Epigenetic modification involves DNA methylation, covalent modification of histones and small inhibitory RNA molecules known as microRNAs (miRNAs) (3
). DNA methylation is a heritable, enzyme-induced modification without changing the nucleotide sequence of the DNA base pairs. DNA methylation involves transfer of a methyl-group to the 5-carbon on the cytosine in a CpG dinucleotide via DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) (4
). Most of the CpG dinucleotides are unevenly distributed within the genome and regions high in these dinucleotides termed CpG islands. The haploid human genome consists of 29,848,753 CpGs and about 6% of all CpGs are located in the CpG islands (5
). These islands are frequently located in the 5′-untranslated region and the first exon of approximately 60% of all genes. Methylation of CpG islands affects the transcriptional activation of genes. It is generally accepted that a high level of promoter CpG island methylation results in gene silencing (6
). In the normal genome, DNA methylation is essential for proper development, chromosomal integrity, maintenance of gene expression states, and X chromosome inactivation (7
). In primary human tumors, methylation patterns are severely altered. This includes hypermethylation of CpG islands and genome-wide hypomethylation (9
). Because DNA methylation has significant effects on gene function and expression, detection of DNA methylation becomes an active area of research for the understanding of normal biological processes and tumorigenesis.
There are several methods available for the determination of methylation patterns and the quantitative assessment of methylation levels in sample tissues (11
). These include methods for interrogating the combined methylation status of several CpG sites in a single gene (methylation-specific polymerase chain reaction or MSP and MethyLight; 12, 13), methods for interrogating methylation status of individual CpG sites present in a gene (combined bisulfite restriction analysis or COBRA and methylation-specific single-nucleotide primer extension or MS-SNuPE; 14, 15), methods for interrogating multiple CpG loci in many genes (methylation-specific oligonucleotide microarray or MSO microarray; 16), and methods for high-throughput, genome-wide epigenetic analysis (differential methylation hybridization or DMH; 17).
A large number of currently available techniques used for studying global methylation differences involve the use of methylation sensitive restriction enzyme(s) thereby limiting these approaches to profile genomic regions containing these restriction site motifs. In this regard, the methylated DNA immunoprecipitation (MeDIP)-chip approach is less biased and therefore more appealing (2
). The general strategy for the MeDIP-chip procedure is outlined in . Genomic DNA is sheared to low molecular weight fragments (average 400 bp) (see Note 1
). Methylated DNAs are immunoprecipitated with the anti-methyl-cytosine antibody, and may be PCR-amplified if source material is limited (see Note 2
). Input and methylated DNA can be subsequently labeled with fluorescent dyes Cy3 (green) and Cy5 (red), pooled, denatured, and hybridized to a microarray slide containing all the annotated human CpG islands (n
= 27,800) or other whole genome or promoter microarray designs (see Note 3
). The slide is scanned using a GenePix 4000B scanner and each image is analyzed with the GenePix Pro 6.0 image analysis software. MeDIP-chip has proven to be an efficient and robust method for analyzing DNA methylation at a genome-wide scale. Recently, several different companies have provided array designs to fit customer needs including whole-genome survey sets and promoter sets so that you can obtain comprehensive data from your methylation samples.
Fig. 10.1 Overview of the MeDIP protocol. The genomic DNA is sonicated into small fragments and then immunoprecipitated with an antibody directed against 5-methylcy-tidine. Input DNA and methylated DNA (black circle) can be differentially labeled with Cy5 (red) (more ...)