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Proof of principle of novel technique of methylation analysis

ABSTRACT: 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 methylcytosine to detect the presence of 5 methylcytosine in genomic DNA from human fibroblasts bearing the karyotype 45 XO. We report that over 2900 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. A method that detects DNA methylation that is not restricted to specific sequences would provide a useful platform to analyse methylation distribution among the genes in any given phenotypic condition.Availability of additional approaches to examine DNA methylation would lead to increase in our understanding of the methylome and would help define the intrinsic and extrinsic factors which govern it. Microarray based high throughput methods have been extensively used for gene expression analysis. The method described uses a microarray to analyse DNA methylation levels in a gene/cDNA/oligo specific manner. Here, genomic DNA is fragmented and used for hybridization to microarray slides. The presence of DNA cytosine methylation in the hybridized DNA is detected by employing Cy3 labelled anti 5mC( 5-methyl cytidine) antibody (monoclonal). The resolution provided by this method is dependent upon the microarray platform used. This method can be adapted to any platform thus enabling seamless integration with existing technology of gene expression analysis

ORGANISM(S): Homo sapiens

PROVIDER: GSE20873 | GEO | 2010/04/09

SECONDARY ACCESSION(S): PRJNA124665

REPOSITORIES: GEO

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Project description:Cytosine methylation in the genome of Drosophila melanogaster has been elusive and controversial: methylcytosine has been detected at very low levels in early embryos, but the genomic location and function of methylation has not been established. We have mapped cytosine methylation genomewide in Stage 5 Drosophila embryo DNA by combining immuno-enrichment for 5-methylcytosine, bisulfite conversion, and deep sequencing. Unlike methylation patterns observed in other eukaryotic species, methylation in Drosophila is punctate and highly strand-asymmetrical; we confirmed this by direct PCR amplification and sequencing of bisulfite-converted DNA. Despite the locally asymmetric nature of methylation, large-scale patterns of methylation are symmetric. Methylated regions make up ~1% of the genome, and within these regions methylation of individual cytosines averages 2-10%. Methylation is concentrated in specific 5-base sequence motifs that are CA- and CT-rich but depleted of guanine. It is depleted from promoters, coding sequences, and most retrotransposons, and enriched in introns and in certain simple sequence repeats containing the commonly methylated motifs. Comparison with available gene expression data indicates that methylation in a gene is associated with lower expression; the X chromosome, which is subject to gene dosage compensation, is more densely methylated than the autosomes. This study firmly establishes the presence of cytosine methylation in Drosophila; the temporal overlap of methylation with the maternal-zygotic transition raises the possibility that methylation participates in the transition to zygotic gene expression. To enrich for rare cytosine methylation in Drosophila at embryonic Stage 5 (2-3 hours post-fertilization), we enriched sonicated Stage 5 genomic DNA for methylcytosine by immunoprecipitation with antibody to 5-methylcytosine. The immunoprecipitated DNA was then bisulfite converted and Illumina sequenced to obtain direct evidence for the presence of methylation. The presence and extent of DNA methylation was confirmed by Illumina sequencing of bisulfite-converted PCR amplicons.

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Proof of principle of novel technique of methylation analysis

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