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Reproduction
An Introductory Guide
Reproduction
An Introductory Guide
Edited by
Cristina Camprubí, PhD

Founder and Director of GenIntegral, Barcelona
and
Assistant Professor of the Department of Cell Biology, Physiology and
Immunology of the Universitat Autònoma de Barcelona, Spain
Joan Blanco, PhD

Senior Scientist of the Genetics of Male Fertility Group
of the Universitat Autònoma de Barcelona
and
Associate Professor of the Department of Cell Biology, Physiology
and Immunology of the Universitat Autònoma de Barcelona, Spain
CRC Press
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Library of Congress Cataloging-in-Publication Data
Names: Camprubi, Cristina, editor. | Blanco, Joan, editor.

Title: Epigenetics and assisted reproduction : an introductory guide / edited
by Cristina Camprubi, and Joan Blanco.
Description: Boca Raton, FL : CRC Press/Taylor & Francis Group, [2019] |
Includes bibliographical references and index.
Identifiers: LCCN 2018013761| ISBN 9781138633094 (hardback : alk. paper) |
ISBN 9781138631632 (paperback : alk. paper) | ISBN 9781315208701 (ebook)
Subjects: | MESH: Epigenesis, Genetic | Reproductive Techniques, Assisted | Epigenomics
Classification: LCC QH450 | NLM QU 475 | DDC 616/.042--dc23
LC record available at https://lccn.loc.gov/2018013761
Visit the Taylor & Francis Web site at

http://www.taylorandfrancis.com
and the CRC Press Web site at
http://www.crcpress.com
Contents
Preface, vii
Contributors, ix
Chapter 1 ◾ A Short Introduction to DNA Methylation 1

Jörg Tost
Chapter 2 ◾ Epigenetic Modifications of Histones 17

George R asti and Alejandro Vaquero
Chapter 3 ◾ Epigenetic Reprogramming in Early Embryo Development 29

Sebastian Canovas and Pilar Coy
Chapter 4 ◾ Epigenetic Reprogramming of Mammalian Primordial

Germ Cells 51
Sebastian Canovas and Susana M. Chuva de Sousa Lopes
Chapter 5 ◾ Laboratory Molecular Methodologies to Analyze DNA

Methylation 69
David Monk and Gavin Kelsey
Chapter 6 ◾ Intrinsic and Extrinsic Factors That Influence Epigenetics 99

Ivan Nalvarte, Joëlle Rüegg, and Carlos Guerrero-Bosagna
Chapter 7 ◾ Epigenetic and Assisted Reproduction Epidemiological

Studies 115
Thomas Eggermann
v
vi ◾ Contents
Chapter 8 ◾ Epigenetics and Assisted Reproduction Experimental

Studies: Sperm Epigenome 131
Joan Blanco and Cristina Camprubí
Chapter 9 ◾ Epigenetic and Assisted Reproduction Experimental

Studies: Sperm ncRNAs 143
Celia Corral-Vazquez and Ester Anton

Studies: Ovarian Stimulation 157
Patricia Fauque

Studies: In Vitro Culture 169
Serafín Pérez-Cerezales, Noelia Fonseca Balvís, Benjamín Planells,
Isabel Gómez-Redondo, Eric Marqués-García, Ricardo Laguna-Barraza,
Eva Pericuesta, R aul Fernández-González, and Alfonso Gutiérrez-Adán
Chapter 12 ◾ Epigenetics: Hope against Genetic Determinism 183

Josep Santaló
Chapter 13 ◾ Future Perspectives 189

Joan Blanco, Cristina Camprubí, and David Monk
INDEX, 193
Preface
H uman fertility depends on a highly orchestrated action of hundreds of genes

playing together to allow the development of highly complex mechanisms such
as gametogenesis and embryogenesis. Abnormal expression of genes involved in these
mechanisms has been associated to infertility affecting the assisted reproduction outcome
of the affected couples. Alteration of gene expression could be caused by genetic alterations
but also by epigenetic causes, which refers to heritable changes in gene expression that occur
without modifications at the DNA sequence level. As contemporary medicine increasingly
aims to personalize the medical approach to a patient’s (epi)genetic profile, the factors that
can affect gene expression also increase in importance and relevance to the clinician.
In the field of Reproductive Medicine, there is growing evidence about the influence of
epigenetics on the reproductive availably of couples to conceive in vitro. The first evidence
came from the early 2000s, when it was proved that the risk for imprinting disorders is
higher in assisted reproduction technology (ART) couples. Since then, the concern of the
clinicians, along with the methodological advances for the study of the epigenome, have
increased our knowledge about the causes and the consequences of epigenetic variations of
gametes and embryos and their effect in the ART outcome.
This handbook has been organized to provide the reader with the clues to understand
the association between epigenetic variations and infertility. The first chapters introduce the
main epigenetic mechanisms, followed by an extensive review of how epigenome variations
of gametes and embryos are related to human infertility. We have also provided an ethical
view of epigenetic variations, and an analysis about where we are and where we go from
here in this field. Overall, this text will give the clinician in Reproductive Medicine a reliable
grounding in current thinking and research on this fast-moving topic.
We would like to thank the book’s contributors for their cooperation in putting together
this excellent text. All of them are recognized scientists in their corresponding areas, and
have provided valuable responses in a complex, changing, and challenging field.
vii
Contributors
Ester Anton Sebastian Canovas

Genetics of Male Fertility Group Department of Physiology
Unitat de Biologia Cel·lular (Facultat de IMIB–Arrixaca
Biociències) Regional Campus of International
Universitat Autònoma de Barcelona Excellence “Campus Mare Nostrum”
Cerdanyola del Vallès, Spain University of Murcia
Murcia, Spain
Noelia Fonseca Balvís
Dpto. de Reproducción Animal Susana M. Chuva de Sousa Lopes
INIA Department of Reproductive
Madrid, Spain Medicine
Ghent University Hospital
Joan Blanco Ghent, Belgium
Genetics of Male Fertility Group
and
Department of Cell Biology, Physiology
and Immunology Department of Anatomy and
Universitat Autònoma de Barcelona Embryology
Bellaterra (Cerdanyola del Vallès), Spain Leiden University Medical
Center
Cristina Camprubí Leiden, The Netherlands
GenIntegral
Barcelona Celia Corral-Vazquez
and Genetics of Male Fertility Group
Department of Cell Biology, Physiology Unitat de Biologia Cel·lular
and Immunology (Facultat de Biociències)
Universitat Autònoma de Barcelona Universitat Autònoma de Barcelona
Bellaterra (Cerdanyola del Vallès), Spain Cerdanyola del Vallès, Spain
ix
x ◾ Contributors
Pilar Coy Alfonso Gutiérrez-Adán

Department of Physiology Dpto. de Reproducción Animal
IMIB–Arrixaca INIA
Regional Campus of International Madrid, Spain
Excellence “Campus Mare Nostrum”
Gavin Kelsey
University of Murcia
The Babraham Institute
Murcia, Spain
Cambridge, United Kingdom
Thomas Eggermann
Ricardo Laguna-Barraza
Institute of Human Genetics
Dpto. de Reproducción Animal
RWTH Aachen University
INIA
Aachen, Germany
Madrid, Spain
Patricia Fauque
Eric Marqués-García
Université Bourgogne Franche-Comté-
Equipe Génétique des Anomalies du
INIA
Développement (GAD)
Madrid, Spain
and
CHU Dijon Bourgogne David Monk
Laboratoire de Biologie de la Reproduction Imprinting and Cancer Group
Dijon, France Cancer Epigenetics and Biology Program
Institut d’Investigació Biomedica de
Raul Fernández-González
Bellvitge
L’Hospitalet de Llobregat
INIA
Barcelona, Spain
Madrid, Spain
Ivan Nalvarte
Isabel Gómez-Redondo
Department of Biosciences and Nutrition
Karolinska Institutet
INIA
Huddinge, Sweden
Madrid, Spain
Serafín Pérez-Cerezales
Carlos Guerrero-Bosagna
Avian Behavioral Genomics and
INIA
Physiology Group
Madrid, Spain
Department of Physics
and Eva Pericuesta
Chemistry and Biology Dpto. de Reproducción Animal
Linköping University INIA
Linköping, Sweden Madrid, Spain
Contributors ◾ xi
Benjamín Planells Josep Santaló

Dpto. de Reproducción Animal Universitat Autònoma de Barcelona
INIA Unitat de Biologia Cel·lular
Madrid, Spain Facultat Biociències
Campus UAB
George Rasti Bellaterra, Spain
Chromatin Biology Laboratory
Cancer Epigenetics and Biology Program Jörg Tost
(PEBC) Laboratory for Epigenetics & Environment
Bellvitge Biomedical Research Institute Centre National de Recherche en
(IDIBELL) Génomique Humaine
L’Hospitalet de Llobregat CEA–Institut de Biologie Francois Jacob
Barcelona, Spain Evry, France
Joëlle Rüegg Alejandro Vaquero

Department of Clinical Neurosciences Chromatin Biology Laboratory
Karolinska Institutet Cancer Epigenetics and Biology Program
Stockholm (PEBC)
and Bellvitge Biomedical Research Institute
Unit of Toxicology Sciences (IDIBELL)
Swetox Karolinska Institutet L’Hospitalet de Llobregat
Södertälje, Sweden Barcelona, Spain
Chapter 1
A Short Introduction
to DNA Methylation
Jörg Tost
CONTENTS
1.1 Introduction 1
1.2 Patterns of DNA Methylation 2
1.3 DNA Methyltransferases 4
1.4 5-Hydroxymethylation and the DNA Demethylation Processes 6
1.5 Transcription and Genome Stability 7
1.6 Epigenetic Changes in Health and Disease 10
1.7 Conclusion 13
References 13
1.1 INTRODUCTION
Almost all cells on an organism share the same genetic material encoded in the DNA
sequence, but display a broad range of morphological and functional diversity. Epigenetics
can be defined as the study of changes of a phenotype such as the gene expression patterns
of a specific cell type not caused by underlying changes in the primary DNA sequence.
These changes are mitotically and maybe in some cases meiotically heritable. Epigenetic
regulation mediates genomic adaption to an environment thereby ultimately contributing
toward the phenotype and “brings the phenotype into being” (1).
Epigenetics consists of a variety of molecular mechanisms including post-transcriptional
histone modifications, histone variants, ATP-dependent chromatin remodeling complexes,
polycomb/trithorax protein complexes, small and other non-coding RNAs including siRNA
and miRNAs, and DNA methylation. These diverse molecular mechanisms have all been
found to be closely intertwined and stabilize each other to ensure the faithful propagation
1
2 ◾ Epigenetics and Assisted Reproduction
of an epigenetic state over time and especially through cell division. Nonetheless epigenetic
states are not static, but change with age in a stochastic manner as well as in response to
environmental stimuli. This review gives a brief introduction to the multiple biological
facets of DNA methylation, probably the best-studied epigenetic modification, and its
potential use in clinical applications.
1.2 PATTERNS OF DNA METHYLATION

DNA methylation is a post-replication modification almost exclusively found on the 5
position of the pyrimidine ring of cytosines, (Figure 1.1), in the context of the dinucleotide
sequence CpG, of which around 29 million are found in the human (haploid) genome (2).
The additional methyl group is located at the major groove edge in a DNA double helix
Cytosine 5-Methylcytosine 5-Hydroxymethyl-

cytosine
N N N
C C C C C
N C N C N C O
C C C C C C
O N O N O N
R R R
Major groove face Major groove face
HN N Me HN N
H O H O
N H N N N H N N
N N C1′ N N C1′
C1′ O H NH C1′ O H NH
Major groove face Major groove face

dC - dG 5mC - dG
FIGURE 1.1 Chemical structure of cytosine, 5-methylcytosine, and 5-hydroxymethylcytosine. R

denotes the sugar moiety, which has been omitted for simplification. Cytosine is incorporated into
the DNA using deoxycytidinetriphosphate as a building block and methylated after its incorporation
by DNA methyltransferases. 5-hydroxymethylcytosine is created by oxidation of methylcytosine by
the TET enzymes. DNA methylation standing out in the major grove of the DNA double helix shows
identical Watson-Crick base pairing compared to cytosine.
A Short Introduction to DNA Methylation ◾ 3
and does not change the Watson-Crick base pairing (Figure 1.1). 5-methylcytosine (5mC)
accounts for ∼1% of all bases, varying slightly in different tissue types and the majority
(60%–80%) of CpG dinucleotides throughout mammalian genomes are methylated
(Figure 1.2). 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, neurons,
and plants, but are generally rare in somatic mammalian/human tissues. DNA methylation
marks are part of the cellular identity and memory and the sequence symmetry of CpG
dinucleotides allows for the transmission of DNA methylation marks through cell division.
CpGs are underrepresented in the genome, as a result of their increased mutation potential
with mutation rates at CpG sites to be about 10–50 times higher than other transitional
Normal tissue
+++
Promoter
CpG island
+++
Promoter
CpG island
Cancer
+++
Promoter
CpG island
+++ Enhancer Exon Repetitive sequence Methylated CpG Unmethylated CpG
FIGURE 1.2 Distribution of DNA methylation in normal tissue and cancer. In the normal tissue,
most promoter CpG islands are free of DNA methylation (indicated by white circles) even if the gene
is not expressed. Repetitive elements as well as interspersed CpG dinucleotides are mostly methylated
(indicated by black circles). Methylation changes at intergenic gene regulatory regions, such as
enhancers, which can change the expression status of the associated gene while the methylation
status of the gene does not change. In tumors, a global loss of DNA methylation (hypomethylation
of the cancer genome) is observed while some promoter CpG islands become methylated in a tumor-
type specific manner. Methylation patterns are dynamic and also change to a lesser extent compared
to cancer with age and in response to environmental factors.
mutations. As the deamination of methylated CpGs to TpGs yields a naturally occurring

DNA base, it is less well corrected. Despite this general trend, relatively CpG rich clusters,
so-called CpG islands, are found in the promoter region and first exons of ∼65% genes
containing about 7% of all CpGs (Figure 1.2) (3). Depending on the employed set of
parameters, a CpG island is defined as having a C + G 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 minimum size of 200 (500) base pairs (4). They are mostly non-methylated in all tissues
and throughout all developmental stages corresponding to an open chromatin structure
and a potentially active state of transcription (Figure 1.2). 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. Binding of transcription factors, exclusion of nucleosomes, and the presence
of H3K4 methylation and the associated histone methyltransferases protect most CpG
islands from DNA methylation. It should be noted that a number of CpG islands have been
identified that are methylated in a tissue-specific manner in normal tissues, but concern
mainly intragenic CpG islands (5,6). CpGs islands associated to genes not expressed in a
specific cell type acquire the repressive histone modification H3K27Me3, but rarely DNA
methylation. In contrast, regions located up- and downstream of CpG islands, termed CpG
island shores, show variable tissue-specific DNA methylation patterns and these are often
altered in tumorigenesis (7). In contrast to CpG islands, gene bodies are commonly highly
methylated, where DNA methylation has been associated with enhanced gene expression
maybe by facilitating transcriptional elongation and preventing initiation of spurious
transcription events (8). Intragenic methylation has in addition been associated with the
repression/use of alternative promoters or different splice variants (6,9).
1.3 DNA METHYLTRANSFERASES

Both local and global epigenetic patterns are dictated by the composition of the genome
depending on CpG spacing as well as sequence motifs and DNA structure (10), while in
turn DNA methylation will have a major influence on DNA shape (11). The transfer of
a methyl-group from the universal methyl donor S-adenosyl-l-methionine (SAMe) is
carried out by DNA methyltransferases. During the methylation reaction a methyl group
is transferred from SAMe to the cytosine, thereby leaving S-adenosylhomocysteine, which
at high concentrations inhibits the action of DNA methyltransferases.
Four DNA methyltransferases have been identified (DNMT1, DNMT3A, DNMT3B, and
DNMT3L) (Figure 1.3) (12). DNMT3L, however, lacks a catalytic domain, but is in complex
with DNMT3A important for maternal genomic imprinting and male spermatogenesis.
Simplified, DNMT1 acts as a maintenance methyltransferase as it prefers hemi-
methylated templates. It is located at the replication fork during the S phase of the cell
DNMT1 (1616 aa; 194 kDa)
DMAPD PBD RFTD CXXC BAH1 BAH2 I IV VI VIII IX X
NLS GK-repeat
DNMT3A (912 aa; 102 kDa)

PWWP ADD I IV VI VIII IX X
GK-repeat
DNMT3B (853 aa; 98 kDa)

PWWP ADD I IV VI VIII IX X
GK-repeat
DNMT3L (387 aa; 42 kDa)

ADD I IV VI VIII
GK-repeat
FIGURE 1.3 Schematic illustration of the domain structure of the mammalian DNMTs. All
DNMTs with the exception of DNMT3L contain a catalytic domain with 10 motifs characteristic
for DNA methylation activity. The DMAPD domain of DNMT1 binds DNMAP1, a factor repressing
transcription through interactions with HDACs. The proliferating cell nuclear antigen (PCNA)
binding domain, binds to the PBD of DNMT1, which recruits DNMT1 to replication foci at the
early and middle stage of the S-phase and binds a number of factors required for replication. The
RFTD domain localizes DNMT1 to the region undergoing replication by interacting with UHRF1,
which recognizes hemimethylated CpGs. The CXXC domain contains two zinc atoms forming
zinc fingers, which bind unmethylated CpGs. However, the exact function of this domain is
currently unclear. The two BAH domains are involved in chromatin remodeling, but their exact
function needs further investigation. The PWWP domain of DNMT3A and 3B is involved in
protein-protein interactions, tethers the DNMTs to chromatin regions including pericentromeric
heterochromatin regions marked with H3K26Me3. The cysteine-rich plant homeodomain (PHD)-
like ADD domain interacts with a multitude of proteins including H3K9 methylases, co-repressors,
and heterochromatin protein 1. DNMT3A and 3B interact with the C-terminal domain of DNMT3L
increasing de novo DNA methylation activity. Abbreviations used: DMAPD: DNA methyltransferase-
associated protein 1 interacting domain; PBD: PCNA-binding domain; NLS: Nuclear localization
signal; RFTD: Replication foci targeting domain; CXXC: CXXC domain; BAH1/2: Bromo-adjacent
homology domain 1 and 2; GK-repeats: Glycine-lysine rich repeats; PWWP: PWWP domain; ADD:
ATRX-DNMT3-DNMT3L domain.
cycle and methylates the newly synthesized DNA strand using the parent strand as a
template with high fidelity (13). The symmetric sequence of CpGs thus allows to pass the
epigenetic information to pass through cell generations. A number of proteins associated
with the local chromatin structure such as LSD1 and URHF1 are required to ensure the
specificity and stability of the DNA methylation reaction associated with DNA replication.
However, DNMT3A and DNMT3B are also required for methylation maintenance (14).
De novo methylation is carried out by the methyltransferases DNMT3A and DNMT3B.
These enzymes have certain preferences for specific targets (e.g., DNMT3A together with
DNMT3L methylates maternal imprinted genes and DNMT3B localizes at minor satellite
repeats as well as the gene bodies of actively transcribed genes), but also work cooperatively
to methylate the genome (12,15). Possible trigger mechanisms to initiate de novo methylation
include preferred target DNA sequences, RNA interference, but mostly chromatin
structures induced by histone modifications and other protein-protein interactions (16,17).
Histone modifications such as H3K9Me3 are thought to initiate heterochromatin formation
and DNA methylation comes in as a secondary molecular alteration to ensure the stable
silencing of the repressed sequences.
1.4 5-HYDROXYMETHYLATION AND THE DNA

DEMETHYLATION PROCESSES
Mechanisms for DNA demethylation mechanism have long been searched for as active
demethylation occurs at different stages of development and a global hypomethylation is
associated with many cancers. DNA demethylation has been proposed to be either passive,
where the 5mC is removed owing to a lack of maintaining the methylation during several
cycles of replication, or as an active process, with direct removal of the methyl group
independently of DNA replication (18). The active process is initiated through the enzymatic
oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) (Figure 1.1) as described below in
this paragraph. However, 5hmC is now considered to be not only an intermediate in oxidative
DNA demethylation, but constitutes a distinct layer in the complex process of epigenetic
regulation with its own distribution and regulatory functions. The reaction yielding 5hmC
is catalyzed by the ten-eleven translocation (TET) methylcytosine dioxygenase family of
enzymes, consisting of three mammalian subtypes, TET1-3 (Figure 1.4) (12,19). 5hmC is
most abundant in human brain tissue and embryonic stem cells, but at levels approximately
tenfold lower than those of 5-methylcytosine (20). TET enzymes are expressed in a tissue/
cell-type and developmental stage dependent manner with 5hmC decreasing during cell
differentiation. Active demethylation of gene regulatory sequences plays a key role in
activating specific genes required for proper tissue-specific gene expression programs. 5hmC
levels do not correlate with 5mC levels of the respective tissue and 5hmC was found enriched
at specific active functional elements of the genome, in particular enhancers, promoters, and
TET1 (2138 aa)
CXXC CysD DSHB
Fe (II)- Low complexity a-KG-

binding insert binding
domain domain
TET2 (2002 aa)
CysD DSHB

TET3 (1660 aa) domain domain
CXXC CysD DSHB

domain domain
FIGURE 1.4 Domain structure of the mammalian TETs. The N-terminal region is involved in
chromatin remodeling and methylation sensing and can directly bind to DNA, while the C-terminal
catalytic domain consisting of a cysteine-rich domain and the double-stranded β-helix dioxygenase
domain, including an Fe(II)-binding HxD motif and an α-ketoglutarate-binding domain separated
by a low complexity spacer of unknown function, recognizes CpGs and oxidizes them. For TET2
the N-terminal domain is provided by a separate protein (IDAX).
gene bodies associating 5hmC with open chromatin and transcriptional activity (20,21).
5hmC levels are globally reduced in cancer and alterations of the TET enzymes have been
reported for various cancers (19,22). This observation suggests that 5hmC alterations may
have a distinct role in the development and progression of malignancies.
In addition to its regulatory function, 5hmC is an intermediate in the active demethylation
process (23), where it is further oxidized to 5-formylcytosine (5fC) and 5-carboxylcytosine
(5caC) again by the TET enzymes, with the latter two modifications being present at barely
detectable levels in the human genome (Figure 1.5). Both the carboxyl and the formyl groups can
be removed enzymatically with or without base excision, generating an unmethylated cytosine.
1.5 TRANSCRIPTION AND GENOME STABILITY

Transcription does not occur on naked DNA but in the context of chromatin, which
critically influences the accessibility of the DNA to transcription factors and the DNA
polymerase complexes. DNA methylation, histone modifications and chromatin
remodeling are closely linked and constitute multiple layers of epigenetic modifications to
control and modulate gene expression through chromatin structure. DNMTs and histone
NH2 NH2 OH NH2

DNMT TET
N N N
N O N O N O
R′ R′ R′
Cytosine 5mC 5hmC
TET
O NH2
N
Base excision repair
N O
TDG
R′
OH 5fC
R′
TET
Abasic site
TDG
O NH2
HO N
N O
R′
5caC
FIGURE 1.5 Demethylation pathway. TET enzymes successively oxidize 5mC to 5hmc, and
5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) are dependent on α-ketoglutarate and
Fe(II). 5fC and 5caC are replaced by unmodified cytosines through a thymine DNA glycosylase
(TDG) initiated base excision repair (BER) mechanism, although additional mechanisms for DNA
demethylation do exist.
deacetylases (HDACs) are found in the same multi-protein complexes and methyl CpG-
binding domain proteins (MBDs) interact with HDACs, histone methyltransferases as well
as with the chromatin remodeling complexes. Furthermore, mutations or loss of members
of the SNF2 helicase/ATPase family of chromatin remodeling proteins such as ATRX or
LSH lead to genome-wide perturbations of DNA methylation patterns and inappropriate
gene expression programs.
Active gene Repressed gene
RNA Pol II
RNA Pol II
HDAC
TF
TF TF MBD
Alternative gene expression
RNA Pol II
TF
RNA Pol II
TF
FIGURE 1.6 Methylation sensitivity of transcription factors. A simplified view how DNA methylation
might influence transcription factor (TF) binding. Some TFs are only binding to unmethylated DNA
and methylation will impede binding. Methylation will on the other hand attract methyl binding
proteins which in complex with HDACS and other repressors will silence gene expression. Other
transcription factors might also have a higher affinity for methylated compared to unmethylated
DNA. Methylation changes at upstream regulatory elements such as enhancers might induce
changes in the TF occupancy of the regulatory element, which can lead to activation of alternative
genes or transcripts.
Transcription may be affected by DNA methylation in several ways (Figure 1.6). First, the
binding of transcriptional activators such as Sp1 and Myc may be inhibited directly by the
methylated DNA through sterical hindrance, while other transcription factors especially
homeodomain transcription factors are attracted by methylated target recognition
sequences (24–26). Methylation of CpG sites in a target sequence can thereby lead to change
in transcription factor occupancy at the same sequence and activation of tissue-specific
genes (27). Second, methylated DNA is bound by specific methyl-CpG binding domain
(MBD1, MBD2, and MBD4) proteins or methyl-CpG binding proteins (MeCP2) as well as
proteins of the Kaiso family (12,28,29). They recruit transcriptional co-repressors such as
histone deacetylating complexes, polycomb proteins and chromatin remodeling complexes,
thereby establishing a repressive closed chromatin configuration (Figure 1.6). Mbd3 binds
specifically hydroxymethylated cytosines.
In many cases, DNA methylation occurs subsequently to changes in the chromatin
structure and is used as a molecular mechanism to permanently and thus heritably lock the
gene in its inactive state. It should be underlined that an unmethylated state of a CpG island
or gene regulatory element does not necessarily correlate with the transcriptional activity
of the gene, but rather that the gene can be potentially activated. The simple presence of
methylation does therefore not necessarily induce silencing of nearby genes. Only when
a specific core region of the promoter that is often—but not necessarily—spanning the
transcription start site becomes hypermethylated, the expression of the associated gene is
modified (30). In CpG-poor intergenic gene regulatory regions DNA methylation is highly
dynamic, CpG dinucleotides are mostly highly methylated, but methylation is reduced
when the region or the methylated CpG is bound by transcription factors (31).
DNA methylation plays an important role in the maintenance of genome integrity by
transcriptional silencing of repetitive DNA sequences and endogenous transposons (32).
DNA methylation may prevent the potentially deleterious recombination events between
non-allelic repeats caused by these mobile genetic elements. In addition, methylation
increases the mutation rate leading to a faster divergence of identical sequences and
disabling many retrotransposons.
The functional relevance of DNA methylation (and other epigenetic changes) can now
be interrogated by epigenetic editing using mainly a nuclease deficient version of the
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated
protein (Cas) 9 system, which allows recruiting chromatin modifying and remodeling
complexes to specific target sequences (33). The fusion of the core catalytic domain of a
DNA methyltransferase (e.g., DNMT3A) or demethylase (e.g., TET1) to a modified nuclease
deficient Cas9 (dCas9) has been shown to induce specific targeted epigenetic changes either
locally if a promoter is targeted or more regionally if a distant gene regulatory element such
as an enhancer is targeted (33–35).
1.6 EPIGENETIC CHANGES IN HEALTH AND DISEASE

Cytosine methylation is essential for normal mammalian development including
X chromosome inactivation and correct setting of genomic imprinting. Epigenetics holds
the promise to explain at least a part of the influences the environment has on a phenotype
as described in Chapter 5 and is an integral part of aging and cellular senescence whereby
the overall content of DNA methylation in the mammalian and human genome decreases
with age in all tissues especially at repetitive elements (36,37). Despite its stochastic nature,
DNA methylation levels at a number of specific loci have been shown to correlate very
well with lifetime (chronological) age and several DNA methylation signatures have been
developed for the accurate prediction of the age through the analysis of DNA methylation
patterns (36,38). Accelerated epigenetic changing has been associated with earlier all-
cause mortality in later life (39), while people with exceptional longevity show a decreased
epigenetic age compared to their chronological age (40).
DNA methylation and chromatin structure are strikingly altered in many complex
diseases. Mutations in genes that are part of the molecular machinery responsible
for correct establishment and propagation of the epigenetic modifications through
development and cell division lead to the neurodevelopmental disorders such as ICF
(immune deficiency, centromeric instability, and facial abnormalities, DNMT3B) (41)
or Rett syndrome (MeCP2) (42), while mutations in TET2 have been found in multiple
hematological malignancies, where they probably among the earliest genetic events of
the disease (43).
Cancer is by far the most studied disease with a strong epigenetic component (44,45). In
tumors, a global loss of DNA methylation (hypomethylation) of the genome is observed.
This hypomethylation has been suggested to initiate and propagate oncogenesis by inducing
aneuploidy, genome instability, activation of retrotransposons, and transcriptional activation
of oncogenes and pro-metastatic genes (46). The overall decrease in DNA methylation is
accompanied by a region- and gene-specific increase of methylation (hypermethylation) of
multiple CpG islands frequently associated with transcriptional silencing of the associated
gene (44,45). This hypermethylation is not random as it occurs primarily at gene promoters
that are targets of the polycomb repressive complex 2 (PRC2) and marked by H3K27Me3 in
(embryonic) stem cells and resembles the DNA methylation changes observed during aging
albeit at a much greater amplitude.
While the contribution of somatic mutations to carcinogenesis has long been recognized,
it has become evident that epigenetic changes leading to transcriptional silencing of tumor
suppressor genes constitute an at least equally contributing mechanism (45,47). DNA
methylation changes and genetic mutations co-occurring in the same tumors show different
dynamics and evolve differently during tumor progression supporting the functional
relevance of epigenetic changes on the phenotype of the tumor (48). Epigenetic changes
occur at higher frequency compared to genetic changes and may be especially important
in early-stage human neoplasia (49).
Examples for the use of DNA methylation-based biomarkers include therefore early
detection with the commercial FDA-approved Epi proColon blood test being a prime
example. This test analyzes the methylation profile of an intronic sequence of the SEPT9 gene
for the population-wide screening of colorectal cancer achieving a sensitivity of 50%–80%
depending of the stage of the cancer and a very high specificity (>95%) (50,51). A number
of DNA methylation changes have been linked to prognosis of different cancers (52). In
addition to the application for early detection of cancers, the analysis of the methylation
status of CpG islands can be used to characterize and classify cancers as for examples
recently demonstrated for subtypes of Ewing sarcoma, which are all characterized by the
same recurrent genetic alteration (53). Furthermore, as the DNA methylation patterns of
distant metastases will at least partly carry the tissue-specific DNA methylation signature
of the primary tumor, analysis of the DNA methylation profile of the metastases has shown
to permit the identification of the tissue of the primary tumor (54). Similarly, the DNA
methylation profile of cell-free DNA from plasma/serum contains the information of the
tissue it is released from and allows detecting the tissue-of-origin of a cancer (55). DNA
methylation changes detect tumor recurrence as well as predict and monitor patients’
response and the effectiveness to a given anti-cancer therapy with the prediction of the
response of patients with glioblastomas to the alkylating agent temozolomide based on
the DNA methylation status of the DNA repair gene MGMT being the prime example
(56). As DNA methylation is a non-mutational and therefore—at least in principle—a
reversible modification, it can be used as point of departure for anti-neoplastic treatment by
chemically induced demethylation (57). Two DNA methyltransferase inhibitors (DNMTis)
(azacytidine [Vidaza] and 5-aza-deoxycytidine [decitabine, Dacogen, Decitibine]) have
been approved for the treatment of several hematological malignancies (58,59). At low
doses azacytidine and decitabine induce their effect through demethylation of silenced
genes associated with reduced apoptosis, cell differentiation and proliferation, whereas
at higher doses the main cytotoxic effect is due to DNA damage after incorporation (60).
Second-generation DNMTis with improved pharmacology and lower toxicity such as
the prodrug SGI-110 show high potential for the use in the treatment of several different
malignancies (58). Epigenetic therapy is rapidly evolving with many combination therapies
now under investigation.
The field of epigenetics of other complex diseases is still relatively young, but epigenetics
may provide the missing link between the genetic susceptibility and the phenotype by
mediating and modulating environmental influences. Several neuropsychiatric disorders
have been linked to epigenetic changes (61). Epigenetic dysregulation in cognitive disorders
such as Alzheimer’s disease as well as age-related memory decline has also been reported
(62,63). DNA methylation patterns are also disturbed in atherosclerosis (64), diabetes (65) as
well as inflammatory, autoimmune, and allergic diseases (66,67). In a number of studies the
epigenetic changes are located in the same genomic region as genetic variation previously
associated with the disease (68,69). It is difficult to infer causality from most of studies, but
at least in some case DNA methylation seems to mediate the effects of genetic variation to
yield the phenotype.
1.7 CONCLUSION
Although our knowledge on the regulation of DNA methylation has been rapidly increasing
over the last years, the gained information has mainly led to the insight that the complexity
of the gene regulatory network, in which DNA methylation plays a pivotal role, had been
underestimated. CpG islands, which had been the focus of research for decades, might
contribute little to the plasticity of the cells necessary for cell-type specific differentiation
and to cope with internal and environmental cues. New functional tools allow now for
the first time to assess the functional consequences of DNA methylation changes during
development and disease. Biomedical applications of DNA methylation as biomarkers for
cancer and other complex diseases, but also for prenatal testing, have become routine and
first assays have been approved by regulatory agencies paving the way for a more widespread
acceptance and use of DNA methylation-based tests.
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Another random document with
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“Oh, I’m not thinking of you, darling; you are as cold and austere as
Diana herself. I do wish you were not so icy to some one—you know
who I mean.”
But Aurea’s expression was not encouraging, and her vivacious
companion continued—
“Isn’t this a darling old place?” rising and looking over the Italian
gardens and sloping lawns. “Somehow I always feel sorry for those
Davenants, and as if we had no business here, and it was still theirs.
We have their heirlooms too—the Davenants’ Vandyke, the
lacquered cabinets, the Chippendale chairs. Dad bought them, as
they matched the place; but we don’t fit in. Dad and mum were far
happier in London; keeping up a great estate and a great position is
an awful strain when one was not caught young. Do you know, the
servants are a frightful trial; they find the country dull. And at the last
ball we had, nearly all the hired waiters were intoxicated; they drank
most of the champagne, and one of them handed a lemonade to
Lord Mottisfont, and said there was no fiz left! The mum was so
mortified she wept, poor dear.”
“Well, everything always seems to go smoothly, quite London
fashion, and without a hitch,” said Aurea consolingly.
“Yes, but not behind the scenes; and the Mum sometimes makes
such horrible blunders in etiquette, such as sending in a baronet’s
wife before a countess—and the countess looked pea-green!
Altogether it’s a fag. When Bertie marries I expect pater will make
him over the place. I wouldn’t mind reigning here myself—would you,
Aurea?”
“What a silly question, Joey! I’m not cut out for reigning anywhere.”
“Only in people’s hearts, eh?” stroking her cheek with a finger. “Isn’t
that a pretty speech? Well, come along, I want to show you the
pretty things I collected abroad—my fans and lace and
embroideries.”
But just at this moment a maid entered, and said—
“If you please, ma’am, I was to say that Miss Parrett’s car is at the
door, and she’s waiting for Miss Morven.”
The drive home was made by another road (in spite of Miss Parrett’s
querulous protestations, and it was evident that the sooner she could
abandon the motor the better she would be pleased). Susan, on the
other hand, was anxious to see more of the country, and make a
detour round by a little town, eight miles away.
“Why, it’s nothing,” she protested; “it’s not worth taking out the car for
a run over to Westmere—one might as well walk!”
“One would think it was your car, to hear you talking, Susan;” and
Miss Parrett threw herself back in the corner, and closed her eyes,
only to open them again immediately, as they sped along the empty,
country roads between hedges already green.
“There’s Hopfield Hill!” she exclaimed, suddenly sitting bolt upright.
“I’m not going down that in a motor, so don’t suppose it, either of
you.”
“But it’s three-quarters of a mile long, and you have a blister on your
heel,” expostulated her sister. “Come, Bella, don’t be foolish.”
“Don’t argue; if it was twenty miles I’d walk it. This thing gives me
palpitation as it is.”
In spite of Aurea’s and Miss Susan’s prayers, vows, and assurances,
Miss Parrett descended at the top of a long hill, insisted that her
companions should accompany her, and together the trio tramped
down in the mud, whilst the chauffeur sped along merrily, and
awaited them at the base. On their way home by a narrow byroad
they nearly met with a nasty accident. A cart, drawn by a young
horse, was coming out of a gate as the motor approached, and there
was an exciting scene. The boy who was driving lost his head, the
horse reared and plunged, Miss Parrett shrieked, and the motor—
which was jammed into the bank—shuddered all over; but, after a
moment—a critical moment—all was well—all but Miss Parrett, who
collapsed into her corner, and announced that she had spasms of
the heart, and was dying!
Ultimately they reached the Manor without further trouble; the dying
lady was restored with brandy and water, and Owen the chauffeur
spent the next two hours in cleaning the muddy car. This was the
part of the job he loathed. Just as he had completed his task, he
beheld, to his discomfiture, the cook stepping delicately across the
yard, carrying a black bottle in one hand, and a wineglass in the
other.
“Good-evening to you, Mr. Owen. My word! you do look hot after all
your fag with the car. Beastly work, ain’t it? I’ve just run over with a
glass of ginger wine—it’s my own.”
“Thank you, Miss Hicks. It’s awfully good of you, but it’s a thing I
never touch,” he answered politely.
“Then what do you say to a pint o’ beer, or a cup o’ tea?”
“No—er—I’m about done,” pulling down his sleeves; “and I’m going.”
“The old girl seems a bit upset,” remarked the cook, who had come
out for conversation; “she’s awful frightened of the car.”
“She needn’t be,” he answered shortly.
“Not with you a-driving, I’m sure, Mr. Owen. I wish I could have a run
in it, eh? There was a chauffeur as I knew in London—rather a pal of
mine—that used to give his friends fine drives, as much as down to
Brighton, when the family was out of town. He were a treat, I can tell
you!”
“Was he? I’d say he was a thief—unless he used his own petrol.”
“Oh, come now, you’re mighty strict and proper, I can see. Chapel, I
suppose?”
“No; you’re wrong there.”
“Look here, what’s the use of being so stand-off and so stiff—it’s
downright silly; you and me, as it were, coming to this cruel place
from the same reference. Won’t you call round and take me for a
nice walk on Sunday afternoon?”
“No; you’re very kind—but I can’t.”
“Why, what else have you to do?” her eyes kindling. What else had
he to do? Lie on his bed and smoke, and read Leila’s papers. And
there were other alternatives; he could take a long stretch, say ten
miles out and back, or he might go to evening service and gaze at
Aurea Morven!
“My word! you are a stupid!” declared Miss Hicks; “even if you have
a young woman up in town, she won’t mind. Have you a young
lady?” and her bold eyes were searching.
Had he? He had! His young lady was Miss Aurea, her mistress’s
niece—Aurea or no other; and as he put on his coat he looked his
tormentor steadily in the face and answered—
“Yes, I have.”
“Oh, so that’s it! I see! And you’re hurrying off to write to her?
Well,”—spitefully—“I can tell you one thing for yer comfort, there’s no
post out of Ottinge before Monday morning!”
“Isn’t there? That’s a pity. Well, good-evening to you, Miss Hicks;”
and he walked off, leaving Miss Hicks gaping after him. She,
however, consoled herself with a couple of glasses of ginger wine,
before re-entering the house.
CHAPTER XII
THE DOGS’ HOTEL
The morning succeeding the motor’s first trip proved depressingly

wet; thick mists of cold spring rain shrouded the outlook from the
Manor, beat down upon the pleasure ground, and made pools in the
hollows of the drive.
Miss Parrett, who was, as the servants expressed it, “dodging” in
and out of the sitting-room, issuing commands and then withdrawing
them, fastened upon the chauffeur the moment he came for orders.
No, the car would not be required, and he could go some errands
into the village.
“Mind you don’t go loitering and gossiping,” she added. “I know your
sort, chattering with the maids. Remember that your time belongs to
me;” and she pointed a stumpy forefinger at her knitted jacket. “I’ve a
note for Miss Morven at the Rectory, and another for Ivy House, and
I want some things at Topham’s shop. I’ll give you a list. You can go
into the schoolroom and wait.”
Calm with excessive rage Wynyard entered the schoolroom, where
he found Miss Susan with a handkerchief tied over her head, and an
apron over her dress, unpacking dusty china from a battered case.
“Such a day!” she exclaimed cheerfully; “and they say it’s going to
last—so we shall be very busy, and make use of you.”
“All right, miss,” he assented shortly; the accusation of “chattering
with maids” still left its sting.
“We are going to get up the cases of old books and china, and
unpack them here. The carpenter is putting shelves in the library; but
he is such a lazy fellow, I don’t expect he will come out in this
weather.”
“There you are as usual, Susan, talking and idling people,” said her
sister, entering with two notes and a list; and in another moment
Wynyard had been dispatched.
First of all he went to the Rectory, and here the door was opened by
Mr. Morven himself, attended by Mackenzie, who immediately
stiffened from head to tail, and growled round the chauffeur’s legs,
evidently recognising in him the ally of his mortal foe. Mr. Morven
was a squarely built elderly man with a grey beard, a benevolent
expression, and the eyes of the dreamer.
As he took the note he glanced at the messenger, and his eyes
dilated with the intentness of a surprised stare. Wynyard’s type was
not common in the parish; somehow Mrs. Hogben’s lodger did not
correspond with his surroundings.
“I see this is for my daughter,” he said, and beckoning to a parlour-
maid he handed it to her. “Just come into my study, will you, till the
answer is written,” leading the way across a wide hall panelled in
oak. Through an open door Wynyard caught a glimpse of the
drawing-room, and was conscious of a faded carpet, fresh chintz,
books, old china, a glowing fire, and a fragrant atmosphere. The
general impression of the Rectory, with its oaken staircase, family
portraits, and bowls of potpourri, was delightful but fleeting; it
seemed a peaceful, flower-scented old house, of spotless neatness.
“You’re a newcomer, I believe?” said the Rector, preceding him into a
room lined with books from floor to ceiling, and seating himself at a
writing-table. “Miss Parrett’s chauffeur?” and he smiled to himself at
some reminiscence. “I see they are making use of you. Church of
England?”
“Yes, sir.”
“If you have any sort of voice—tenor, baritone, or bass—we shall be
glad to have you in the choir; our tenor is getting on; he must be
close on seventy.”
“I’m afraid I’m not much good, sir.”
“Well, if you don’t sing, you look like a cricketer, eh? I must get
something out of you, you know;” and he laughed pleasantly.
“Oh yes, I can play cricket all right.”
“If you can bowl a bit, with Miss Parrett’s leave, I’ll put you into the
village club; we rather fancy ourselves, and a young man of your
stamp will be an acquisition.” At this moment Aurea entered, carrying
an enormous cardboard box.
“Good-morning,” she said. “I see aunt sent you for the lampshade,
and here it is.”
“What a size!” exclaimed her father. “Why, you must have robbed
your best hat! I declare it’s not fair to a man to ask him to be seen
with such a thing going through the village.”
“Not half so bad as seeing people go down the street with a black
bottle in either hand!” retorted his daughter.
“I don’t mind, sir,” said Wynyard, taking up the box as he spoke.
“Please tell Aunt Bella I will be after you in two or three minutes,”
said Aurea; then to her father, “She wants to unpack grandpapa’s
books at last!”
“You mean that she wants you to unpack the books,” corrected Mr.
Morven; “you might steal a few for me, eh? I suppose you will be
away all day?” and he looked at her rather wistfully.
“No, no, dear, I’ll be back soon after tea.” To Owen: “Straight on, it’s
an easy door.”
As Wynyard turned in the hall and backed out, box in hand, he had a
vision of pretty Miss Aurea perched on the arm of his chair, with her
arm round her father’s neck. Lucky old beggar!
His next errand was to the shop—Topham’s—and as he lingered
irresolutely in the rain, staring up and down the street, he was
overtaken by a brisk figure in an aquascutum and motor cap.
“I see you are searching for our emporium,” she began, “and I’ll
show it to you—in fact, I’m going in myself to get some brass-headed
carpet nails.”
The shop stood sideways to the street, as if anxious for
concealment, and was the most astonishing place of its kind that
Wynyard had ever entered. A stall in an Indian bazaar was tame and
tidy in comparison. The house was old and low, the shop of narrow
dimensions; it widened out as it ran back, and lost itself in a sort of
tumbledown greenhouse. The smell was extraordinary, so varied,
penetrating, and indescribable—and small wonder, he said to
himself, when he had inspected the stock!
An oldish woman with a long nose (the Ottinge nose) stood stiffly
behind the counter; at her left the window was full of stale
confectionery, biscuit tins, sticky sweets in glass bottles, oranges
and apples in candle boxes; heaps of Rickett’s blue, and some fly-
blown advertisements.
Behind Mrs. Topham were two shelves dedicated to “the library,”
which consisted of remarkably dirty and battered sixpenny novels;
these she hired to the village at the generous price of a penny a
volume for one week. To the left of the entrance were more shelves,
piled with cheap toys, haberdashery, and china; and here ended the
front of the shop. Concealed by a low screen were tins of oil, a barrel
of ginger ale on tap, and a large frying-pan full of dripping. The
remainder of the premises was abandoned to the greengrocery
business on a large scale—onions, potatoes, and cabbages in
generous profusion.
“Good-morning, Mrs. Topham,” said Miss Morven. “What a wet day!
How is your cough?”
“Oh, I’m amongst the middlings, miss. What can I do for you?”
“I want some brass-headed carpet nails, and my aunts have sent a
list;” and she motioned to Wynyard.
Mrs. Topham seized upon it with her long, yellow fingers (they
resembled talons)—the Manor were good customers.
“You can send over the things, Mrs. Topham; but I want the nails
now.”
“I’m sure, miss, I’ve got ’em, but I can’t just rightly think where they
be.”
As she spoke, she turned out a drawer and rummaged through it
violently, and then another; the contents of these gave one an idea
of what is seriously understood by the word “chaos”: wool, toffee,
night-lights, dog biscuits, and pills were among the ingredients.
“Try the blue box,” suggested Aurea, who was evidently acquainted
with the resources of the establishment.
The blue box yielded nothing but a quantity of faded pink ribbon, a
few postcards of the church and Drum, a dozen tennis balls, some
small curling-pins, and several quires of black-edged paper.
“Why, if that isn’t the very thing I was looking for last week!”
exclaimed Mrs. Topham, as she pounced on the paper. “And now
Miss Jakes she’s bin and got it over at Brodfield; ’tis a cruel chance
to be near a big town—and so there’s for you!”
As the search for nails promised to be protracted, Miss Morven
turned to Wynyard and said—
“You need not wait; please take the lampshade on, and say that I’m
coming.”
But before returning to the Manor he had yet another errand to fulfil
—a note for Mrs. Ramsay at Ivy House. Here he rang repeatedly, he
even gave heavy single knocks with the bulbous brass knocker, but
received no reply beyond the distant barking of indignant dogs. At
last he went round and discovered a large paved yard, but no human
being. Then he ventured to approach one of the sitting-room
windows and peered in—a comfortable dining-room with a cheerful
fire, but empty. No, just underneath the window on a sofa lay an
elderly man fast asleep. He wore grey woollen socks on his
slipperless feet, an empty tumbler stood on a chair beside him—and
this at eleven o’clock in the morning. (True, O. Wynyard, but it had
contained no stronger drink than hot water.)
He had the intention of rapping at the pane, but changed his mind
and retired to the door, and as he waited he heard a voice above him
calling out in a rich brogue—
“Bad scran to ye, Fanny, if there isn’t a young gentleman below wid a
big band-box, and he is afther pullin’ out the bell by the roots; ’tis a
shame to lave him standin’ in all the pours of rain! An’ such a lovely
big man!”
At this moment the hall door was opened by a tall dark woman in a
mackintosh and motor cap, with two frantic fox-terriers on the lead,
and a self-possessed French bulldog in dignified attendance.
“I’m afraid you’ve been waiting,” she said, in a soft brogue. “I was
away at the kennels, the servants were upstairs, and the Captain is
asleep.” Then, opening the note (as well as the fox-terriers would
permit), she glanced over it, and the messenger glanced at her—a
woman of thirty-five, with a thin, well-bred face, black hair, and very
long lashes. When she lifted them, he saw that her eyes were of a
blue-black shade, both sad and searching—the whole expression of
her face seemed to be concentrated in their pupils.
“Please tell Miss Parrett I’ll come to tea. I’ve no time to write. I have
to take the dogs out.” The fox-terriers were straining hard at their
leash. “They must have exercise; and when these come back, there
are three more.”
As she spoke, Wynyard could hear the injured yelping of their
disappointed companions.
“Now, don’t open the little dogs’ room,” she called to an elderly
woman in the background, who gave the amazing answer—
“And what would ail me?”
“And mind that the Captain has his broth at twelve.” Then she
stepped out into the beating rain, and Wynyard was surprised to find
that Mrs. Ramsay was about to accompany him.
“I’m going your way,” she explained; “it’s the safest. These two are
new dogs, and I’m rather afraid to go near the Rectory; their
Aberdeen is such a quarrelsome beast—always trailing his coat.”
“Mackenzie?”
“Ah, and so you know him?” she said, with a smile; “you weren’t long
in making his acquaintance.”
Wynyard exhibited his left hand, and a severe bite.
“I suppose he was trying to kill Joss; that’s his profession—a killer of
other dogs.”
“You seem to have a good many of them,” as an afterthought,
“ma’am.”
“Yes; they are not all my own. I take in boarders—only six at a time,
and they must be small, no invalids accepted. I look after them for
people who go abroad, or from home for a few weeks. I am fond of
dogs, so I combine business and pleasure.”
“Yes, ma’am; but they must be a trouble and a responsibility—other
people’s pets.”
“I have to take my chance! Some are so nice, it just breaks my heart
to part with them. Indeed, there’s Tippy here, the bulldog, I’m
pretending he is sick—isn’t it a shame of me? Some are surly, others
so sporting, that half my time is spent in scouring the country, and
looking into rabbit holes. Others are quarrelsome, or chase, snap,
and kill fowl and get me into great trouble. I never keep them on an
hour after their time is up. You are the Miss Parretts’ chauffeur, aren’t
you?”
“Yes, ma’am.”
“Is this your first situation?” eyeing him keenly.
“Yes, ma’am.”
Why did she ask such a question? Did she, to use the good old
expression, “smell a rat”?
“I’m afraid you will find Ottinge terribly dull. I wonder how you
discovered a place so far from everywhere—just the back of
beyond?” and she looked at him interrogatively—her dark blue eyes
were extraordinarily piercing.
To this impertinent remark no reply was necessary, as it brought
them precisely to the Manor gate. The lady nodded, and walked on
quickly—a slim, active, resolute figure, with the straining fox-terriers
dragging at her hands, the little bulldog trotting sedately at her heels.
The group passed steadily out towards the open country, with the
light rain drifting down upon them. What queer people one came
across in Ottinge! Miss Parrett, the ill-tempered old bully, the Hon.
Mrs. Ramsay, with her soft voice and expressive eyes, eking out a
living by making herself a slave to strangers’ dogs.
“Oh, so she sent a verbal message, did she?” snorted Miss Parrett.
“Well, when I was a girl,”—turning to her sister—“and people asked
me out, I always wrote them a proper note; but manners are not
what they were in my day. Oh, if my dear, courteous father could
only know of some of the things that are done, he would turn in his
grave!”
Miss Parrett was fond of quoting the old Colonel, and insisting upon
his devotion to herself; whilst, if the truth were known, they had been
bitterly antagonistic to one another during his lifetime, and the Manor
was the frequent scene of acrimonious quarrelling, unfilial gibes, and
furious rejoinders.
It was fully a quarter of an hour later when Miss Morven arrived with
the brass-headed carpet nails.
“I knew she had them!” she declared triumphantly; “for she got a lot
for us last winter, so I ransacked the shop, and, after a long search,
where do you think I found them, Susan?”
“In her pocket, to be sure!”
“No, not quite—probably I shall next time. In one of the brown
teapots she has on sale! She was surprised—I wasn’t! She is getting
quite dotty, and won’t have help; and there is Dilly, her pretty, flighty
granddaughter, with nothing to do but flirt!”
All that day Wynyard worked zealously, assisting the carpenter (who
had come after all) and in unpacking and dusting books that had not
seen daylight for thirty years. On this occasion, in spite of Miss
Parrett’s condescending invitation, he dined at Holiday Cottage.
That very same evening Mrs. Ramsay came to tea at the Manor, and
was fervent in her admiration of the drawing-room, which praise Miss
Parrett absorbed with toothless complacency, saying in her
quavering bleat—
“I’m so glad you like it. Of course it was my taste, and my ideas, and
they are my things; but Aurea and Susan helped me—yes, and the
chauffeur made himself useful.”
Wynyard, who was working close by, felt inclined to laugh out loud. It
seemed to him that he was everything but a chauffeur: window-
cleaner, carpet-layer, messenger, and assistant carpenter—a good
thing he was naturally pretty handy. And although all these extra
burdens had been laid upon him, the first impulse to throw up the
situation had died away; he did not mind what jobs the old lady set
him to do, but would take them as all in the day’s work, for he had no
intention of leaving Ottinge at present—he must have some
consideration for Leila!
After tea, when Miss Parrett was engaged in scolding her domestics
and writing violent postcards to her tradesmen, Mrs. Ramsay drew
Aurea into the drawing-room.
“Well, me dear,” and her dark eyes danced, “I did not say a word
before your aunts, but I’ve seen the remarkable chauffeur! I assure
you, when I opened the door and found him standing there with a
large box, you might have knocked me down with the traditional
feather! I was taking the new dogs out for a run, and so we walked
together to this gate.”
“What do you think of him?” asked Aurea, carelessly, as she
rearranged some daffodils in a blue bowl.
“What do I think? I think—although he scarcely opened his lips—that
there is some mystery attached to him, and that he is a gentleman.”
“Why do you say so?” inquired the girl, anxious to hear her own
opinion endorsed. “He is not a bit smarter than the Woolcocks’ men.”
“Oh, it’s not exactly smartness, me dear, it’s the ‘born so’ air which
nothing can disguise. His matter-of-course lifting his cap, walking on
the outside, opening the gate, and, above all, his boots.”
“Boots!”
“Yes, his expensive aristocratic shooting boots; I vow they come from
Lobbs. Jimmy got his there—before he lost his money.”
“Perhaps the chauffeur bought them second-hand?” suggested
Aurea.
Mrs. Ramsay ignored the remark with a waving hand.
“I cannot think what has induced a man of his class to come and
bury himself here in this God-forsaken spot.”
“Ottinge-in-the-Marsh is obliged to you!”
“Now, you know what I mean, Aurea. You are a clever girl. I put the
question to him, and got no satisfactory answer. Is it forgery, murder,
piracy on the high seas, somebody’s wife—or what?” She rested her
chin on her hand, and nodded sagaciously at her companion. “I
understand that he has been working indoors a good deal, and
helping you and Miss Susan.” She paused significantly. “You must
have seen something of him. Tell me, darling, how did you find him?”
“Most useful, wonderfully clever with his hands, strong, obliging, and
absolutely speechless.”
“Ah! Does he have his meals here?”
“No.”
“Dear me, what a cruel blow for the maid-servants! Did he come
from a garage?”
“No; a friend of Aunt Bella’s found him.”
“A woman friend?”
“Yes; she gave him an excellent character.”
“And what of hers?”
“Oh, my dear Kathleen, she is Lady Kesters, a tremendously smart
Society lady, awfully clever, too, and absolutely sans reproche!”
“Is that so?” drawled Mrs. Ramsay. “Well, somehow or other, I’ve an
uneasy feeling about her protégé. There is more than meets the eye
with respect to that young man’s character, believe me. My woman’s
instinct says so. I’m sorry he has come down and taken up your
aunt’s situation, for I seem to feel in me bones that he will bring
trouble to some one.”
“Oh, Kathleen! You and your Irish superstitions!” and Aurea threw up
her hands, clasping them among her masses of hair, and stared into
her friend’s face and laughed.
“Well, dear, if he does nothing worse, he will have half the girls in
love with him, and breaking their hearts. It’s too bad of him, so good-
looking, and so smart, coming and throwing the ‘comether’ over this
sleepy little village. Believe me, darlin’, he has been turned out of his
own place; and it would never surprise me if he was just a nice-
looking young wolf in sheep’s clothing!”
“Oh, what it is to have the nice, lurid, Celtic imagination!” exclaimed
Aurea. “I don’t think the poor man would harm a fly. Joss has taken
to him as a brother—and——”
“Miss Morven as—a sister?”
“Now, what are you two conspiring about?” inquired Miss Susan,
entering, brisk, smiling, and inquisitive.
“I’m only discussing your chauffeur, me darlin’ Miss Susan. I notice
that several of the village girls drop in on Mrs. Hogben—you see I
live opposite—and they expose their natural admiration without
scruple or reserve.”
“Owen is a useful young man, if he is a bit ornamental—isn’t he,
Aurea? I’m going to get him to help me in the greenhouse, for I don’t
believe, at this rate, that we shall ever use the car.”
CHAPTER XIII
THE DRUM AND ITS PATRONS
Mrs. Hogben had lost no time in giving her lodger explicit

instructions as to what was expected of him in Ottinge! Her lecture
assumed a negative form. He was not to take out any one’s girl, or
there’d be trouble; he was not to talk too much politics, or there’d be
more trouble; he was not to drink and get fuddled and fighting, or
there was the Bench and a fine; as to amusement, there was cricket,
Mrs. Topham’s Library, and the Drum Inn, for his evenings.
The good woman said to herself, “The motor is always washed and
put away by six o’clock, and if he comes here, he must either sit in
his room or in the kitchen, and she wasn’t a-goin’ to have that
blocked up with young girls, and never a chair for herself and her
own friends.”
Wynyard readily took the hint; at Ottinge one must do as Ottinge did,
and he cheerfully accompanied Tom over to the Drum a few
evenings after his arrival.
“What sort of liquor do they keep, Tom?” he asked, as they crossed
the street.
“Well, some be better nor some, but there’s no bad beer; the old stuff
here is rare and strong, but it comes pretty dear.”
The low, wainscoted taproom, with its sanded floor, was full of day-
labourers, herds, ploughmen, cow-men, and carters taking their bit of
pleasure, talking loudly and disjointedly, drinking beer in mugs, or
playing the ever-popular game of “ring.” Here, for the first time in his
life, Wynyard was brought into personal contact, as man to man, with
the agricultural world as it is. In the more exclusive bar were to be
found farmers, owners of certain comfortable red houses scattered
up and down the street, the organist, the schoolmaster, the grocer—
in short, the moneyed patrons of the hostelry. Several were talking
over village affairs, discussing politics, racing, artificial manures, or
cattle. Some were playing draughts, some were reading the daily
papers, others were doing nothing. Of these, one was a bent,
gentlemanly individual in a grey tweed suit, with a grey moustache, a
grey, sunken, vacant face, who sat aloof smoking a brier pipe—his
eyes staring into vacancy. Another was a white-haired, shrunken old
man, who wore green carpet slippers, and occupied a cushioned
arm-chair, and the best seat near the fire. This was Joe Thunder, the
oldest inhabitant, ninety-three years of age his last birthday. Once
upon a time he had seen the world—and other worlds; now he was
comfortably moored in a fine, substantial cottage with a garden back
and front, kept bees, was an authority on roses, and filled the post of
the patriarch of Ottinge.
All newcomers were formally presented to Daddy Thunder, and as
Tom pushed Wynyard in his direction he said—
“This be the Parrett ladies’ new man, daddy.” To Owen, “Daddy,
here, he knows the place well, and can tell ye all about it, better nor
any, though he wasn’t Ottinge born.”
Daddy slowly removed his long clay pipe, and inspected the stranger
with a pair of shrewd little grey eyes. He had rosy cheeks, a
benevolent, even sweet expression, and looked fifteen years
younger than his age.
“Ye come fra’ London?” he began agreeably.
“Yes, sir, three days ago. It’s a good long journey.”
“Ay, mister,” nodding his white head expressively. “Ye don’t belong to
us. Yer speech—like the Bible chap—bewrayeth ye—y’re no working
man!”
“I am, indeed,” rejoined Wynyard quickly, “and working for my bread
the same as the rest of the company; it’s all I have to look to—my
two hands.”
“Nay, is that so?” and he glanced at him incredulously. “Well, I’ve bin
here a matter o’ twenty year, and I never see one o’ your make a-
comin’ in and settin’ in the Drum. There’s ’im,” and he indicated the
bent figure in the corner, whose pipe was in his hand, his eyes
riveted on the stranger with a look of startled inquiry.
“That’s the Captain, but ’e’s no account. ’E comes in and ’e sits and
maybe listens; ’e never speaks. They do say ’e ’ad a soort o’ stroke
in India, and ’is brain ’as melted like, but ’e is ’armless enough—
anyhow, ’is lady won’t put ’im away.”
“I suppose you’ve lived here a long time?” said Wynyard, drawing
forward a chair, and placing it so as to sit with his back to the said
Captain, whose stare was disagreeably steady.
“Twenty year, more or less. I am a south country man, and my
daughter she married and settled ’ere, and ’er ’usband died; an’ as
there was only the two on us, I come along to keep ’er company, and
to die ’ere, since I was gettin’ pretty old, being over seventy; but, Lor’
bless ye! that’s twenty-two year ago, and ’ere I be still gettin’ about,
and doin’ a bit o’ gardenin’. The air is grand—nothing ails me but
gout,” holding out a crippled hand. “This isn’t the place to die in—it’s
the place to live in. It keeps ye alive. Why, I’m ninety-three. Oh, it’s
what ye may call a terrible lively place.”
This was not his listener’s opinion, who would have instituted instead
the word “deadly.”
“You must have seen a great deal in ninety-three years,” said
Wynyard, lighting his pipe.
“Lor’ bless ye, yes; and I’ve a wunnerful memory.”
“Do you remember the days of Napoleon?”
“What—old Bony! Nay,” a little offended, “I’m not as old as that; but I
do mind a talk o’ ’is funeral in France.”
“I beg your pardon, I’m an awful duffer at dates. You remember
Wellington?”
“Oh ay, ’e was only the other day, so to speak.”
“And what else do you remember?”
“Well, as a lad, I remember I was terrible afeerd o’ the press gang.”
“The press gang?”
“Ay; that come pokin’ round after able-bodied men for the Navy, and
kidnappin’ ’em away to sea, and keepin’ them there, whether or no,
for years, and their families at home starvin’.”
“I say, what times!”
“Ay, so they was. I’ve seen two men ’angin’ in chains on Camley
Moor when I was about ten—it were for sheep-stealin’, and put the
fear o’ death on me. Surely I can ’ear them chains a-clankin’ now!”
Wynyard felt as if he had been suddenly precipitated into another
world. Here he was, sitting talking to a live man, who discoursed
familiarly of hanging in chains, and the press gang!
“Would you take something, sir?” he asked. “I’d like to drink your
health.”
“Ay, ay, I don’t mind ’avin’ a glass wi’ ye. Ginders! Ginders!” raising
his voice, “give us a taste of yer old beer, the best—two half-pints;”
and, as they were brought, he looked at Wynyard, and said, “To ye,
young sir, and good luck to ye in Ottinge; may ye live as long as I
do!”
“Thank you; have you any prescription for your wonderful health?”
“Ay, I have so. Look ’ere, I’ve not tasted medicine for fifty year. I don’t
hold wi’ doctors. I only eat twice a day—my breakfast at eight, and
my dinner at two. My daughter she do mike me a cup o’ tea at six,
but I don’t want it, and it’s only to oblige her. Work—work’s the thing
when yer young. I mind bein’ in the train one day, and a great heavy
man complainin’ o’ his pore ’ealth, and ’is inside, and another says, ‘I
can tell ye o’ a cure, master, and a sure one.’ ‘What’s that?’ ses ’e,
all alive. ‘Rise of a mornin’ at four o’clock, and mow an acre before
ye break yer fast, and go on mowing all day—that will cure ye—ye’ll
be a new man.’ ‘I’d be a dead un,’ ses ’e. My advice is: no medicine,
short commons, lots of work, and there ye are, and ye’ll live to
maybe a hundred.”
“But what about cuts and wounds? How do you doctor them?”
“Oh, just a plaster o’ earth, or a couple o’ lily leaves. One is as good
as t’other. Well, I’m a-goin’,” struggling to his feet; “an old gaffer like
me keeps early hours.”
As Wynyard handed him his stick, he slapped him smartly on the
back, and it was evident from this accolade that the “shover” was
now made free of the Drum.
The newcomer looked about him, some were playing dominoes,
some cards, one or two were reading the day’s papers, and all the
time the Captain sat immovable in a corner, and his eyes never
moved from Wynyard. Such cold, impassive staring made him feel
uncomfortable, and settling his reckoning he presently followed old
Thunder’s example and went home.
Captain Ramsay, whose fixed attention had made the stranger so
uneasy, had once been a popular officer in a popular regiment, and
when quartered in India had fallen in love with and married the Hon.
Kathleen Brian (daughter of an impoverished viscount) who was on a
visit to relatives in Simla. The first year was rapturously happy for
both of them, and then one day, when out pig-sticking near
Cawnpore, Captain Ramsay had his topee knocked off, and in the
excitement of the chase galloped on, with the result that he was
knocked over by a sunstroke. Sunstroke was followed by brain fever,
and he nearly died. Ultimately he was invalided home, and, owing to
ill-health, obliged to leave the Service. Nor was this all. He seemed
to become another man, his character underwent a complete
change; he was quarrelsome and morose, fought with his own
family, insulted his wife’s people, and developed into an Ishmael. He
invested his money in the maddest ventures, and rapidly dispersed
his entire fortune (Kathleen was penniless), and now nothing
remained but his small pension. Year by year he became more
disagreeable, restless, and strange. The couple wandered from
place to place, from lodging to lodging. Vainly his wife’s relatives
implored her to leave him; he was “impossible,” her health was
suffering; she, who had been so pretty, at twenty-seven looked
prematurely faded and haggard; but Kathleen was obstinate, and
would go her own way and stick to her bad bargain. Her brothers did
not know, and would never know, the Jimmy she had married—so

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Epigenetics and Assisted

Reproduction: An Introductory Guide

Subfertility Reproductive Endocrinology and Assisted

IVF and Assisted Reproduction: A Global History Sarah

Controversies in Assisted Reproduction 1st Edition

Manual of Embryo Culture in Human Assisted Reproduction

Infertility and Non Traditional Family Building From

Applied Social Science Methodology An Introductory

Epigenetics and Health: A Practical Guide 1st Edition

International Sports Law An Introductory Guide 1st

Cristina Camprubí, PhD

Joan Blanco, PhD

Library of Congress Cataloging-in-Publication Data

Names: Camprubi, Cristina, editor. | Blanco, Joan, editor.

Visit the Taylor & Francis Web site at

Chapter 1 ◾ A Short Introduction to DNA Methylation 1

Chapter 2 ◾ Epigenetic Modifications of Histones 17

Chapter 3 ◾ Epigenetic Reprogramming in Early Embryo Development 29

Chapter 4 ◾ Epigenetic Reprogramming of Mammalian Primordial

Chapter 5 ◾ Laboratory Molecular Methodologies to Analyze DNA

Chapter 6 ◾ Intrinsic and Extrinsic Factors That Influence Epigenetics 99

Chapter 7 ◾ Epigenetic and Assisted Reproduction Epidemiological

Chapter 8 ◾ Epigenetics and Assisted Reproduction Experimental

Chapter 9 ◾ Epigenetic and Assisted Reproduction Experimental

Chapter 10 ◾ Epigenetic and Assisted Reproduction Experimental

Chapter 11 ◾ Epigenetic and Assisted Reproduction Experimental

Chapter 12 ◾ Epigenetics: Hope against Genetic Determinism 183

Chapter 13 ◾ Future Perspectives 189

H uman fertility depends on a highly orchestrated action of hundreds of genes

Ester Anton Sebastian Canovas

Pilar Coy Alfonso Gutiérrez-Adán

Benjamín Planells Josep Santaló

Joëlle Rüegg Alejandro Vaquero

1.2 PATTERNS OF DNA METHYLATION

Cytosine 5-Methylcytosine 5-Hydroxymethyl-

Major groove face Major groove face

Major groove face Major groove face

FIGURE 1.1 Chemical structure of cytosine, 5-methylcytosine, and 5-hydroxymethylcytosine. R

+++ Enhancer Exon Repetitive sequence Methylated CpG Unmethylated CpG

mutations. As the deamination of methylated CpGs to TpGs yields a naturally occurring

1.3 DNA METHYLTRANSFERASES

DNMT1 (1616 aa; 194 kDa)

DMAPD PBD RFTD CXXC BAH1 BAH2 I IV VI VIII IX X

DNMT3A (912 aa; 102 kDa)

DNMT3B (853 aa; 98 kDa)

DNMT3L (387 aa; 42 kDa)

1.4 5-HYDROXYMETHYLATION AND THE DNA

TET1 (2138 aa)

CXXC CysD DSHB

Fe (II)- Low complexity a-KG-

Fe (II)- Low complexity a-KG-

CXXC CysD DSHB

Fe (II)- Low complexity a-KG-

1.5 TRANSCRIPTION AND GENOME STABILITY

NH2 NH2 OH NH2

Active gene Repressed gene

Alternative gene expression

1.6 EPIGENETIC CHANGES IN HEALTH AND DISEASE

The morning succeeding the motor’s first trip proved depressingly

Mrs. Hogben had lost no time in giving her lodger explicit

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Chapter 1 ◾ A Short Introduction to DNA Methylation 1

Chapter 2 ◾ Epigenetic Modifications of Histones 17

Chapter 3 ◾ Epigenetic Reprogramming in Early Embryo Development 29

Chapter 4 ◾ Epigenetic Reprogramming of Mammalian Primordial

Chapter 5 ◾ Laboratory Molecular Methodologies to Analyze DNA

Chapter 6 ◾ Intrinsic and Extrinsic Factors That Influence Epigenetics 99

Chapter 7 ◾ Epigenetic and Assisted Reproduction Epidemiological

Chapter 8 ◾ Epigenetics and Assisted Reproduction Experimental

Chapter 9 ◾ Epigenetic and Assisted Reproduction Experimental

Chapter 10 ◾ Epigenetic and Assisted Reproduction Experimental

Chapter 11 ◾ Epigenetic and Assisted Reproduction Experimental

Chapter 12 ◾ Epigenetics: Hope against Genetic Determinism 183

Chapter 13 ◾ Future Perspectives 189

1.4 5-HYDROXYMETHYLATION AND THE DNA