HELP (HpaII Tiny Fragment Enrichment by Ligation-Mediated PCR) Assay for DNA Methylation Profiling of Primary Normal and
The role of cytosine methylation in the regulation of gene expression during normal development and malignant transformation is currently under intense investigation. An ever increasing body of evidence demonstrates that carcinogenesis is associated with
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ethylation is found on position five of the cytosine ring at CpG dinucleotides and plays a diverse set of roles, from embryogenesis and imprinting in development to gene silencing in Sridar V. Chittur (ed.), Microarray Methods for Drug Discovery, Methods in Molecular Biology, vol. 632, DOI 10.1007/978-1-60761-663-4_12, © Humana Press, a part of Springer Science+Business Media, LLC 2010
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disease (12, 13). Cytosine methylation is associated with repressed chromatin state and gene silencing (14–16). It cooperates with histone-tail modifications to achieve chromatin remodeling and to affect protein expression. Cells contain an evolutionaryconserved machinery of DNA methyltransferases and methylbinding proteins, which bind methylated cytosine and interact with the transcription machinery (17–19). DNA methylation undergoes dramatic changes during tumorigenesis, resulting in global losses in methylation from repetitive sequences as well as in promoter-specific gain of methylation (20–24). Khulan et al. demonstrated the utility of HELP assay by interrogating 6.2 Mb of mouse genome at 1,399 HpaII sites (25). By comparing mouse brain tissue and spermatogenic cells, they were able to identify 223 new tissue-specific differentially methylated regions. In addition, the assay allowed defining the profile of methylation: with most of the genome being methylated with clusters of hypomethylated areas at promoters and CpG islands. Figueroa et al. applied HELP to the study of human leukemias. In the subset of Acute Myelogenous Leukemias and Precursor B Lymphoblastic Leukemias, it was possible to correctly subclassify 2 subtypes of leukemias based on their HELP profiles, capturing the methylation signature of the two disease states (26). This study also demonstrated that integration of gene expression profiling with methylation profiling allowed to capture genes differentially expressed between two leukemia subtypes that were not identified by gene expression alone. This work demonstrates the utility of whole-genome profiling approach to study epigenomic signatures. Study of a subgroup of Myeloid Leukemias with the common gene expression profile, but with different CEBPA-locus status (mutated vs. hypermethylated wild type) also demonstrated the sensitivity and utility of the HELP assay in revealing the presence of underlying DNA methylation differences (27). The HELP assay is based on the principle of comparative isoschizomer profiling of cytosine methylation in the HpaII Tiny Fragment (HTF) fraction of the genome (25). This approach allows effective profiling of DNA methylation in CpG rich regions of the genome such as promoter associated CpG islands or CG clusters (28, 29). The assay is based on using 2 isoschizomer enzymes: HpaII, which is methylation-sensitive and only cuts unmethylated CCGG, and MspI, which cuts CCGG whether it is methylated or not. Purified, high molecular weight genomic DNA is digested to completion in 2 separate reactions with HpaII and MspI, after which linker oligo
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