Skip to main content
SpringerLink
Log in

High-resolution mapping of DNA methylation in human genome using oligonucleotide tiling array

  • Original Investigation
  • Published:
Human Genetics Aims and scope Submit manuscript

Abstract

DNA methylation is an epigenetic mark crucial in regulation of gene expression. Aberrant DNA methylation causes silencing of tumor suppressor genes and promotes chromosomal instability in human cancers. Most of previous studies for DNA methylation have focused on limited genomic regions, such as selected genes or promoter CpG islands (CGIs) containing recognition sites of methylation-sensitive restriction enzymes. Here, we describe a method for high-resolution analysis of DNA methylation using oligonucleotide tiling arrays. The input material is methylated DNA immunoprecipitated with anti-methylcytosine antibodies. We examined the ENCODE region (∼1% of human genome) in three human colorectal cancer cell lines and identified over 700 candidate methylated sites (CMS), where 24 of 25 CMS selected randomly were subsequently verified by bisulfite sequencing. CMS were enriched in the 5′ regulatory regions and the 3′ regions of genes. We also compared DNA methylation patterns with histone H3 and H4 acetylation patterns in the HOXA cluster region. Our analysis revealed no acetylated histones in the hypermethylated region, demonstrating reciprocal relationship between DNA methylation and histone H3 and H4 acetylation. Our method recognizes DNA methylation with little bias by genomic location and, therefore, is useful for comprehensive high-resolution analysis of DNA methylation providing new findings in the epigenomics.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Bernstein BE, Kamal M, Lindblad-Toh K, Bekiranov S, Bailey DK, Huebert DJ, McMahon S, Karlsson EK, Kulbokas EJ III, Gingeras TR, Schreiber SL, Lander ES (2005) Genomic maps and comparative analysis of histone modifications in human and mouse. Cell 120:169–181

    Article  PubMed  CAS  Google Scholar 

  • Bird A (2002) DNA methylation patterns and epigenetic memory. Genes Dev 16:6–21

    Article  PubMed  CAS  Google Scholar 

  • Bolstad BM, Irizarry RA, Astrand M, Speed TP (2003) A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19:185–193

    Article  PubMed  CAS  Google Scholar 

  • Cawley S, Bekiranov S, Ng HH, Kapranov P, Sekinger EA, Kampa D, Piccolboni A, Sementchenko V, Cheng J, Williams AJ, Wheeler R, Wong B, Drenkow J, Yamanaka M, Patel S, Brubaker S, Tammana H, Helt G, Struhl K, Gingeras TR (2004) Unbiased mapping of transcription factor binding sites along human chromosomes 21 and 22 points to widespread regulation of noncoding RNAs. Cell 116:499–509

    Article  PubMed  CAS  Google Scholar 

  • Cooper SJ, Trinklein ND, Anton ED, Nguyen L, Myers RM (2006) Comprehensive analysis of transcriptional promoter structure and function in 1% of the human genome. Genome Res 16:1–10

    Article  PubMed  CAS  Google Scholar 

  • Ding S, Gong BD, Yu J, Gu J, Zhang HY, Shang ZB, Fei Q, Wang P, Zhu JD (2004) Methylation profile of the promoter CpG islands of 14 “drug-resistance” genes in hepatocellular carcinoma. World J Gastroenterol 10:3433–3440

    PubMed  CAS  Google Scholar 

  • Ehrlich M (2002) DNA methylation in cancer: too much, but also too little. Oncogene 21:5400–5413

    Article  PubMed  CAS  Google Scholar 

  • ENCODE Project Consortium (2004) The ENCODE (ENCyclopedia Of DNA Elements) Project. Science 306:636–640

    Google Scholar 

  • Feinberg AP, Ohlsson R, Henikoff S (2006) The epigenetic progenitor origin of human cancer. Nat Rev Genet 7:21–33

    Article  PubMed  CAS  Google Scholar 

  • Fiegl H, Millinger S, Mueller-Holzner E, Marth C, Ensinger C, Berger A, Klocker H, Goebel G, Widschwendter M (2005) Circulating tumor-specific DNA: a marker for monitoring efficacy of adjuvant therapy in cancer patients. Cancer Res 65:1141–1145

    Article  PubMed  CAS  Google Scholar 

  • Gardiner-Garden M, Frommer M (1987) CpG islands in vertebrate genomes. J Mol Biol 196:261–282

    Article  PubMed  CAS  Google Scholar 

  • Hagihara A, Miyamoto K, Furuta J, Hiraoka N, Wakazono K, Seki S, Fukushima S, Tsao MS, Sugimura T, Ushijima T (2004) Identification of 27 5′ CpG islands aberrantly methylated and 13 genes silenced in human pancreatic cancers. Oncogene 23:8705–8710

    Article  PubMed  CAS  Google Scholar 

  • Hatada I, Fukasawa M, Kimura M, Morita S, Yamada K, Yoshikawa T, Yamanaka S, Endo C, Sakurada A, Sato M, Kondo T, Horii A, Ushijima T, Sasaki H (2006) Genome-wide profiling of promoter methylation in human. Oncogene 25:3059–3064

    Article  PubMed  CAS  Google Scholar 

  • Hayashizaki Y, Hirotsune S, Okazaki Y, Hatada I, Shibata H, Kawai J, Hirose K, Watanabe S, Fushiki S, Wada S, Sugimoto T, Kobayakawa K, Kawara T, Katsuki M, Shibuya T, Mukai T (1993) Restriction landmark genomic scanning method and its various applications. Electrophoresis 14:251–258

    Article  PubMed  CAS  Google Scholar 

  • http://www.genome.ucsc.edu/cgi-bin/hgGateway, UCSC genome browser

  • Huang TH, Perry MR, Laux DE (1999) Methylation profiling of CpG islands in human breast cancer cells. Hum Mol Genet 8:459–470

    Article  PubMed  CAS  Google Scholar 

  • Ishikawa S, Komura D, Tsuji S, Nishimura K, Yamamoto S, Panda B, Huang J, Fukayama M, Jones KW, Aburatani H (2005) Allelic dosage analysis with genotyping microarrays. Biochem Biophys Res Commun 333:1309–1314

    Article  PubMed  CAS  Google Scholar 

  • Jones PA, Baylin SB (2002) The fundamental role of epigenetic events in cancer. Nat Rev Genet 3:415–428

    Article  PubMed  CAS  Google Scholar 

  • Jones PA, Martienssen R (2005) A blueprint for a human epigenome project: the AACR human epigenome workshop. Cancer Res 65:11241–11246

    Article  PubMed  CAS  Google Scholar 

  • Kampa D, Cheng J, Kapranov P, Yamanaka M, Brubaker S, Cawley S, Drenkow J, Piccolboni A, Bekiranov S, Helt G, Tammana H, Gingeras TR (2004) Novel RNAs identified from an in-depth analysis of the transcriptome of human chromosomes 21 and 22. Genome Res 14:331–342

    Article  PubMed  CAS  Google Scholar 

  • Kaneda A, Kaminishi M, Nakanishi Y, Sugimura T, Ushijima T (2002) Reduced expression of the insulin-induced protein 1 and p41 Arp2/3 complex genes in human gastric cancers. Int J Cancer 100:57–62

    Article  PubMed  CAS  Google Scholar 

  • Kapranov P, Cawley SE, Drenkow J, Bekiranov S, Strausberg RL, Fodor SP, Gingeras TR (2002) Large-scale transcriptional activity in chromosomes 21 and 22. Science 296:916–919

    Article  PubMed  ADS  CAS  Google Scholar 

  • Katou Y, Kaneshiro K, Aburatani H, Shirahige K (2006) Genomic approach for the understanding of dynamic aspect of chromosome behavior. Methods Enzymol 409:389–410

    PubMed  Google Scholar 

  • Kim TH, Barrera LO, Qu C, Van Calcar S, Trinklein ND, Cooper SJ, Luna RM, Glass CK, Rosenfeld MG, Myers RM, Ren B (2005) Direct isolation and identification of promoters in the human genome. Genome Res 15:830–839

    Article  PubMed  CAS  Google Scholar 

  • Kmita M, Tarchini B, Zakany J, Logan M, Tabin CJ, Duboule D (2005) Early developmental arrest of mammalian limbs lacking HoxA/HoxD gene function. Nature 435:1113–1116

    Article  PubMed  ADS  CAS  Google Scholar 

  • Li Q, Peterson KR, Fang X, Stamatoyannopoulos G (2002) Locus control regions. Blood 100:3077–3086

    Article  PubMed  CAS  Google Scholar 

  • Liu CL, Schreiber SL, Bernstein BE (2003) Development and validation of a T7 based linear amplification for genomic DNA. BMC Genomics 4:19

    Article  PubMed  CAS  Google Scholar 

  • Lorincz MC, Dickerson DR, Schmitt M, Groudine M (2004) Intragenic DNA methylation alters chromatin structure and elongation efficiency in mammalian cells. Nat Struct Mol Biol 11:1068–1075

    Article  PubMed  CAS  Google Scholar 

  • Müller HM, Oberwalder M, Fiegl H, Morandell M, Goebel G, Zitt M, Mühlthaler M, Öfner D, Margreiter R, Widschwendter M (2004) Methylation changes in faecal DNA: a marker for colorectal cancer screening? Lancet 363:1283–1285

    Article  PubMed  Google Scholar 

  • Munroe SH (2004) Diversity of antisense regulation in eukaryotes: multiple mechanisms, emerging patterns. J Cell Biochem 93:664–671

    Article  PubMed  CAS  Google Scholar 

  • Palmisano WA, Divine KK, Saccomanno G, Gilliland FD, Baylin SB, Herman JG, Belinsky SA (2000) Predicting lung cancer by detecting aberrant promoter methylation in sputum. Cancer Res 60:5954–5958

    PubMed  CAS  Google Scholar 

  • Poon LL, Leung TN, Lau TK, Chow KC, Lo YM (2002) Differential DNA methylation between fetus and mother as a strategy for detecting fetal DNA in maternal plasma. Clin Chem 48:35–41

    PubMed  CAS  Google Scholar 

  • Rada-Iglesias A, Wallerman O, Koch C, Ameur A, Enroth S, Clelland G, Wester K, Wilcox S, Dovey OM, Ellis PD, Wraight VL, James K, Andrews R, Langford C, Dhami P, Carter N, Vetrie D, Ponten F, Komorowski J, Dunham I, Wadelius C (2005) Binding sites for metabolic disease related transcription factors inferred at base pair resolution by chromatin immunoprecipitation and genomic microarrays. Hum Mol Genet 14:3435–3447

    Article  PubMed  CAS  Google Scholar 

  • Rakyan VK, Hildmann T, Novik KL, Lewin J, Tost J, Cox AV, Andrews TD, Howe KL, Otto T, Olek A, Fischer J, Gut IG, Berlin K, Beck S (2004) DNA methylation profiling of the human major histocompatibility complex: a pilot study for the human epigenome project. PLoS Biol 2:e405

    Article  PubMed  Google Scholar 

  • Rollins RA, Haghighi F, Edwards JR, Das R, Zhang MQ, Ju J, Bestor TH (2005) Large-scale structure of genomic methylation patterns. Genome Res 16:157–163

    Article  PubMed  Google Scholar 

  • Ropero S, Setien F, Espada J, Fraga MF, Herranz M, Asp J, Benassi MS, Franchi A, Patino A, Ward LS, Bovee J, Cigudosa JC, Wim W, Esteller M (2004) Epigenetic loss of the familial tumor-suppressor gene exostosin-1 (EXT1) disrupts heparan sulfate synthesis in cancer cells. Hum Mol Genet 13:2753–2765

    Article  PubMed  CAS  Google Scholar 

  • Santini S, Boore JL, Meyer A (2003) Evolutionary conservation of regulatory elements in vertebrate Hox gene clusters. Genome Res 13:1111–1122

    Article  PubMed  CAS  Google Scholar 

  • Schmitt JF, Millar DS, Pedersen JS, Clark SL, Venter DJ, Frydenberg M, Molloy PL, Risbridger GP (2002) Hypermethylation of the inhibin alpha-subunit gene in prostate carcinoma. Mol Endocrinol 16:213–220

    Article  PubMed  CAS  Google Scholar 

  • Schubeler D, Lorincz MC, Cimbora DM, Telling A, Feng YQ, Bouhassira EE, Groudine M (2000) Genomic targeting of methylated DNA: influence of methylation on transcription, replication, chromatin structure, and histone acetylation. Mol Cell Biol 20:9103–9112

    Article  PubMed  CAS  Google Scholar 

  • Shiraishi M, Sekiguchi A, Oates AJ, Terry MJ, Miyamoto Y (2002) HOX gene clusters are hotspots of de novo methylation in CpG islands of human lung adenocarcinomas. Oncogene 21:3659–3662

    Article  PubMed  CAS  Google Scholar 

  • Song F, Smith JF, Kimura MT, Morrow AD, Matsuyama T, Nagase H, Held WA (2005) Association of tissue-specific differentially methylated regions (TDMs) with differential gene expression. Proc Natl Acad Sci USA 102:3336–3341

    Article  PubMed  ADS  CAS  Google Scholar 

  • Suzuki H, Watkins DN, Jair KW, Schuebel KE, Markowitz SD, Chen WD, Pretlow TP, Yang B, Akiyama Y, Van Engeland M, Toyota M, Tokino T, Hinoda Y, Imai K, Herman JG, Baylin SB (2004) Epigenetic inactivation of SFRP genes allows constitutive WNT signaling in colorectal cancer. Nat Genet 36:417–422

    Article  PubMed  CAS  Google Scholar 

  • Takai D, Jones PA (2002) Comprehensive analysis of CpG islands in human chromosomes 21 and 22. Proc Natl Acad Sci USA 99:3740–3745

    Article  PubMed  ADS  CAS  Google Scholar 

  • Ushijima T, Morimura K, Hosoya Y, Okonogi H, Tatematsu M, Sugimura T, Nagao M (1997) Establishment of methylation-sensitive-representational difference analysis and isolation of hypo- and hypermethylated genomic fragments in mouse liver tumors. Proc Natl Acad Sci USA 94:2284–2289

    Article  PubMed  ADS  CAS  Google Scholar 

  • Warnecke PM, Stirzaker C, Song J, Grunau C, Melki JR, Clark SJ (2002) Identification and resolution of artifacts in bisulfite sequencing. Methods 27:101–107

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Widschwendter A, Muller HM, Fiegl H, Ivarsson L, Wiedemair A, Muller-Holzner E, Goebel G, Marth C, Widschwendter M (2004) DNA methylation in serum and tumors of cervical cancer patients. Clin Cancer Res 10:565–571

    Article  PubMed  CAS  Google Scholar 

  • Yelin R, Dahary D, Sorek R, Levanon EY, Goldstein O, Shoshan A, Diber A, Biton S, Tamir Y, Khosravi R, Nemzer S, Pinner E, Walach S, Bernstein J, Savitsky K, Rotman G (2003) Widespread occurrence of antisense transcription in the human genome. Nat Biotechnol 21:379–386

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Hiroko Meguro and Kaori Shiina for technical assistance. This study was supported by Grants-in-Aid for Scientific Research (S) 16101006 from the Ministry of Education, Culture, Sports, Science and Technology, the Program of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO), by NFAT project of New Energy and Industrial Technology Development Organization (NEDO) and by Special Coordination Fund for Science and Technology from Ministry of Education, Culture, Sports, Science and Technology.

Author information

Authors and Affiliations

  1. Genome Science Division, Research Center for Advanced Science and Technology (RCAST), The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8904, Japan

    Hiroshi Hayashi, Genta Nagae, Shuichi Tsutsumi, Kiyofumi Kaneshiro, Atsushi Kaneda & Hiroyuki Aburatani

  2. Department of Technology Development and Assessment, IVD, SRL Inc., Tokyo, 191-0002, Japan

    Hiroshi Hayashi, Takazumi Kozaki & Hajime Sugisaki

Authors
  1. Hiroshi Hayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Genta Nagae
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shuichi Tsutsumi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kiyofumi Kaneshiro
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Takazumi Kozaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Atsushi Kaneda
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hajime Sugisaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Hiroyuki Aburatani
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hiroyuki Aburatani.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hayashi, H., Nagae, G., Tsutsumi, S. et al. High-resolution mapping of DNA methylation in human genome using oligonucleotide tiling array. Hum Genet 120, 701–711 (2007). https://doi.org/10.1007/s00439-006-0254-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00439-006-0254-6

Keywords

Search

Navigation