Several biological phenomena depend on differential methylation of chromosomal strands. While understanding the role of these processes requires information on allele-specific methylation, the available methodologies are not quantitative or labor-intensive. We describe a novel, rapid method to quantitate allele-specific DNA methylation based on the combination of bisulfite PCR and Pyrosequencing™. In this method, DNA is first treated with sodium bisulfite, which converts cytosine but not 5-methylcytosine to uracil. Genes of interest are subsequently amplified using PCR. Allele-specific methylation can then be determined by pyrosequencing each allele individually using sequencing primers that incorporate single nucleotide polymorphisms (SNPs) that allow differentiation between the two parental alleles. This allele-specific methylation methodology can potentially afford quantitative analyses relevant to the regulation of X chromosome inactivation, allele-specific expression of genes in the immune system, repetitive elements, and genomic imprinting. As an illustration of our new method, we quantitated allele-specific methylation of the differentially methylated region of the H19 gene, which is imprinted. Although we could reliably determine allele-specific methylation with our technique, additional studies will be required to confirm the ability of our assay to measure loss of imprinting.
Variation in allele-specific expression is common in humans (1,2) and underlies normal human variability and predisposition to human diseases (3). Genomic imprinting is an example of allele-specific expression, where only one chromosomal allele is expressed. In imprinting, this monoallelic expression is dependent on the parental origin of the chromosome (4). Since normal genomic imprinting is regulated, in part, by methyl groups on cytosines within cytosine-guanine (CpG) dinucleotides, normal or loss of imprinting (LOI) can be predicted by DNA methylation in regions that are differentially methylated between parental alleles (differentially methylated regions; DMRs) (5,6). As a proxy for parent-specific expression, there are two current methodologies used to analyze DNA methylation. The first is bisulfite genomic sequencing (7) of multiple alleles, a process that is labor-intensive. The second is bisulfite pyrosequencing, which does not assess allele-specific methylation levels, but instead, averages methylation levels of both alleles in a highly quantitative manner (8). Our new method uses both bisulfite PCR and allele-specific pyrosequencing to measure DNA methylation of a single allele. To illustrate our new methodology, we analyzed allele-specific methylation of the H19 gene, a gene that is normally imprinted and expressed only from the maternal allele.Outline of the Allele-Specific Methylation Methodology
To obtain the methylation status of the CpG sites of interest, we adapted a standard method known as bisulfite genomic sequencing. In brief, this method is based on the selective deamination of cytosine to uracil by treating DNA with bisulfite and the sequencing of subsequently generated PCR products. In contrast to cytosine, 5-methylcytosine does not get converted by bisulfite and remains as cytosine. This inherent selectivity of bisulfite to induce a primary sequence change can be used to distinguish DNA methylation status (7).
DNA methylation at a specific CpG dinucleotide can then be quantitated as a ratio of cytosine to thymine, for which several methods have been developed (8,9,10,11,12,13,14,15,16). To amplify the H19 imprinting center, specifically the region harboring 19 CpG sites within and surrounding a CTCF binding site, primers specific to the bisulfite-converted template that are not dependent on the methylation status were used.
To measure DNA methylation of each allele individually, we used Pyrosequencing™, with sequencing primers that were directed to a heterozygous single nucleotide polymorphism (SNP). Thus, a pair of sequencing primers was used to measure methylation of each allele separately. Hence, the sequencing primers incorporate the polymorphic information of the parent-specific mark (Figure 1). We demonstrate that this methodology correctly identified the percentage of allele-specific methylation within a 3% margin of error.
An immediate health application of this allele-specific methylation methodology is to classify persons as to whether or not they have LOI in prospective epidemiologic studies. As a proof of principle, we present the application of this methodology to study a methylation-dependent imprinting center: the H19 imprinting center, which regulates the paternally imprinted noncoding RNA H19 (17,18,19). We can distinguish the methylation levels of each individual allele by exploiting a SNP in the upstream region of the H19 gene (rs2071094; dbSNP build 124) (20) (Figure 1). By doing so, we can quantitate methylation on each allele at a single base resolution for persons who are heterozygous for the SNP.Materials and Methods Cell Lines and Human Tissue Samples
The Colo205 colorectal carcinoma cell line was cultured in Dulbecco's modified essential medium at 37°C in a humidified atmosphere with 5% CO2. Tissue samples were collected through the University of Southern California Pathology Core Laboratory. Informed written consent was obtained, and the collection was approved by the University of Southern California Institutional Review Board. The informative samples for the allele-specific methylation assay are samples heterozygous for a SNP in the H19 imprinting center. Genomic DNA was extracted using standard phenol-chloroform methodology.Sodium Bisulfite Modification of Genomic DNA
Sodium bisulfite modification of genomic DNA was performed as described previously by Frommer et al. (7), with minor modifications. In brief, DNA (approximately 2 µg) suspended in 50 µL distilled water was denatured in 5.5 µL 0.2 M NaOH at 37°C for 10 min. After the addition of 30 µL 10 mmol hydroquinone and 520 µL 3 M sodium bisulfite, pH 5.0, the DNA was incubated at 50°C for 16 h. The bisulfite-modified DNA was purified with Wizard® Plus kits (Promega, Madison, WI, USA) according to manufacturer's instructions. NaOH (5.5 µL 3M) was added and allowed to incubate for 5 min at room temperature. The bisulfite-treated DNA was precipitated with 0.6 volumes 10 M sodium acetate and 3.0 volumes ethanol using glycogen as a carrier. The final precipitate was resuspended in 20 µL water.