Location, location, location: methylation analysis for epigenetic research

Epigenetics

No, we’re not talking about real estate! In research, location is key to how DNA methylation works. Epigenetics, the study of chromatin-modifying marks that do not alter DNA sequences, has recently undergone a paradigm shift regarding the effect of DNA methylation on gene transcription. In the human genome, the most commonly studied epigenetic mark is 5-methylcytosine, often found in the sequence CpG. Methylation has been extensively studied in CpG islands near the transcriptional start site of genes, and the effect on transcription has been evaluated in this context (1). However, recent genome-wide methylation capabilities have changed this view considerably. Methylation within the gene body may actually stimulate transcriptional elongation and may also affect RNA splicing (2). The effect of methylation at other sites in the genome, such as enhancers, insulators and other regulatory elements, is currently under investigation.

The standard workflow for methylation analysis consists of sample collection, DNA purification, methylation-dependent DNA treatment, amplification (if necessary) and DNA analysis. One of the most critical steps in the workflow is DNA treatment which differentially modifies methylated versus unmethylated DNA. This can be accomplished by three methods – endonuclease digestion, affinity enrichment and bisulfite treatment (1). Endonuclease digestion is the classic technique for methylation analysis, and relies upon methylation-sensitive restriction enzymes such as HpaII, which only cleave unmethylated sites. The resulting pattern of cut versus uncut DNA provides inforamtion about methylation status at each restriction site. With this method, incomplete digestion will generate false positives that are unrelated to methylation. Affinity enrichment utilizes methylation-specific antibodies for chromatin immunoprecipitation (ChIP), followed by array hybridization or next-generation sequencing (NGS). Input DNA must be compared to immunoprecipitated DNA for comparison and to confirm enrichment.

Bisulfite treatment transformed DNA methylation analysis in the 1990s (1). Bisulfite converts unmethylated cytosine to uracil, leaving 5-methylcytosine intact. The resulting modification in DNA sequence can be read by standard techniques. DNA must be denatured before conversion, and the reaction is performed in high-salt, high-temperature conditions, followed by purification to remove contaminants. The bisulfite reaction should be complete in order to obtain accurate and reliable results.

The analysis of methylated DNA can be accomplished by several methods. Individual loci can be examined by PCR. In the case of bisulfite-treated DNA, methylation-dependent sequence differences can be used to design specific primers or probes (in the case of real-time PCR). Array-based techniques can be methylation-specific or, alternatively, a comparative sample of untreated DNA may be used. NGS, including methyl-seq, is emerging as a favored technique over array-based analysis. Continuing improvements in genome-wide methylation analysis have revolutionized our understanding of the role methylation plays in gene regulation.

References

1. Laird, P.W. (2010) Principles and challenges of genomewide DNA methylation analysis. Nat. Rev. Genet. 11, 191.

2. Jones, P.A. (2012) Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat. Rev. Genet. 13, 484.

Abhishek Sharma, Msc., MBA

Senior Global Market Manager, Discovery Sciences

Abhishek Sharma trained as a biochemist and has hands-on experience in nucleic acid and protein purification, tissue culturing and recombinant DNA technology. Previously, he was as a market analyst on emerging technologies in life science research. Sharma also worked in a USA-based healthcare consultancy on the discovery, development and commercialization of new disease treatments across multiple therapeutic areas. Currently, he’s involved with managing QIAGEN’s sample preparation portfolio, specializing in RNA technologies.

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