We use a protocol of DNA methylation profiling that uses the unmethylated fraction of the genome. This protocol works with most microarray types, as exemplified here with CpG island- and oligonucleotide microarrays. A detailed description of how specific 'epigenetic' microarrays are designed and how the slides are processed can be found in Schumacher et al., 2006 (1). DNA samples can either be interrogated as pairs (tester and control) on one array, as described in this protocol, or hybridized independently with only one fluorescent dye (e.g. for Affymetrix tiling arrays).
The key principles and some technical details of this method are described in our recent article (1) and the strategy for enrichment of unmethylated portions of the genome is presented in Figure 1. Briefly, genomic DNA (gDNA) is digested with the methylation-sensitive restriction enzymes such as HpaII and Hin6I. Whereas methylated restriction sites remain unaltered, the sites containing unmethylated CpGs are cleaved by the enzymes, and DNA fragments with 5'-CpG protruding ends are generated. In the next step, the double-stranded adapter CG-1 is ligated to the CpG-overhangs. At this point, it is expected that most of the relatively short and amplifiable DNA fragments derive from the unmethylated DNA regions. Some ligation fragments, however, may still contain methylated cytosines. A large proportion of these fragments are eliminated by treatment with McrBC, thereby increasing the specificity of the enrichment of the unmethylated DNA fraction. McrBC cleaves DNA containing methylcytosine on one or both strands, recognizing two half-sites of the form (G/A)mC; these half-sites can be separated by up to 3 kb, but the optimal separation is 55-103 base pairs. The remaining pool of unmethylated DNA fragments is then enriched by aminoallyl-PCR amplification that uses primers complementary to the adapter CG-1. An important advantage of using protruding ends in the adapter-ligation step is that degraded gDNA fragments will not be ligated and amplified and, therefore, will not interfere with DNA methylation analysis (which is especially useful when analyzing tissues with relatively long post-mortem interval or paraffin-embedded samples).
Fig 1: Schematic outline of the microarray-based method for identification of DNA methylation differences in genomic DNA. Samples are cleaved by methylation-sensitive restriction endonucleases, such as HpaII and Hin6I, ligated to the CpG-overhang specific adapter, and then cut by McrBC to eliminate residual methylated DNA fragments. The resulting unmethylated DNA fragments are then selectively enriched by adapter-specific aminoallyl-PCR, labelled, and hybridized to microarrays.
The enriched unmethylated DNA fractions are then labelled with fluorescent dyes and hybridized to microarrays. Several different types of microarrays can be used for epigenetic analysis, for example oligonucleotide arrays of individual genes or microarrays containing relatively larger DNA fragments of gene regulatory regions, such as CpG islands (1). Yet, it is evident that epigenetic profiling should be performed in a systematic, unbiased fashion and not limited to the traditionally preferable regions, such as CpG islands. Numerous other genomic loci exist that may be sites for important epigenetic modification, including enhancers, imprinting control elements or the regions that encode regulatory RNA elements. It is therefore beneficial to use high-density tiling-arrays that can cover entire chromosomes and even entire genomes represented by millions of oligonucleotides on glass chips. Whole genome tiling arrays are already available for several species and will soon be available for the entire human genome (1-3).
This Methylation Analysis provides the following benefits:
* No need for tedious cloning of PCR products
* Whole genome coverage
* No bisulfite conversion necessary.
* Rapid, semi-quantitative assessment of the degree of methylation
* Detection of methylation levels as low as 5% in sample mixtures
* Results may be obtained from various sample types, e.g. blood, frozen tissue, mouth swabs or paraffin embedded tissue
* High precision, reproducibility and throughput on an established microarry platform
References 1. Schumacher, A., Kapranov, P., Kaminsky, Z., Flanagan, J., Assadzadeh, A., Yau, P., Virtanen, C., Winegarden, N., Cheng, J., Gingeras, T., and Petronis, A. (2006) Microarray-based DNA methylation profiling: technology and applications. Nucleic Acids Res 34, 528-542.
2. Cheng, J., Kapranov, P., Drenkow, J., Dike, S., Brubaker, S., Patel, S., Long, J., Stern, D., Tammana, H., Helt, G., Sementchenko, V., Piccolboni, A., Bekiranov, S., Bailey, D. K., Ganesh, M., Ghosh, S., Bell, I., Gerhard, D. S., and Gingeras, T. R. (2005) Transcriptional maps of 10 human chromosomes at 5-nucleotide resolution. Science 308, 1149-1154.
3. Schumacher, A., and Petronis, A. (2006) Epigenetics of complex disease: from general theory to laboratory praxis. Curr Top Microbiol Immunol 310, 81-115.
4. Schumacher, A. (2001) Mechanisms and brain specific consequences of genomic imprinting in Prader-Willi and Angelman syndromes. Gene Funct. Dis. 1, 7-25.
5. Schumacher, A., Arnhold, S., Addicks, K., and Doerfler, W. (2003) Staurosporine is a potent activator of neuronal, glial, and "CNS stem cell-like" neurosphere differentiation in murine embryonic stem cells. Mol Cell Neurosci 23, 669-680.
6. Yamamoto, F., and Yamamoto, M. (2004) A DNA microarray-based methylation-sensitive (MS)-AFLP hybridization method for genetic and epigenetic analyses. Mol Genet Genomics 271, 678-686.
7. Li, J., Protopopov, A., Wang, F., Senchenko, V., Petushkov, V., Vorontsova, O., Petrenko, L., Zabarovska, V., Muravenko, O., Braga, E., Kisselev, L., Lerman, M. I., Kashuba, V., Klein, G., Ernberg, I., Wahlestedt, C., and Zabarovsky, E. R. (2002) NotI subtraction and NotI-specific microarrays to detect copy number and methylation changes in whole genomes. Proc Natl Acad Sci U S A 99, 10724-10729.
8. Schumacher, A., Friedrich, P., Schmid, J., Ibach, B., Eisele, T., Laws, S. M., Foerstl, H., Kurz, A., and Riemenschneider, M. (2006) No association of chromatin-modifying protein 2B with sporadic frontotemporal dementia. Neurobiol Aging, epub.
9. Rakyan, V. K., Hildmann, T., Novik, K. L., Lewin, J., Tost, J., Cox, A. V., Andrews, T. D., Howe, K. L., Otto, T., Olek, A., Fischer, J., Gut, I. G., Berlin, K., and Beck, S. (2004) DNA methylation profiling of the human major histocompatibility complex: a pilot study for the human epigenome project. PLoS Biol 2, e405.
