during embryonic and ESC differentiation. Funded by Pfizer.