METHYLATION ANALYSIS BY GENOMIC SEQUENCING OF 5' REGION OF MOUSE PGK-1 GENE AND A CAUTIONARY NOTE CONCERNING THE METHOD

We have used genomic sequencing aided by ligation-mediated PCR (LMPCR) to assay for 5-methylcytosine in the CpG-rich promoter region of the mouse X-linked phosphoglycerate kinase gene (Pgk-1). Earlier studies showed that there was very heavy methylation of CpG dinucleotides in the CpG-rich promoter of the human PGK1 gene on the inactive X chromosome (the Xi), but that these same sites were completely unmethylated on the active X chromosome (the Xa). For mouse Pgk-1, previous restriction enzyme analysis had shown apparently complete methylation of only one cytosine in the promoter region on the Xi, at HpaII site H7, which is located in the untranslated region, 28 nucleotides upstream of the translation start site. We analyzed this potentially critical region by combining the use of HpaII with LMPCR, and find that the CpG dinucleotides near H7 are either unmethylated or only partially methylated on the Xi. LMPCR analysis of male and female DNA over a 490-bp sequence including the promoter and enhancer extend the finding of relative hypomethylation on the mouse Xi to include all CpG dinucleotides in this region. These results are relevant to the role of DNA methylation in stabilizing the inactive state of chromatin. In addition, we find that caution must be exercised in using LMPCR for methylation analysis of some sequences. A DNA concentration-dependent band-suppression artifact can incorrectly suggest methylation of both CpG and nonCpG dinucleotides.