Abstract
Alteration in epigenetic regulation of gene expression is a common event in human cancer and developmental disease. CpG island hypermethylation and consequent gene silencing is observed for many genes involved in a diverse range of functions and pathways that become deregulated in the disease state. Comparative profiling of the methylome is therefore useful in disease gene discovery. The ability to identify epigenetic alterations on a global scale is imperative to understanding the patterns of gene silencing that parallel disease progression. Methylated DNA immunoprecipitation (MeDIP) is a technique that isolates methylated DNA fragments by immunoprecipitating with 5′-methylcytosine-specific antibodies. The enriched methylated DNA can then be analyzed in a locus-specific manner using PCR assay or in a genome-wide fashion by comparative genomic hybridization against a sample without MeDIP enrichment. This article describes the detailed protocol for MeDIP and hybridization of MeDIP DNA to a whole-genome tiling path BAC array.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Esteller M, Herman JG. (2002) Cancer as an epigenetic disease: DNA methylation and chromatin alterations in human tumours. J Pathol. 196, 1–7.
Feinberg AP. (2007) Phenotypic plasticity and the epigenetics of human disease. Nature 447, 433–440.
Ozanne SE, Constancia M. (2007) Mechanisms of disease: the developmental origins of disease and the role of the epigenotype. Nat Clin Pract Endocrinol Metab. 3, 539–546.
Shames DS, Minna JD, Gazdar AF. (2007) DNA methylation in health, disease, and cancer. Curr Mol Med. 7, 85–102.
Jones PA, Baylin SB. (2002) The fundamental role of epigenetic events in cancer. Nat Rev Genet. 3, 415–428.
Feinberg AP, Ohlsson R, Henikoff S. (2006) The epigenetic progenitor origin of human cancer. Nat Rev Genet. 7, 21–33.
Glasspool RM, Teodoridis JM, Brown R. (2006) Epigenetics as a mechanism driving polygenic clinical drug resistance. Br J Cancer. 94, 1087–1092.
Chekhun VF, Lukyanova NY, Kovalchuk O, Tryndyak VP, Pogribny IP. (2007) Epigenetic profiling of multidrug-resistant human MCF-7 breast adenocarcinoma cells reveals novel hyper- and hypomethylated targets. Mol Cancer Ther. 6, 1089–1098.
Shames DS, Girard L, Gao B, Sato M, Lewis CM, Shivapurkar N, Jiang A, Perou CM, Kim YH, Pollack JR, Fong KM, Lam CL, et al. (2006) A genome-wide screen for promoter methylation in lung cancer identifies novel methylation markers for multiple malignancies. PLoS Med. 3, e486.
Gryn J, Zeigler ZR, Shadduck RK, Lister J, Raymond JM, Sbeitan I, Srodes C, Meisner D, Evans C. (2002) Treatment of myelodysplastic syndromes with 5-azacytidine. Leuk Res. 26, 893–897.
Weber M, Davies JJ, Wittig D, Oakeley EJ, Haase M, Lam WL, Schubeler D. (2005) Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet. 37, 853–862.
Wilson IM, Davies JJ, Weber M, Brown CJ, Alvarez CE, MacAulay C, Schubeler D, Lam WL. (2006) Epigenomics: mapping the methylome. Cell Cycle 5, 155–158.
Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW, Molloy PL, Paul CL. (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci U S A 89, 1827–1831.
Gonzalgo ML, Liang G, Spruck CH, 3rd, Zingg JM, Rideout WM, 3rd, Jones PA. (1997) Identification and characterization of differentially methylated regions of genomic DNA by methylation-sensitive arbitrarily primed PCR. Cancer Res. 57, 594–599.
Huang TH, Laux DE, Hamlin BC, Tran P, Tran H, Lubahn DB. (1997) Identification of DNA methylation markers for human breast carcinomas using the methylation-sensitive restriction fingerprinting technique. Cancer Res. 57, 1030–1034.
Livak KJ, Schmittgen TD. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408.
Coe BP, Ylstra B, Carvalho B, Meijer GA, Macaulay C, Lam WL. (2007) Resolving the resolution of array CGH. Genomics 89, 647–653.
Khojasteh M, Lam WL, Ward RK, MacAulay C. (2005) A stepwise framework for the normalization of array CGH data. BMC Bioinformatics 6, 274.
Branham WS, Melvin CD, Han T, Desai VG, Moland CL, Scully AT, Fuscoe JC. (2007) Elimination of laboratory ozone leads to a dramatic improvement in the reproducibility of microarray gene expression measurements. BMC Biotechnol. 7, 8.
Chi B, DeLeeuw RJ, Coe BP, MacAulay C, Lam WL. (2004) SeeGH – a software tool for visualization of whole genome array comparative genomic hybridization data. BMC Bioinformatics. 5, 13.
Chari R, Lockwood WW, Coe BP, Chu A, Macey D, Thomson A, Davies JJ, MacAulay C, Lam WL. (2006) SIGMA: a system for integrative genomic microarray analysis of cancer genomes. BMC Genomics 7, 324.
Acknowledgments
The authors wish to thank Bradley Coe, Chad Malloff, and Spencer Watson for useful discussion and assistance with this manuscript. This work was supported by funds from the Canadian Institutes for Health Research, Canadian Breast Cancer Research Alliance, Genome Canada/British Columbia, and National Institute of Dental and Craniofacial Research (NIDCR) grant R01 DE15965.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Vucic, E.A., Wilson, I.M., Campbell, J.M., Lam, W.L. (2009). Methylation Analysis by DNA Immunoprecipitation (MeDIP). In: Pollack, J. (eds) Microarray Analysis of the Physical Genome. Methods in Molecular Biology™, vol 556. Humana Press. https://doi.org/10.1007/978-1-60327-192-9_10
Download citation
DOI: https://doi.org/10.1007/978-1-60327-192-9_10
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-191-2
Online ISBN: 978-1-60327-192-9
eBook Packages: Springer Protocols