Table Of ContentMethods in
Molecular Biology 2198
Alexey Ruzov
Martin Gering Editors
DNA
Modifi cations
Methods and Protocols
M M B
ethods in olecular iology
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK
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DNA Modifications
Methods and Protocols
Edited by
Alexey Ruzov
Centre for Biomolecular Sciences, University Park, University of Nottingham, Nottingham, UK
Martin Gering
School of Life Sciences, Queen’s Medical Centre, University of Nottingham, Nottingham, UK
Editors
Alexey Ruzov Martin Gering
Centre for Biomolecular Sciences School of Life Sciences
University Park Queen’s Medical Centre
University of Nottingham University of Nottingham
Nottingham, UK Nottingham, UK
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-0716-0875-3 ISBN 978-1-0716-0876-0 (eBook)
https://doi.org/10.1007/978-1-0716-0876-0
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Cover Caption: Pictured is a mouse 1-cell embryo double-stained with an anti-5mC antibody (red) and an anti-5fC
antibody (green) at the prometaphase during the first mitosis of life. The chromosomes inherited from the father have
undergone oxidation of 5mC, as shown by 5fC staining, whereas the chromosomes inherited from the mother are rich
in 5mC. Both sets of condensing chromosomes form a cartwheel structure. In the paternal cartwheel, a 5mC-rich,
oxidation-resistant circle appears to organize the 5fC-rich, radially arranged chromosomes. Image courtesy of Tie-Bo
Zeng and Piroska E. Szabó (Center for Epigenetics, Van Andel Research Institute, Grand Rapids, MI, USA).
This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer
Nature.
The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.
Preface
Until recently, cytosine methylation (5-methylcytosine, 5mC) was considered to be virtu-
ally the only DNA modification with a biological function that is present in significant
quantities in eukaryotic genomes. This view has changed in the last decade which has wit-
nessed an unprecedented flurry of research activity focused on other noncanonical DNA
nucleotides. This activity has produced a number of paradigm-shifting publications demon-
strating the importance of oxidized forms of 5mC (5-hydroxymethylcytosine, 5hmC;
5-formylcytosine, 5fC; and 5-carboxylcytosine, 5caC) for active DNA demethylation and
their potential roles as bona fide “epigenetic” marks in mammals. Together with the recent
discovery of N6-methyldeoxyadenine (6mA) in metazoan DNA and advances in decipher-
ing the genomic distribution and potential functions of 5-hydroxymethyluracil (5hmU)
and deoxyuridine in eukaryotes, these studies have considerably widened the field of DNA
modifications, making it both exciting and exceptionally complex.
This volume of the Methods in Molecular Biology series provides an overview of methods
and experimental protocols that are currently used to analyze the presence and abundance
of noncanonical DNA nucleotides in different biological systems. It focuses particularly on
the newly discovered and less studied DNA modifications that are enzymatically produced
and are likely to play specific roles in various biological processes. Specifically, the book
describes chromatography- and mass spectrometry-based techniques for the detection and
quantification of DNA modifications, antibody-based approaches to study their spatial dis-
tribution in different cells and tissues, and methods to analyze their genomic distribution
with the help of bioinformatics tools that interrogate the corresponding datasets.
Part I of this book consists of introductory chapters that outline the diversity of modi-
fied forms of cytosine and their functions in eukaryotes (Chapter 1), summarize current
experimental evidence for noncytosine epigenetic DNA modifications in multicellular organ-
isms (Chapter 2), provide a synopsis of applications of mass spectrometry (MS) for detection
and quantification of noncanonical DNA nucleosides (Chapter 3), and sketch out a sum-
mary of current approaches for the mapping of DNA methylation in mammals (Chapter 4).
In Part II, there are a number of protocols describing different aspects of detection and
quantification of modified deoxynucleosides using mass spectrometry (MS)-based tech-
niques, the gold standard method for global quantitation of DNA modifications. Chapter
5 describes the detection of modified bases in the genomic DNA of bacteriophages that
contain the largest chemical diversity in naturally occurring DNA modifications observed
to date; Chapter 6 provides a protocol for the analysis of modified forms of cytosine using
liquid chromatography–mass spectrometry; and Chapter 7 is dedicated to the detection of
different forms of DNA methylation (including 6mA, 5mC and N4-methylcytosine, 4mC)
by Triple Quadrupole Liquid Chromatography coupled with tandem Mass Spectrometry
(QQQ-LC-MS/MS). The following chapters in this section focus on the use of two-
dimensional ultraperformance liquid chromatography–tandem mass spectrometry
(2D-UPLC–MS/MS) for the quantification of different DNA modifications (Chapter 8),
application of this technique for analysis of modified deoxyribonucleosides in urine and
other body fluids (Chapter 9), and preparation of internal standards for this method
v
vi Preface
(Chapter 10). As the MS techniques require specialist equipment, they are often time- and
resource-consuming and, therefore, are not accessible to all researchers; the last protocol in
this section describes the detection of oxidized forms of 5mC using capillary gel electro-
phoresis as a rapid and quantitative alternative to the MS-based methods of their analysis
(Chapter 11).
While the techniques presented in Part II of our book are quantitative and highly accu-
rate, they do not provide any information on the cell type, tissue, or nuclear compartment
specificity of DNA modifications. Therefore, the next part of our volume (Part III) con-
tains a range of protocols that allow assessment of the levels and spatial distribution of
modified nucleotides using antibodies. Here, we present a number of immunostaining pro-
cedures optimized for the detection of the modified cytosine species in mammalian preim-
plantation embryos (Chapter 12) and amphibian lampbrush chromosomes (Chapter 13),
as well as for visualization of low abundant DNA modifications in mammalian cells (Chapter
14), rodent brain (Chapter 15), zebrafish embryos (Chapter 16), and plant tissues (Chapter
17). These protocols are complemented by texts that examine antigen retrieval techniques
used to immunostain modified cytosines (Chapter 18) and computational analysis of DNA
modifications in confocal images (Chapter 19). A method that combines three-dimensional
analysis of chromosome positioning with immunochemical detection of 8-oxoguanine in
murine sperm nuclei is described in Chapter 20. In addition to these different variants of
immunostaining, Chapter 21 describes a protocol for the analysis of 5hmU levels using flow
cytometry.
The following section of this volume (Part IV) aims to demonstrate a variety of differ-
ent approaches that are currently used to map DNA modifications in the genome. In this
part, such classical long-established techniques as hairpin-bisulfite PCR, which allows
assessment of DNA methylation patterns on complementary strands of individual DNA
molecules (Chapter 22), and methylated DNA immunoprecipitation (MeDIP) (Chapter
23) are presented together with recently developed protocols for studying the genomic
distribution of oxidized forms of 5mC. Thus, Chapter 24 describes a procedure for 5caC
DNA Immunoprecipitation (DIP), Chapter 25 is dedicated to the base-resolution analysis
of 5hmC by TET-assisted bisulfite sequencing, and Chapter 26 presents an experimental
and computational protocol for oxidative bisulfite sequencing that permits genome-wide
mapping of this modification together with 5mC. A novel bisulfite-free approach for 5hmC
sequencing, designated as APOBEC-Coupled Epigenetic Sequencing (ACE-Seq), is
described in Chapter 27. The final chapter of this part, Chapter 28, presents a procedure
for single-nucleotide-resolution mapping of 6mA that couples crosslinking of 6mA-specific
antibodies to the DNA fragments with subsequent exonuclease treatment.
Methods described in Part V of this volume go beyond the mapping of existing DNA
modifications and seek to manipulate their genomic distribution at particular loci. The aim
is to alter the level of expression of a target gene by using DNA modification-specific
transcription-activator-like effectors (Chapter 29) and CRISPR/Cas9 (Chapter 30) genome
targeting approaches. Finally, the last section of this volume (Part VI) is comprised of four
chapters that cover different aspects of bioinformatics analysis of DNA modifications
sequencing data. Chapter 31 discusses the experimental setup and appropriate controls for
enrichment-based genome-wide DIP studies, while Chapter 32 provides a detailed proto-
col for the bioinformatics analysis of bisulfite sequencing data. A procedure for the unified
simultaneous analysis of multiple peak datasets is presented in Chapter 33. The final c hapter,
Chapter 34, gives a step-by-step guide to examine the relationship between DNA methyla-
tion/hydroxymethylation and gene expression using original software tools.
Preface vii
We are grateful to all the authors of this book for their contributions and hope that,
collectively, their protocols will complement each other, providing a useful overview of the
current state of DNA modification studies. While we understand that this volume is far
from being a complete compendium that covers the full range of methods employed in this
field, we believe it will provide orientation to research scientists and PhD students in this
rapidly developing discipline and, thus, will ultimately contribute to deciphering the roles
of noncanonical DNA nucleotides in different biological systems.
Nottingham, UK Alexey Ruzov
Nottingham, UK Martin Gering
Contents
Preface............................................................. v
Contributors...................................................... xiii
Part I I ntroductIon
1 Modified Forms of Cytosine in Eukaryotes: DNA
(De)methylation and Beyond . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Maria Eleftheriou and Alexey Ruzov
2 Evidence for Noncytosine Epigenetic DNA Modifications
in Multicellular Eukaryotes: An Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Paige Lowe, Ryszard Olinski, and Alexey Ruzov
3 Mass Spectrometry-Based Analysis of DNA Modifications:
Potential Applications in Basic Research and Clinic . . . . . . . . . . . . . . . . . . . . . . . 27
Ryszard Olinski, Rafal Rozalski, and Daniel Gackowski
4 Mapping DNA Methylation in Mammals: The State of the Art . . . . . . . . . . . . . . 37
Antonio Lentini and Colm E. Nestor
Part II detectIon and QuantIfIcatIon of dna
ModIfIcatIons usIng Mass sPectroMetry
and caPIllary gel electroPhoresIs
5 Detection of Modified Bases in Bacteriophage Genomic DNA. . . . . . . . . . . . . . . 53
Yan-Jiun Lee and Peter R. Weigele
6 Liquid Chromatography–Mass Spectrometry Analysis
of Cytosine Modifications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Nan Dai and Ivan R. Corrêa Jr
7 Detection of DNA Methylation in Genomic DNA
by UHPLC-MS/MS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Konstantinos Boulias and Eric Lieberman Greer
8 Quantification of DNA Modifications Using Two-Dimensional
Ultraperformance Liquid Chromatography Tandem
Mass Spectrometry (2D-UPLC-MS/MS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Marta Starczak, Maciej Gawronski, Ryszard Olinski, and Daniel Gackowski
9 MS Analysis of DNA Modifications in Urinary/Body Fluids . . . . . . . . . . . . . . . .109
Aleksandra Skalska, Agnieszka Siomek-Gorecka, Ryszard Olinski,
and Rafal Rozalski
10 Preparation of Internal Standards for 2D-UPLC-MS/MS
Quantification of Noncanonical DNA Bases . . . . . . . . . . . . . . . . . . . . . . . . . . . .123
Marta Starczak, Aleksandra Skalska, Rafal Rozalski, Ryszard Olinski,
and Daniel Gackowski
ix
x Contents
11 Detection of TET-Oxidized 5-Methylcytosine Bases by Capillary
Gel Electrophoresis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .137
Romualdas Vaisvila, Lise Hunault, and Lana Saleh
Part III a ssessIng the levels and sPatIal dIstrIbutIon
of dna ModIfIcatIons usIng antIbody-based aPProaches
12 Immunochemical Detection of Modified Cytosine Species
in Mammalian Preimplantation Embryos. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .147
Tie-Bo Zeng and Piroska E. Szabó
13 Immunochemical Detection of Modified Cytosine Species
in Lampbrush Chromatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .159
Garry T. Morgan
14 Detection of Low-Abundance DNA Modifications Using
Signal Amplification-Based Immunocytochemistry . . . . . . . . . . . . . . . . . . . . . . .169
Abdulkadir Abakir and Alexey Ruzov
15 Immunohistochemical Detection of Modified Cytosine
Bases in Rodent Brain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .183
Marcus D. Wallis and Rebecca C. Trueman
16 Immunohistochemical Detection of 5-Hydroxymethylcytosine
and 5-Carboxylcytosine in Sections of Zebrafish Embryos. . . . . . . . . . . . . . . . . .193
Peter Jessop and Martin Gering
17 Immunochemical Detection of Modified Species of Cytosine
in Plant Tissues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .209
Marcos Viejo, Igor Yakovlev, and Carl Gunnar Fossdal
18 Antigen Retrieval for Immunostaining of Modified Cytosine Species. . . . . . . . . .217
Selcen Celik-Uzuner
19 Computational Analysis of DNA Modifications in Confocal Images. . . . . . . . . . .227
Seema Rajani, Christopher Gell, Abdulkadir Abakir, and Robert Markus
20 Three-Dimensional Confocal Analysis of Chromosome
Positioning Coupled with Immunofluorescence in Mouse Sperm Nuclei. . . . . . .255
Alexandre Champroux, Chantal Goubely, Joëlle Henry-Berger,
Joël R. Drevet, and Ayhan Kocer
21 Analysis of 5-Hydroxymethyluracil Levels Using Flow Cytometry . . . . . . . . . . . .269
Lidia Gackowska, Anna Labejszo, and Daniel Gackowski
Part Iv MaPPIng dna ModIfIcatIons
22 Hairpin-Bisulfite PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .287
Reinhard Stöger
23 Genome-Wide Mapping of DNA Methylation 5mC by Methylated
DNA Immunoprecipitation (MeDIP)-Sequencing. . . . . . . . . . . . . . . . . . . . . . . .301
Millissia Ben Maamar, Ingrid Sadler-Riggleman, Daniel Beck,
and Michael K. Skinner
24 Analysis of 5-Carboxylcytosine Distribution Using DNA
Immunoprecipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .311
Abdulkadir Abakir, Fahad Alenezi, and Alexey Ruzov
