We have developed a novel one tube assay, termed MB-PCR, to detect genomic DNA fragments according to their level of CpG methylation. The novel technique requires little amounts of DNA and allows the rapid screening of multiple loci. MB-PCR is particularly useful to screen for methylation levels of candidate genes in tumor tissue or tumor cells as exemplified for acute myeloid leukemia in this report. It may, however, also be useful to detect changes in DNA methylation in other situations, including normal cellular differentiation and aging.
Comparison with existing methods
At present, mainly two technical approaches are used to detect the level of CpG methylation of known candidate gene loci: methylation-sensitive restriction or bisulfite treatment of DNA (29
Isoschizomers of bacterial restriction endonucleases with different sensitivities for 5-methylcytosine can be used to determine the methylation status of specific CpG-dinucleotides (29
). The use of methylation-sensitive restriction enzymes, however, has several limitations. Apart from the fact that incomplete restriction digests may complicate the analysis, the greatest disadvantage is that methylation-sensitive restriction merely informs on the methylation status of the cytosine residues which are recognized by the methylation-sensitive restriction enzymes used.
A global picture of the methylation pattern in a candidate gene locus may be obtained by bisulfite sequencing as originally described by Frommer et al
). The treatment of double-stranded genomic DNA with sodium bisulfite leads to the deamination of unmethylated cytosine residues (but not 5-methyl cytosine) into uracil residues. DNA treated with bisulfite can be used directly in PCR in which uracil residues (previously unmethylated cytosine) and thymidine residues are amplified as thymidine and only 5-methylcytosine residues are amplified as cytosine residues (20
). Depending on the application, the primers used for the PCR differentiate between methylated and unmethylated sequences or amplify fragments independently of the methylation status (29
). PCR fragments which have been amplified using non-discriminating primers can, for instance, be sequenced directly to determine the position of methylated and unmethylated CpGs. Other methodical approaches that allow high-throughput analyses utilize the differences in sequence for the specific amplification of methylated and unmethylated sequences by discriminating primers or probes (e.g. methylation-specific PCR, MethyLight) (29
). In contrast to the methylation-sensitive restriction enzymes, the DNA treated with bisulfite can potentially provide information on the methylation status of several CpG residues in an amplified genomic fragment. The detection of CpG methylation by using discriminating primers or probes, however, is limited to the methylation status of single (or few) cytosine residues. Hence, the information provided by all currently known assays that are suitable for high-throughput methylation analysis of single gene loci is limited to one or only a few CpG residues within the gene of interest.
Rather than analyzing single CpG residues, MB-PCR analyses target DNA fragments according to their methylation degree. The information provided by MB-PCR will be at least as relevant as that obtained with other existing PCR techniques—the methylation density of a proximal promoter may actually correlate better with the transcriptional status of a gene than the methylation status of a single CpG residue within the region. We believe that the high methyl-CpG affinity of MBD2 combined with the bivalent, antibody-like structure of the recombinant MBD-Fc protein greatly increases its binding capacity, enabling the efficient retention of a DNA fragment on the basis of its degree of methylation.
A comparable approach discriminating DNA fragments on the basis of their methylation density was developed in the laboratory of A. Bird already 10 years ago (26
). A recombinant MeCP2 protein bound to a matrix was used in this and a number of recent studies for binding and enriching highly methylated DNA through affinity chromatography (26
). Although we have not tested recombinant MeCP2, it is possible that it may also work as methyl-binding polypeptide in MB-PCR.
The company Panomics introduced recently a commercially available kit that differentiates promoters with methylated groups from unmethylated promoters. In principle, the company's method consists of a spin column affinity purification using MeCP2. This method also appears to be rapid, however, it requires a relatively large amount of starting material. It is not clear, to which extent the information can be quantified or whether the amount of isolated fragments correlates with the degree of promoter methylation. A recent report by Klose et al
) clearly demonstrated that MeCP2 requires an A/T run adjacent to the methylated CpG dinucleotide for efficient DNA binding, suggesting that all methods based on MeCP2-affinity chromatography, including the Panomics kit, will be biased towards certain CpG motifs. Therefore, it is not clear, whether MeCP2 will be able to detect every methylated CpG island fragment. No binding requirements or preferences of MBD2 were detected in this and previous studies (30
). Owing to its binding properties, MeCP2 may be better suited to detect non-CpG island promoters with a lower CpG-density, e.g. CD14, IFNγ or IL-4 Promoters (as demonstrated in the user manual of the commercially available kit).
An important aspect of MB-PCR is the fragmentation of the genomic DNA. We have used the restriction enzyme MseI (T/TAA) in our study; however, other methylation-insensitive restriction enzymes such as Csp6I (G/TAC) or Tsp509I (/AATT) may also be used (either alone or in combination) to achieve an appropriate fragmentation of the target gene. Most informative (with respect to the effects on transcription) and clearest results (in terms of noise and background) are obtained when a target gene fragment contains only the proximal promoter within the CpG island. In addition to enzyme restriction, DNA fragmentation may also be achieved by mechanical means, e.g. ultra-sonication.
As demonstrated, our current approach allowed the distinction between strong (>30–40% methylation), intermediate methylation levels (>10%) or no methylation. Owing to the limitations of standard PCR, a more detailed grading is technically difficult. In most cases, however, standard MB-PCR will be sufficiently informative to detect aberrant methylation in a tumor sample. A standardization of individual experiments may be achieved by using a series of mixtures of methylated and unmethylated DNA as a standard curve for each experiment. As a control for the completeness of restriction digestion as well as the washing procedure, a DNA fragment is amplified that contains no CpG residues and therefore should not be retained and amplified. Although we have not yet tested, it is conceivable that MB-PCR may also be run as a real-time PCR application, which may allow the quantification of amplified products and a better correlation with methylation levels in a sample.
Since the surface area of the PCR tube and hence its binding capacity for the recombinant methyl-DNA-binding polypeptide is limited, it is important to avoid the use of an excess amount of genomic DNA for the assay. We found that MB-PCR produces consistent results using 160 pg to 10 ng of restricted genomic DNA. The little amount of DNA required for MB-PCR is actually a great advantage of this technique, allowing the methylation analysis of candidate genes from very limited cell numbers which may include biopsy samples or cells collected by laser-mediated microdissection.
Screening for aberrant CpG methylation
To test the usefulness of MB-PCR for methylation analysis, we initially analyzed CpG island promoters that were known to us or described in the literature to be methylated or unmethylated in particular cell lines, including the promoters of CDKN2B (p15INK4b) and ESR1 genes. Since initial results obtained by MB-PCR from several leukemia cell lines corresponded with previously published observations, we selected a number of novel putative candidate genes (ICSBP, ETV3 and DDX20) for further analyses that are involved in cell-cycle arrest or cellular differentiation and represented good candidates as tumor suppressor genes. Using MB-PCR we show that the CpG island promoter of ICSBP is methylated in many leukemia cell lines and a subset of patients with AML. MB-PCR results were independently confirmed by bisulfite sequencing and methylation of the promoter correlated with the absence or down-regulation of ICSBP transcription in the leukemia cell lines. Our data suggest that epigenetic silencing may contribute significantly to the observed down-regulation of ICSBP in human myeloid leukemia. The CpG island promoters of ETV3 and DDX20 were not methylated in any of the samples tested so far. It will be interesting to analyze the methylation status of these genes, especially ICSBP, in other malignancies, e.g. CML or non-myeloid malignancies.
In summary, our report describes a rapid and sensitive procedure for detecting methylated DNA target sequences from limited sample material. MB-PCR will be particularly useful in screening methylation levels of candidate genes not only in tumor tissue but also in tumor cells. Our study also suggests that the promoter of ICSBP is hypermethylated in a subgroup of patients with myeloid leukemia, which may serve as a molecular marker for disease.