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DNA Methylation Methods and Protocols 2nd Edition
Jörg Tost (Auth.) Digital Instant Download
Author(s): Jörg Tost (auth.), Jörg Tost (eds.)
ISBN(s): 9781597455220, 1597455229
Edition: 2
File Details: PDF, 16.96 MB
Year: 2009
Language: english
DNA Methylation
 M E T H O D S I N M O L E C U L A R B I O L O G Y TM
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 TM
METHODS IN MOLECULAR BIOLOGY

DNA Methylation
Methods and Protocols

Second Edition

Edited by

Jörg Tost, PhD

CEA - Institut de Génomique, Evry, France
Editor
Jörg Tost
CEA - Institut de Génomique
Evry
France
[email protected]

Series Editor
John M. Walker
University of Hertfordshire
Hatfield, Herts
UK

ISSN: 1064-3745 e-ISSN: 1940-6029

ISBN: 978-1-934115-61-9 e-ISBN: 978-1-59745-522-0
DOI 10.1007/978-1-59745-522-0

Library of Congress Control Number: 2008937946

c 2009 Humana Press, a part of Springer Science+Business Media, LLC

All rights reserved. This work may not be translated or copied in whole or in part without the written permission of
the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013,
USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of
information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology
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The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified
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Printed on acid-free paper

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Preface
Epigenetics can be defined as the study of heritable changes in gene expression without
alteration of the DNA sequence itself. This means that epigenetic variants are stable alter-
ations that are heritable during somatic cell divisions (and possibly transmitted through
germ line transmissions in some occasions) but do not involve mutations of the DNA
itself. Epigenetic phenomena are mediated by various molecular mechanisms, including
histone modifications and core histone variants; ATP-dependent chromatin-remodeling
complexes; polycomb/trithorax protein complexes; small RNAs, including siRNA and
miRNAs as well as other noncoding RNAs; and last but not least DNA methylation. This
volume in the Methods in Molecular BiologyTM series focuses entirely on protocols for
the analysis of DNA methylation, which is the only genetically programmed DNA mod-
ification in mammals occurring almost exclusively at the carbon 5 position of cytosines
followed by a guanine.
Realizing the importance of epigenetic changes in development and disease, a variety
of techniques for the study of DNA methylation have been developed over the last few
years. Figure 1 gives an overview of many of the commonly used technologies, but many
more methods and variants of the named assays do exist. No single method has emerged
as the “gold” standard technique unifying quantitative accuracy and high sensitivity or
possibilities for whole genome analysis and precise investigations of individual CpG posi-
tions. The choice of the method mainly depends on the desired application. Although
by no means complete, this second edition of “DNA methylation” gives a comprehen-
sive overview of available technologies together with detailed step-by-step protocols for
all experimental procedures required to successfully perform DNA methylation analysis.
This is the second edition of the DNA methylation protocols; however, the field has
dramatically changed within the 6 years that have passed since the first edition edited by
K.I. Mills and B.H. Ramsahoye was published. As DNA methylation technologies and
our knowledge of DNA methylation patterns have been advancing at a breathtaking pace
over the past few years and most of the techniques described in the first edition have been
further optimized and/or replaced by novel, easier, refined, and/or more quantitative
technologies, I have entirely remodeled the contents of this book. The increase in available
methods is also reflected in the great expansion of the number of chapters within this
book. While the first edition contained 14 chapters, this second edition consists now of
27 chapters. Only three chapters have been retained from the first edition and these have
been completely rewritten by the authors to accommodate the changes and improvements
made in the last years. The analysis of gene-specific DNA methylation patterns has been
complemented or superseded by genome-wide approaches and epigenomics has taken a
central place in many laboratories.
The selection of different technologies enables the analysis of the global DNA methy-
lation content as well as precise quantitative data on single CpG positions. Methods for
the high-resolution analysis of CpG positions within a target region identified by one of
the multiple available genome-wide technologies are presented, and emphasis has been
placed on array-based approaches that permit a hypothesis-free-driven research to identify

v
vi Preface

Fig. 1. An overview of the different technologies used for the analysis of DNA methylation. MS: Methylation sen-
sitive; HPLC: High-performance Liquid Chromatography; TLC: Thin-layer Chromatography; MS-AFLP: Methylation-
sensitive Amplified Fragment Length Polymorphism; MIAMI: Microarray-based Integrated Analysis of Methylation by
Isochizomers; HELP: HpaII tiny fragment Enrichment by Ligation-mediated PCR; MSNP: Methylation Single Nucleotide
Polymorphism; MS-AP-PCR: Methylation-sensitive Arbitrarily-primed PCR; MSRF: Methylation-sensitive Restriction Fin-
gerprinting; MS-RDA: Methylation-sensitive Representational Difference Analysis; MCA-RDA: Methylated CpG island
Amplification—Representational Difference Analysis; AIMS: Amplification of intermethylated Sites; RLGS: Restriction
Landmark Genomic Scanning; MeDIP: Methylated DNA ImmunoPrecipitation; MIRA: Methylated CpG Island Recovery
Assay; MSO: Methylation-specific Oligonucleotide array; MALDI: Matrix-assisted Laser Desorption/Ionization mass spec-
trometry; COBRA: Combined Bisulfite Restriction Analysis, MS-SNuPE: Methylation-sensitive Single Nucleotide Primer
extension; QAMA: Quantitative Analysis of Methylated Alleles. Reproduced with permission from Tost, J. (2008) Methods
for the genome-wide and gene-specific analysis of DNA methylation levels and patterns. In: Epigenetics (Tost, J., ed.),
Horizon Scientific Press, Norwich, UK, pp 63–103.

DNA methylation patterns of interest. In the final chapters of this book, more specialized
applications like the sensitive detection of aberrant methylation patterns in body fluids,
prevention of contamination, and whole genome amplification of bisulfite-treated DNA
are described. Methods requiring special instruments are presented along technologies
that can be performed with a simple thermocycler. This volume of the Methods in Molec-
ular BiologyTM series contains widely used methods, such as cloning and sequencing and
methylation-specific PCR as well as novel and promising techniques such as the immun-
odetection array that have only very recently passed the proof-of-principle stage.
This book is addressed to postdoctoral investigators and research scientists that are
implicated in the different aspects of genetics and cellular and molecular biology as well
as to clinicians involved in diagnostics or choice of treatment of diseases that have an epi-
genetic component. The presentation in this volume is equally suited for laboratories that
already have a great deal of expertise in a certain technology to analyze DNA methylation,
but might want to obtain other or complementary data using a second technique, and
for genetics/genomics/biology groups that want to initiate research in this exciting area
and want to identify the method best suited to answer their question. Notes and tips from
Preface vii

the experts and/or pioneers of the different methods will enable a rapid implementation
of the different protocols in the laboratory and avoid time-consuming and cost-intensive
mistakes. With the tools and protocols available, our knowledge and understanding of
DNA methylation will increase rapidly, and this book will contribute to spreading of the
“savoir faire” to analyze DNA methylation.
I am indebted to all the authors for their hard work and outstanding contributions to
this second edition of “DNA methylation”. It was a pleasure to work with them on this
project. I hope that the protocols described in detail in this volume will help to accelerate
the analysis and description of the “methylome” of different species and will enhance our
understanding of the molecular processes that determine the genomic DNA methylation
landscape.

Evry, March 2008 Jörg Tost

Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii

PART I. INTRODUCTION
1. DNA Methylation: An Introduction to the Biology
and the Disease-Associated Changes of a Promising Biomarker
Jörg Tost . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

PART II. GLOBAL METHYLATION LEVELS

2. Quantification of Global DNA Methylation by Capillary Electrophoresis
and Mass Spectrometry
Marı́a Berdasco, Mario F. Fraga, and Manel Esteller . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3. Methyl Group Acceptance Assay for the Determination of Global DNA
Methylation Levels
Kenneth P. Nephew, Curt Balch, and David G. Skalnik . . . . . . . . . . . . . . . . . . . . . . . . . 35

PART III. METHODS FOR WHOLE GENOME ANALYSIS OF DNA

METHYLATION PATTERNS
4. Immunodetection Array
Johannes Pröll, Christian Wechselberger, Mathilde Födermayr, Otto Zach,
and Dieter Lutz . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
5. Methylated DNA Immunoprecipitation (MeDIP)
Fabio Mohn, Michael Weber, Dirk Schübeler, and Tim-Christoph Roloff . . . . . . . . . . 55
6. The MIRA Method for DNA Methylation Analysis
Tibor A. Rauch and Gerd P. Pfeifer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
7. The HELP Assay
Mayumi Oda and John M. Greally . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
8. Differential Methylation Hybridization: Profiling DNA Methylation
with a High-Density CpG Island Microarray
Pearlly S. Yan, Dustin Potter, Daniel E. Deatherage, Shili Lin,
and Tim H.-M. Huang . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
9. Analysis of DNA Methylation by Amplification
of Intermethylated Sites (AIMS)
Mireia Jordà, Jairo Rodriguez, Jordi Frigola, and Miguel A. Peinado . . . . . . . . . . . 107
10. Methylation-Sensitive Representational Difference Analysis (MS-RDA)
Toshikazu Ushijima and Satoshi Yamashita . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

ix
x Contents

11. Restriction Landmark Genomic Scanning: Analysis of CpG Islands

in Genomes by 2D Gel Electrophoresis
Joseph F. Costello, Dominic J. Smiraglia, Chibo Hong,
and Christoph Plass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
12. GoldenGate
R Assay for DNA Methylation Profiling
Marina Bibikova and Jian-Bing Fan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
13. 5 -Azacytidine Expression Arrays
Paul Cairns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165

PART IV. GENE-SPECIFIC METHYLATION ANALYSIS

14. DNA Methylation Analysis by Bisulfite Conversion, Cloning,
and Sequencing of Individual Clones
Yingying Zhang, Christian Rohde, Sascha Tierling,
Heinrich Stamerjohanns, Richard Reinhardt, Jörn Walter,
and Albert Jeltsch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
15. Identification and Quantification of Differentially Methylated Loci
by the PyrosequencingTM Technology
Emelyne Dejeux, Hafida El abdalaoui, Ivo Glynne Gut, and Jörg Tost . . . . . . . . . . . 189
16. Mass Spectrometric Analysis of Cytosine Methylation by Base-Specific
Cleavage and Primer Extension Methods
Dirk van den Boom and Mathias Ehrich . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
17. Melting Curve Assays for DNA Methylation Analysis
Tomasz K. Wojdacz and Alexander Dobrovic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
18. Methylation SNaPshot: A Method for the Quantification
of Site-Specific DNA Methylation Levels
Zachary Kaminsky and Arturas Petronis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
19. Bio-COBRA: Absolute Quantification of DNA Methylation
in Electrofluidics Chips
Romulo Martin Brena and Christoph Plass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
20. Restriction Digestion and Real-Time PCR (qAMP)
Christopher C. Oakes, Sophie La Salle, Jacquetta M. Trasler,
and Bernard Robaire . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
21. MethylQuant: A Real-Time PCR-Based Method to Quantify DNA
Methylation at Single Specific Cytosines
Claire Dugast-Darzacq and Thierry Grange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
22. Methylation-Specific PCR
Julien D. F. Licchesi and James G. Herman . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
23. MethyLight
Mihaela Campan, Daniel J. Weisenberger, Binh Trinh,
and Peter W. Laird . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
24. Quantification of Methylated DNA by HeavyMethyl Duplex PCR
Jürgen Distler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
Contents xi

PART V. SPECIAL APPLICATIONS

25 Analysis of Methylated Circulating DNA in Cancer Patients’ Blood
Eiji Sunami, Anh-Thu Vu, Sandy L. Nguyen, and Dave S. B. Hoon . . . . . . . . . . . . . . 349
26 Prevention of PCR Cross-Contamination by UNG Treatment
of Bisulfite-Treated DNA
Reimo Tetzner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
27 Profiling DNA Methylation from Small Amounts of Genomic DNA
Starting Material: Efficient Sodium Bisulfite Conversion
and Subsequent Whole-Genome Amplification
Jonathan Mill and Arturas Petronis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
Contributors
CURT BALCH • Department of Cellular and Integrative Physiology, Medical Sciences
Program, Indiana University School of Medicine, Bloomington, IN, USA; Indiana
University Cancer Center, Indianapolis, IN, USA
MARÍA BERDASCO • Epigenetics Group, Molecular Pathology Program, Spanish National
Cancer Center (C.N.I.O.), Madrid, Spain
MARINA BIBIKOVA • Illumina, Inc., San Diego, CA, USA
ROMULO MARTIN BRENA • Division of Human Cancer Genetics, The Ohio State
University, Columbus, OH, USA
PAUL CAIRNS • Department of Surgical Oncology, Fox Chase Cancer Center,
Philadelphia, PA, USA
MIHAELA CAMPAN • Departments of Surgery and of Biochemistry and Molecular Biology,
University of Southern California, Keck School of Medicine, Norris Comprehensive
Cancer Center, Los Angeles, CA, USA
JOSEPH F. COSTELLO • Department of Neurological Surgery, University of California
San Francisco Comprehensive Cancer Center, San Francisco, CA, USA
DANIEL E. DEATHERAGE • Human Cancer Genetics Program, The Ohio State University
Comprehensive Cancer Center, Columbus, OH, USA
EMELYNE DEJEUX • Laboratory for Epigenetics, Centre National de Génotypage, CEA –
Institut de Génomique, Evry, France
JÜRGEN DISTLER • Epigenomics AG, Berlin, Germany
ALEXANDER DOBROVIC • Molecular Pathology Research and Development Laboratory,
Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, Australia;
Department of Pathology, University of Melbourne, Parkville, Australia
CLAIRE DUGAST-DARZACQ • Institut Jacques Monod du CNRS, Université Paris 7,
Paris, France
MATHIAS EHRICH • SEQUENOM, Inc., San Diego, CA, USA
HAFIDA EL ABDALAOUI • Laboratory for Epigenetics, Centre National de Génotypage,
CEA – Institut de Génomique, Evry, France
MANEL ESTELLER • Epigenetics Group, Molecular Pathology Program, Spanish National
Cancer Center (C.N.I.O.), Madrid, Spain
JIAN-BING FAN • Illumina, Inc., San Diego, CA, USA
MATHILDE FÖDERMAYR • Elisabethinen Hospital, 1st Department of Internal Medicine,
Linz, Austria
MARIO F. FRAGA • Epigenetics Group, Molecular Pathology Program, Spanish National
Cancer Center (C.N.I.O.), Madrid, Spain
JORDI FRIGOLA • Institut de Medicina Predictiva i Personalitzada del Càncer (IMPPC),
Barcelona, Spain
THIERRY GRANGE • Institut Jacques Monod du CNRS, Université Paris 7, Paris, France
JOHN M. GREALLY • Departments of Medicine (Hematology) and Molecular Genetics,
Albert Einstein College of Medicine, Bronx, NY, USA

xiii
xiv Contributors

IVO GLYNNE GUT • Department of Technology Development, Centre National de

Génotypage, CEA – Institut de Génomique, Evry, France
JAMES G. HERMAN • Cancer Biology Program, Sidney Kimmel Comprehensive Cancer
Center, Johns Hopkins, Baltimore, MD, USA
CHIBO HONG • Department of Neurological Surgery, University of California,
San Francisco Comprehensive Cancer Center, San Francisco, CA, USA
DAVE S. B. HOON • Department of Molecular Oncology, John Wayne Cancer Institute
and Breast Center, Saint John’s Health Center, Santa Monica, CA, USA
TIM H.-M. HUANG • Human Cancer Genetics Program, The Ohio State University
Comprehensive Cancer Center, Columbus, OH, USA
ALBERT JELTSCH • School of Engineering and Science, Jacobs University Bremen, Bremen,
Germany
MIREIA JORD À • Institut de Medicina Predictiva i Personalitzada del Càncer (IMPPC),
Barcelona, Spain
ZACHARY KAMINSKY • The Krembil Family Epigenetics Laboratory, Centre for Addiction
and Mental Health, Toronto, Ontario, Canada and University of Toronto, Toronto,
Ontario, Canada
PETER W. LAIRD • Departments of Surgery and of Biochemistry and Molecular Biology,
University of Southern California, Keck School of Medicine, Norris Comprehensive
Cancer Center, Los Angeles, CA, USA
SOPHIE LA SALLE • Department of Pharmacology and Therapeutics and The Montreal
Children’s Hospital Research Institute, McGill University, Montreal, Quebec, Canada
JULIEN D. F. LICCHESI • Cancer Biology Program, Sidney Kimmel Comprehensive
Cancer Center, Johns Hopkins, Baltimore, MD, USA
SHILI LIN • Department of Statistics, The Ohio State University, Columbus, OH, USA
DIETER LUTZ • Elisabethinen Hospital, 1st Department of Internal Medicine, Linz,
Austria
JONATHAN MILL • The Krembil Family Epigenetics Laboratory, Centre for Addiction
and Mental Health, Toronto, Ontario, Canada and Institute of Psychiatry, London, UK
FABIO MOHN • Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland
KENNETH P. NEPHEW • Department of Cellular and Integrative Physiology, Medical
Sciences Program, Indiana University School of Medicine, Bloomington, IN, USA;
Indiana University Cancer Center, Indianapolis, IN, USA
SANDY L. NGUYEN • Department of Molecular Oncology, John Wayne Cancer Institute
and Breast Center, Saint John’s Health Center, Santa Monica, CA, USA
CHRISTOPHER C. OAKES • Department of Pharmacology and Therapeutics
and The Montreal Children’s Hospital Research Institute, McGill University, Montreal,
Quebec, Canada
MAYUMI ODA • Departments of Medicine (Hematology) and Molecular Genetics, Albert
Einstein College of Medicine, Bronx, NY, USA
MIGUEL A. PEINADO • Institut de Medicina Predictiva i Personalitzada del Càncer
(IMPPC), Barcelona, Spain
ARTURAS PETRONIS • The Krembil Family Epigenetics Laboratory, Centre for Addiction
and Mental Health, Toronto, Ontario, Canada and University of Toronto, Toronto,
Ontario, Canada
GERD P. PFEIFER • Division of Biology, Beckman Research Institute of the City of Hope,
Duarte, CA, USA
Contributors xv

CHRISTOPH PLASS • Toxicology and Cancer Risk Factors, Division C010, German
Cancer Research Center (DKFZ), Heidelberg, Germany
DUSTIN POTTER • Human Cancer Genetics Program, The Ohio State University
Comprehensive Cancer Center, Columbus, OH, USA
JOHANNES PR ÖLL • Red Cross Transfusion Service of Upper Austria, Linz and
Elisabethinen Hospital, 1st Department of Internal Medicine, Linz, Austria
TIBOR A. RAUCH • Division of Biology, Beckman Research Institute of the City of Hope,
Duarte, CA, USA
RICHARD REINHARDT • Max Planck Institute for Molecular Genetics, Berlin, Germany
BERNARD ROBAIRE • Department of Pharmacology and Therapeutics and Department
of Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada
JAIRO RODRIGUEZ • Institut de Medicina Predictiva i Personalitzada del Càncer
(IMPPC), Barcelona, Spain
CHRISTIAN ROHDE • School of Engineering and Science, Jacobs University Bremen,
Bremen, Germany
TIM-CHRISTOPH ROLOFF • Friedrich Miescher Institute for Biomedical Research, Basel,
Switzerland
DIRK SCH ÜBELER • Friedrich Miescher Institute for Biomedical Research, Basel,
Switzerland
DAVID G. SKALNIK • Indiana University Cancer Center, Indianapolis, IN, USA
and Department of Biochemistry and Molecular Biology, Herman B. Wells Center for
Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA
DOMINIC J. SMIRAGLIA • Roswell Park Cancer Institute, Buffalo, NY, USA
HEINRICH STAMERJOHANNS • School of Engineering and Science, Jacobs University
Bremen, Bremen, Germany
EIJI SUNAMI • Department of Molecular Oncology, John Wayne Cancer Institute and
Breast Center, Saint John’s Health Center, Santa Monica, CA, USA
REIMO TETZNER • Epigenomics AG, Berlin, Germany
SASCHA TIERLING • Institut für Genetik, FB Biowissenschaften, Universität des
Saarlandes, Saarbrücken, Germany
JÖRG TOST • Laboratory for Epigenetics, Centre National de Génotypage, CEA – Institut
de Génomique, Evry, France
JACQUETTA M. TRASLER • Department of Pharmacology and Therapeutics, Department
of Pediatrics, Department of Human Genetics and The Montreal Children’s Hospital
Research Institute, McGill University, Montreal, Quebec, Canada
BINH TRINH • Departments of Surgery and of Biochemistry and Molecular Biology,
University of Southern California, Keck School of Medicine, Norris Comprehensive
Cancer Center, Los Angeles, CA, USA
TOSHIKAZU USHIJIMA • Carcinogenesis Division, National Cancer Center Research
Institute, Tokyo, Japan
DIRK VAN DEN BOOM • SEQUENOM, Inc., San Diego, CA, USA
ANH-THU VU • Department of Molecular Oncology, John Wayne Cancer Institute
and Breast Center, Saint John’s Health Center, Santa Monica, CA, USA
JÖRN WALTER • Institut für Genetik, FB Biowissenschaften, Universität des Saarlandes,
Saarbrücken, Germany
MICHAEL WEBER • Institut de Génétique Moléculaire, CNRS UMR 5535, 1919 Route de
Mende, 34293 MONTPELLIER Cedex 5, France
xvi Contributors

CHRISTIAN WECHSELBERGER • Upper Austrian Research GmbH, Center

for Biomedical Nanotechnology, Linz, Austria
DANIEL J. WEISENBERGER • Departments of Surgery and of Biochemistry and Molecular
Biology, University of Southern California, Keck School of Medicine, Norris
Comprehensive Cancer Center, Los Angeles, CA, USA
TOMASZ K. WOJDACZ • Institute of Human Genetics, University of Aarhus, Aarhus,
Denmark
SATOSHI YAMASHITA • Carcinogenesis Division, National Cancer Center Research
Institute, Tokyo, Japan
PEARLLY S. YAN • Human Cancer Genetics Program, The Ohio State University
Comprehensive Cancer Center, Columbus, OH, USA
OTTO ZACH • Elisabethinen Hospital, 1st Department of Internal Medicine, Linz,
Austria
YINGYING ZHANG • School of Engineering and Science, Jacobs University Bremen,
Bremen, Germany
 Part I

Introduction
 Chapter 1

DNA Methylation: An Introduction to the Biology

and the Disease-Associated Changes
of a Promising Biomarker
Jörg Tost

Abstract
DNA methylation occurring on the 5 position of the pyrimidine ring of cytosines in the context of the
dinucleotide sequence CpG forms one of the multiple layers of epigenetic mechanisms controlling and
modulating gene expression through chromatin structure. It closely interacts with histone modifications
and chromatin-remodeling complexes to form the genomic chromatin landscape. DNA methylation is
essential for proper mammalian development, crucial for imprinting, and plays a role in maintaining
genomic stability as well as in dosage compensation. DNA methylation patterns are susceptible to change
in response to environmental stimuli such as diet or toxins whereby the epigenome seems to be most
vulnerable during early in utero development. Aberrant DNA methylation changes have been detected
in several diseases, particularly cancer where genome-wide hypomethylation coincides with gene-specific
hypermethylation. DNA methylation patterns can be used to detect cancer at very early stages, to classify
tumors as well as predict and monitor the response to antineoplastic treatment. As a stable nucleic acid-
based modification with limited dynamic range that is technically easy to handle, DNA methylation is a
promising biomarker for many applications.

Key words: DNA methylation, nutritional epigenetics, environmental epigenetics, complex disease,
epigenetics, imprinting, cancer.

1. Introduction

All cells of a multicellular organism carry the same genetic

material coded in their DNA sequence, but cells obviously display
a broad morphological and functional diversity. This heterogene-
ity is caused by differential expression of genes. Epigenetics can
be defined as the study of heritable changes of a phenotype such
as the gene expression patterns of a specific cell type that are not

Jörg Tost (ed.), DNA Methylation: Methods and Protocols, Second Edition, vol. 507

C 2009 Humana Press, a part of Springer Science+Business Media
DOI 10.1007/978-1-59745-522-0 1 Springerprotocols.com

3
4 Tost

caused by changes in the nucleotide sequence of the genetic code

itself.
These changes are mitotically and in some cases meiotically
heritable. Epigenetic regulation mediates genomic adaption to an
environment, thereby ultimately contributing toward the pheno-
type. They “bring the phenotype into being” as said by the devel-
opmental biologist Conrad H. Waddington in the 1940s (1).
Epigenetic phenomena are mediated by a variety of molecular
mechanisms including posttranslational histone modifications,
histone variants, ATP-dependent chromatin remodeling com-
plexes, polycomb/trithorax protein complexes, small and other
noncoding RNAs, including siRNA and miRNAs, and DNA
methylation, as described in detail in (2). These diverse molec-
ular mechanisms have all been found to be closely intertwined
and stabilize each other to ensure the faithful propagation of
an epigenetic state over time and especially through cell divi-
sion. Nonetheless, epigenetic states are not definitive and changes
occur with age in a stochastic manner as well as in response to
environmental stimuli. Chromatin modulations play a central role
to shape the epigenome and delineate a functional chromatin
topology, which serves as the platform forming regulatory cir-
cuits in all cells. Open (euchromatin) and closed (heterochro-
matin) chromatin states are controlled by histone modifications
and histone composition in close cross talk with the binding of a
myriad of nonhistone proteins. The basic building block of chro-
matin is the nucleosome which is formed of an octamer of histone
proteins containing an H3–H4 tetramer, flanked on either side
with an H2A–H2B dimer around which 146 base pairs of DNA
are spooled in a 1.65 left-handed superhelical turn. The protrud-
ing N-terminal tails of these histones are extensively modified by
various modifications such as acetylation, methylation, phospho-
rylation, and ubiquitylation (3). The combination of different
N-terminal modifications and the incorporation of different his-
tone variants, which have distinct roles in gene regulation, have
led to the proposition of a regulatory histone code which deter-
mines at least partly the transcriptional potential for a specific gene
or a genomic region (4). DNA methylation is highly related to
certain chromatin modifications; and enzymes that modify DNA
and histones have been shown to directly interact and constitute
links between local DNA methylation and regional chromatin
structure.
This chapter briefly describes the DNA methylation landscape
and the enzymes responsible for adding and potentially removing
methyl groups to the DNA and touches upon the various biolog-
ical processes in which DNA methylation plays a key role. Differ-
ent pathologies for which changes in DNA methylation patterns
have been investigated will be mentioned with a certain empha-
sis on cancer, as most of the disease-associated DNA methylation
 DNA Methylation 5

literature concerns changes in tumorigenesis. This chapter con-

cludes with the reasons why DNA methylation is a very promising
biomarker. Due to space restrictions and the large field of research
described in this introduction, oversimplifications and omissions
are inevitable. This chapter is addressed to scientists who have not
been in close contact with this field before, and most of the refer-
ences direct the reader to more exhaustive review articles. People
with experience in DNA methylation research can directly jump
to the protocol of their choice.

2. The Biology of
DNA Methylation
DNA methylation is the only genetically programmed DNA mod-
ification in mammals. This postreplication modification is almost
exclusively found on the 5 position of the pyrimidine ring of
cytosines in the context of the dinucleotide sequence CpG (5).
5-Methylcytosine accounts for ∼1% of all bases, varying slightly
in different tissue types and the majority (75%) of CpG dinu-
cleotides throughout mammalian genomes are methylated. Other
types of methylation such as methylation of cytosines in the
context of CpNpG or CpA sequences have been detected in
mouse embryonic stem cells and plants, but are generally rare in
somatic mammalian/human tissues. CpGs are underrepresented
in the genome, probably because they act as a mutation hotspot
(deamination of methylated CpGs to TpGs). Mutation rates at
CpG sites have been estimated to be about 10–50 × higher
than other transitional mutations, as the mutation product is a
naturally occurring DNA base which may not be appropriately
repaired. The elevated mutation rate has led to depletion of the
dinucleotide during evolution. Despite this general trend, rela-
tively CpG-rich clusters of approximately 1–4 kb in length—so-
called CpG islands—are found in the promoter region and first
exons of many genes. They are mostly nonmethylated corre-
sponding to the maintenance of an open chromatin structure and
a potentially active state of transcription (6). There are around
30,000 CpG islands in the human genome. As CpG islands are
mainly unmethylated in the germline, they are less susceptible to
deamination and have therefore retained the expected frequency
of CpGs. It should be noted that a growing number of CpG
islands have been identified that are methylated in nonpatho-
logical somatic tissues (7). Depending on the employed set of
parameters, a CpG island is defined as having a G+C content
of more than 50% (55%), an observed versus expected ratio for
the occurrence of CpGs of more than 0.6 (0.65) and a min-
imum size of 200 (500) bp (8). About three-quarters of tran-
scription start sites and 88% of active promoters are associated
6 Tost

with CpG-rich sequences and might be regulated by DNA

methylation. Promoter CpG islands of genes differ in their sus-
ceptibility to become methylated during normal development as
well as during carcinogenesis, which might be due to intrin-
sic sequence properties (9). This regulation is controlled in a
tissue- and developmental-stage-specific manner and is main-
tained throughout the life of an individual.

2.1. DNA The composition of the genome is reflected in and dictates the
Methyltransferases epigenetic machinery to establish particular local and global epi-
and Methyl-Binding genetic patterns using both CpG spacing as well as sequence
Proteins motifs and DNA structure (10, 11). Mammalian one-carbon
metabolism provides the methyl group for all biologic methyla-
tion reactions. These are dependent on methyl donors (methio-
nine and choline) and cofactors (folic acid, vitamin B12, and
pyridoxal phosphate) to synthesize the universal methyl donor
S -adenosyl- L -methionine (SAM) (12). During the methylation
reaction, a methyl group is transferred from SAM to the DNA
leaving S-adenosylhomocysteine which at high concentrations
inhibits the action of DNA methyltransferases.
So far, four DNA methyltransferases have been identified
(DNMT1, DNMT2, DNMT3A, and DNMT3B) as well as
a DNMT-related protein (DNMT3L) (13). They catalyze the
transfer of a methyl group from SAM to the cytosine base. With
the exception of DNMT2 which acts probably as RNA methyl-
transferase in vivo, all Dnmts are essential for embryonic viability
as homozygous mutant mice die early. Simplified DNMT1 acts as
maintenance methyltransferase as it prefers hemimethylated tem-
plates. It is located at the replication fork during the S phase of
the cell cycle and methylates the newly synthesized DNA strand
using the parent strand as a template. Consequently, it passes
the epigenetic information through cell generations. De novo
methylation is carried out by the methyltransferases DNMT3A
and DNMT3B. These enzymes not only have certain preferences
for specific targets (e.g., Dnmt3a together with Dnmt3L methy-
lates maternal imprinted genes and Dnmt3b localizes at minor
satellite repeats), but also work cooperatively to methylate the
genome. Possible trigger mechanisms to initiate de novo methyla-
tion include preferred target DNA sequences, RNA interference,
certain chromatin structures induced by histone modifications,
and other protein–protein interactions (13).
DNA methyl-binding domain (MBD1-4) proteins or methyl
CpG-binding proteins (MeCP2) recognize and bind to methy-
lated DNA. They recruit transcriptional corepressors such
as histone-deacetylating complexes, polycomb proteins, and
chromatin-remodeling complexes, and attract chromodomain-
binding proteins. Besides the structurally related MBD proteins,
methylated DNA can also be bound by some zinc finger proteins
Other documents randomly have
different content
serious as is Sam the Book-keeper's. But Fitz-Adams is a young man,
barely thirty, I should say. Almost his earliest memory is that of
being a mule-driver in one of the mines near Wilkesbarre. From this
he went to picking slate in a breaker. Now he is a jobber, employing
a large crew, and undertaking contracts which involve considerable
sums of money. There has been offered to him, and it is still open,
the position of overseer in a far larger enterprise than his own,
where, personally, he would run none of the business risk; but he
has confided to me that he does not dare to accept the place owing
to his lack of even elementary education. In this connection he once
asked me whether I thought that he might yet go to school. I did
think so with emphasis, and I gave him so many reasons for this
opinion, and cited so many examples of men as old as he and older
who were at school, that he really warmed to it as a practicable
plan.
* * * * * * * *
The rain stopped hours ago, and it is turning very cold, and snow
has begun to fall. Fitz-Adams got back from English Centre long
before dinner, and there is evidence that he has not been drinking. I
have consulted him on the matter of leaving, and he has urged me
to stay, and has offered me permanent employment; but he says
that, if I must be off, and am bent on going westward, I would
better get as far as Hoytville as soon as possible, else I may run the
risk of encountering roads blocked with snow. Then, for the first
time, he introduced the subject of wages, and asked me what I
thought was "right." I said that before coming to the camp, I had
worked for a farmer, and had been given seventy-five cents a day
and my keep; and I added that, if this rate of wage seemed fair to
him, it would suit me perfectly. He agreed at once, and now I am a
capitalist. Soon I shall set out for Hoytville, which is, I judge, a
matter of two or three hours' walk from here. Fitz-Adams has given
me careful directions about the road, and has shown the deepest
interest in my plan of getting West, and has urged me to write to
him.
The crew are all gone to work, and I shall not see them. They were
off as soon as the storm slackened. All were keen to go, and so be
spared the misery of a day of enforced idleness, all except "Old
Pete," and he is past being keen. He is over sixty, and has a strongly
marked Celtic face, deeply furrowed with the lines of age and pain.
He works with the crew, but in camp he sits alone on the bench
opposite the stove, with the overalls and shirts hanging over him.
When not at work he sits there hour after hour, his large, muscular
frame bent forward, and his elbows resting on his knees, and there
he endures, in the dumb agony of animal pain, the torment of
rheumatism in his legs. He seldom speaks, and never of his
sufferings—only sometimes in comically sententious response to
something that has interested him. And the men let him alone,
knowing by a true intuition that he prefers it so.
After the rain let up I happened to pass through the lobby as the
men were starting for their work. Old Pete was the last to move. I
watched him rising slowly to his feet. In spite of him, his face drew
the picture of the hideous pain he bore, but through it shone the
clear courage of a man, and his eyes reflected the grim humor of a
thought that touched his native sense, and he smiled as he said:
"We don't have to work; we can starve."
* * * * * * * *
I have spent three Sundays in the woods. On the first I fled cravenly
into the forest, hugging a book from out my pack, and the hours
flew swiftly along the pages. The second Sunday was another
glorious autumn day. By that time I had won a modest place in
camp, and could hold up my head with due respect among the men.
I asked several of them whether there was any church service at
English Centre. They thought that there was, but they would take no
stock at all in my plan of discovery.
Alone I set out for the village. There was perfect quiet in the
mountains, no sound of axe or saw, nor crash of falling trees, nor
rumble of bark-wagons; only the tuneful flow and splash of the run,
which caught the living sunlight, and flashed it back in radiance
through the flushing air, that quivered in the ecstasy of buoyant life.
The fire of life flamed in the glowing hues of autumn, and burned
with white heat in the hoar-frost which clung to the shaded crevices
in the rocks, and along the blades of seared grass, and on the fringe
of fallen leaves. And I was free, as free and careless as the
mountain-stream, and before me was a blessed day of rest!
Every foot of the road was strangely familiar, but the familiarity lay in
an intimate association with some distant past, as of earliest
childhood. There was the camp by the dam, and there the
Irishman's cabin, where the cow was still munching straw, and the
sow wallowing in the mire. Then I came to the fork in the road,
where one way led to Wolf's Run. It was a lifetime since I had gone
up that way, feeling as cocky as a wedding-guest, and soon had
come down again "a sadder and a wiser man." I felt like another Rip
Van Winkle as I re-entered the village, but the marvel lay in there
being no change at all, except in the Sunday calm which now
possessed the place.
The post-office is in a private house, and I knocked in some
uncertainty of being able to get my letters; but the postmistress
gave them to me with obliging readiness, and with them a cordial
invitation to attend the Sunday-school, which, she said, was the only
service of that morning. Her invitation was more welcome than she
knew, for it was the first of its kind to reach me as a proletaire.
I read my letters, and then went to the church, which stands at the
end of the village street. The service was beginning. As
superintendent the postmistress was in charge. There were no men
present. About thirty women and girls, and half a dozen boys, made
up the school. The conduct of the service I thought intensely
interesting. The superintendent was entirely at home in her place,
and she valued the opportunity.
When the classes grouped themselves for the study of the lesson, a
teacher was lacking. I was asked to take the place, and was startled
at finding myself in charge of a class of village belles. What their
feeling toward the arrangement was, I could only guess; but it was
clear that they were not accustomed to being taught by an
unshaven, unshorn woodsman, in rough clothes, and boots covered
with patches. But the lesson was in my favor; it was the incident of
the washing of the disciples' feet at the last Passover. I soon forgot
my embarrassment in the interest of the text, and in an atmosphere
of serious study.
Last Sunday I went again to the Sunday-school, and I had my
former class to teach. Some preparation had been possible during
the week, and the hour passed successfully. Among the
announcements was one of a prayer-meeting to be held that night.
I reached the church at the hour of the evening service. I opened
the door, and there sat a crowded congregation in waiting. The back
seats on both sides of the aisle were solid ranks of men, lumbermen,
and teamsters, and tannery hands, many of them in their working-
clothes. There were women and children scattered through the pews
farther up, and some boys had overflowed upon the pulpit steps, but
most of the company were men.
There was no one in the minister's seat, but the postmistress was in
place at the organ, and as I entered, she nodded to me in evident
expectation of my joining her. I walked forward, and she stepped out
into the aisle to meet me.
"It's time to begin," she said, quietly.
"Is your minister not come yet?" I asked.
"Oh, you're going to speak to-night, you know."
I did not know. For an instant I knew only that there was a cold,
hard grip upon my heart which seemed to hold it still, and that in my
brain there had begun a mad dance of all that I ever thought I
knew. But from out the turmoil a sane thought emerged: "This is a
company of working-people who are come to hear a fellow-workman
speak to them about our deepest needs." In another moment I was
cooler, and a strange, unreasoning peace ensued.
I asked the postmistress to select some hymns. She handed me a
list, chosen with perfect knowledge of those which the congregation
most enjoyed. The people were soon singing, thinly at first; but the
familiar melody spread, and carried with it a sense of solidarity, in
which self was merged and lost, and the swelling sound rolled on,
deepening with the voices of the men. Soon it recalled college-
chapel, with the students in a mood to sing, and "Ein' Feste Burg"
mounting in the majesty of that deep-toned hymn, until the vaulted
ceilings rock, and the archangels above the chancel seem to join in
the splendid volume of high praise!
But more helpful to me than the singing was the sight of familiar
faces. Black Bob stood towering like another Saul above the mass of
men; and at his side was one of our teamsters who lives in the
village, and with whom I had often loaded bark. Near the door—I
was not quite sure at first, but there could be no mistake—near the
door was Fitz-Adams, and not far from him Long-nosed Harry and
Phil the Farmer stood together.
I was trembling when I began to speak, trembling with awful fear, a
fear that was yet a solemn joy; for I had vision then of human
hearts hungering to be fed, and, as a sharer in their need, I knew
that it was given to me to point them to the Bread of Life.
I could speak to them now, for with greater clearness I could see
these fellow-workers as they were—strong, brave men who win the
mastery which comes to those who clear the way for progress,
giving play, in their natural living, to the forces which make men
free, and growing strong in heart and in the will to do, as they grow
strong of arm and catch the rough cunning of their trade; men of
many races, yet meeting on the common ground of men all free and
under equal chance to make their way; knowing no differences but
those of personality, and winning their places in the crew, each man
according to his kind, and his rewards according to his skill.
Such were they in their outward lives, the physical life within them
growing in living ways, and making them the true, efficient workmen
that they were. But of the inner life that makes us men, that life
wherein we act from choice, and must "give account of the deeds
done in the body," that range of action which we call moral, where
conscience speaks to us in words of command, there they knew no
mastery at all, and, least of all, the mastery of the moralist.
To them God was a moral ruler, dwelling afar from the daily life of
men, and righteousness was a slavish obedience to His laws, and
religion a mystic somewhat which was good for women and children
and weak men.
And yet deep in their own hearts was their supremest need. Life as
they knew it brought to them no satisfaction for its craving want. It
was not so in other things; they knew their work; and in the
overcoming of its difficulties, they had felt the fierce joy of conquest.
But confronted with temptations, the difficulties of their inner life,
there they had no strength; and lust and passion mastered them,
and left their real desire unsatisfied. Here, in respect of mastery,
they were slaves, and as regards life, they were dead, having only
the need of life.
There, then, was their want; it was for Life, abundant, victorious
Life.
And now I could speak to them of God; of Him "who is not far from
every one of us, for in Him we live, and move, and have our being;"
the living God who reveals Himself in all life, and who became
incarnate in the Son of Man, and who speaks to us in human words
which go straight to our seeking hearts: "I am the way, the truth,
and the life." "I am come that ye might have life, and that ye might
have it more abundantly." "The words that I speak unto you, they
are life."
"Strong Son of God!" whose living words quicken us from the death
of sin and set us free. By whose grace we are "renewed in the whole
man after His image, and enabled, more and more, to die unto sin
and live unto righteousness." Who was "made sin for us, who knew
no sin; that we might be made the righteousness of God in Him."
"Who His own self bare our sins in His own body on the tree, that
we, being dead to sin, should live unto righteousness." Whose death
was not a reconcilement of God to us, but was "God in Christ
reconciling the world unto Himself." Whose Gospel is the glad tidings
of this reconciliation, and we are become "ambassadors for Christ,
as though God did beseech you by us; we pray you in Christ's stead,
be ye reconciled to God."
And then we prayed, confessing our sinful state, our bondage, our
death in sin, and pleading that we might be "transformed by the
renewing of our minds, that we might prove what is that good, and
acceptable, and perfect will of God."
* * * * * * * *
Now that I am on the eve of leaving Fitz-Adams's Camp, I cannot
hide from myself my eagerness to go. I have real regrets; for while
two weeks and as many days do not constitute a long period, yet
time is purely relative, and I shall have a livelier memory of the
camp and of certain of the men, and a keener interest in them, than
I have for places and men with whom my association has been
much longer.
But of the feelings of which I am conscious at leaving, I am
surprised at the intensity of the longing to know what has happened
during the three weeks, nearly, since I have seen a newspaper from
the great world. I thought little of it as the days passed, but now I
am all aglow with desire for news about the progress of the
campaigns in New York and Massachusetts and Ohio. And then the
last word from abroad had piqued one's curiosity to the utmost as to
possible results. Mr. Smith, the leader of the House of Commons, I
know is dead; and as I was leaving Williamsport for the woods, I
saw upon the bulletin-boards the announcement of Mr. Parnell's
sudden death; but of the political effect of these events no word has
reached me. Has Mr. Balfour or Mr. Goschen succeeded to the
leadership of the House? And if Mr. Balfour became the First Lord of
the Treasury, does he retain the Chief Secretaryship for Ireland? And
has the death of Mr. Parnell brought about a reunion between
Parnellites and. M'Carthyites, or is the breach as hopeless as ever?
It will be intensely interesting to find answers to these questions and
to many more; but after all I am sincerely sorry to leave the camp,
and as I go up now to say good-by to Fitz-Adams, who is in his
office, it is with the knowledge that I am parting from a man whom
it is an inspiration to have known.
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