A novel method to assess the full genome methylation profile using monoclonal antibody combined

REsEARch pApER

Epigenetics 4:6, 415-420; August 16, 2009; © 2009 Landes Bioscience

A novel method to assess the full genome methylation profile using monoclonal antibody combined with the high throughput based

microarray approach

Ashwin Kelkar1 and Deepti Deobagkar2,*

1

Senior Research Fellow; Molecular Biology Research Laboratory; 2Department of Zoology; (Centre for Advanced Studies); University of Pune; Pune, India

Keywords: DNA methylation, epigenetics, methylation detection, 5 methyl cytosine, microarray

In this work we demonstrate a novel method of methylation detection that utilises immunoaffinity to detect the presence of methylated DNA hybridised to a cDNA microarray. We use a monoclonal antibody specific to 5 methyl cytidine to detect the presence of 5 methyl cytosine in genomic DNA from human fibroblasts bearing the karyotype 45 XO. We report that over 2,900 genes show the presence of methylation in this condition. We also report that 165 genes are consistently methylated in all replicates of these experiments. The methylated genes show a uniform distribution over all the chromosomes. The gene ontology of these also indicates no functional correlation between the genes that are methylated. We detect the presence of methylation in IGF2, an imprinted gene and thus known to harbour DNA methylation. The method is extremely specific and offers a quick and efficient way to analyse the methylation landscape on a high throughput scale. This method uses existing technology to assess methylation and thus can integrate very efficiently into any platform used.

Introduction

It has long been known that the genotypic variation is not the only reason for phenotypic variation. Epigenetic changes like DNA and histone modifications have been shown to be associ-ated with gene expression in humans.1 DNA methylation changes have been associated with a plethora of biological functions rang-ing from development,2 cancer biology3 X chromosome inactiva-tion to imprinting.4 Thus the measurement of DNA methylation levels in a genome wide manner would be useful in studying the mechanisms of epigenetic control that are involved in gene regu-lation and imprinting. Unlike other epigenetic modifications, DNA methylation adds an additional level of information on the primary DNA sequence itself. DNA cytosine methylation shows features of memory and heritability between generations and alterations in methylation patterns can lead to drastic changes in cellular function. An example of this phenomenon is the aberrant methylation of oncogenes in malignant cells.3 DNA methylation has been implicated in development, nutrigenomics, oncogenesis, imprinting and appears to have the ability to influence pheno-types.2-4 DNA methylation thus can act as an important signal in the ability of an organism to adapt to changing environments.There are many methods of assessing DNA methylation such as DNA melting analysis and semi quantitative PCR, restriction analysis followed by PCR, the use of specific primers to detect methylation on DNA sequences coupled with techniques like

bisulphite sequencing.5-7 CpG island amplification in combina-tion with microarrays is a very effective way to determine methy-lation of promoters.8 Although CpG islands have been implied to play a very important role in gene regulation in mammalian genomes, only 50–70% of the genes in the genome harbour CpG islands in their promoters.8,9 In addition to this, there is evidence of non CpG methylation that is present in the human genome. Any a method that determines DNA methylation at CpG islands while providing valuable information, may not be enough to give a through landscape of DNA methylation.10,11 Use of CpG island arrays for methylation analysis would therefore exclude methylation of genes that do not have detectable CpG islands.8 Alternatively enrichment of methylated DNA using immunoaf-finity or the use of methyl sensitive restriction enzymes are also extensively used to study methylation patterns. MSP or methyla-tion specific PCR is also used in clinical situations to assess the methylation of oncogenes in cases of cancer.

However, MSP is limited to detecting methylation in a speci-fied DNA sequence for example, MSP and bisulphite sequencing can detect methylation only in the region for which the amplifi-cation primers are designed and therefore requires prior knowl-edge of the sequence within the genes where methylation is to be assessed. Even though high throughput techniques like mas-sive parallel bisulphite sequencing of CG rich regions exist and has revealed voluminous data,12 the method is restricted to CpG methylation. In spite of availability of a variety of methods to

*Correspondence to: Deepti Deobagkar; Email: dddeo@unipune.ernet.in; deepti.deobagkar@gmail.comSubmitted: 08/06/09; Accepted: 08/18/09

Previously published online: doc.guandang.net

Epigenetics

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