2-3 pages summary

2-3 pages summary focusing on the environmental aspect of Epigenetics using the 2 provided papers 

Epigenetics, Plasticity, and
Evolution: How do We Link
Epigenetic Change to Phenotype?
ELIZABETH J. DUNCAN1,
PETER D. GLUCKMAN2, AND PETER K. DEARDEN1*
1Genetics Otago and Gravida, The National Centre for Growth and Development, Biochemistry
Department, University of Otago, Dunedin, New Zealand
2Liggins Institute and Gravida, The National Centre for Growth and Development, University of
Auckland, Auckland, New Zealand

The term “epigenetics” has a complex history. Originally meant to
refer to the mechanisms that link gene to phenotype (Wadding-
ton, ’42), ithas, inrecentyears, becomemore narrowlydefined to refer
only to modifications of the DNA and chromatin that do not change
the underlying DNA sequence. This has led to a focus on DNA
modifications, such as the reversible addition of a methyl group to a
cytosine residue to generate 5‐methylcytosine, and post‐translational
modification of histone proteins (Fig. 1). These epigenetic mecha-
nisms, which are linked to more familiar aspects of gene regulation by
proteins such as transcription factors, act to regulate gene expression
in cells. Through regulation of gene expression, epigenetic
mechanisms have the potential to define and alter cell phenotype

s

and, as the epigenome can be altered by the environment (i.e., Dolinoy
et al., 2007; Sinclair et al., 2007; Kucharski et al., 2008; Seong
et al., 2011; Gertz et al., 2012; Herb et al., 2012; Wang et al., 2012), may
also orchestrate dynamic regulation of the genome in response to
changes in the environment. Epigenetic mechanisms also mediate
dosage compensation, chromosomal silencing and imprinting
(Trescot et al., 2006; Wutz and Gribnau, 2007; Abramowitz and
Bartolomei, 2012; Gertz et al., 2013).

Epigenetic mechanisms are intimately linked with cell differ-
entiation (reviewed in Reik, 2007). In vitro experiments have
demonstrated that as cells move from a pluripotent to a terminally
differentiated state, epigenetic marks change across the genome
(Hochedlinger and Plath, 2009). In vertebrates these epigenetic
marks are found across all regions of the genomic landscape
including enhancer, promoter and intergenic regions of the
genome, as well as in exons and introns. How and when these

ABSTRACT Epigenetic mechanisms are proposed as an important way in which the genome responds to the
environment. Epigenetic marks, including DNA methylation and Histone modifications, can be
triggered by environmental effects, and lead to permanent changes in gene expression, affecting the
phenotype of an organism. Epigenetic mechanisms have been proposed as key in plasticity, allowing
environmental exposure to shape future gene expression. While we are beginning to understand how
these mechanisms have roles in human biology and disease, we have little understanding of their
roles and impacts on ecology and evolution. In this review, we discuss different types of epigenetic
marks, their roles in gene expression and plasticity, methods for assaying epigenetic changes, and
point out the future advances we require to understand fully the impact of this field. J. Exp. Zool.
(Mol. Dev. Evol.) 322B:208–220, 2014.©2014 The Authors.

J. Exp. Zool. (Mol. Dev. Evol.)

published by
Wiley Periodicals, Inc.

How to cite this article: Duncan EJ, Gluckman PD, Dearden PK. 2014. Epigenetics, plasticity and
evolution: How do we link epigenetic change to phenotype?

J. Exp. Zool. (Mol. Dev. Evol.)

322B:208–220.

J. Exp. Zool.
(Mol. Dev. Evol.)
322B:208–220,
2014

Grant sponsor: Gravida, National Centre for Growth and Development;
grant number: MP04; grant sponsor: Gravida, and a Royal Society of New
Zealand Marsden; grant number: 11‐UOO‐124.

�Correspondence to: Peter K. Dearden, Biochemistry Department,
University of Otago, P.O. Box 56, Dunedin, New Zealand.
E‐mail: peter.dearden@otago.ac.nz

Received 22 November 2013; Revised 13 March 2014; Accepted 15
March 2014

DOI: 10.1002/jez.b.22571
Published online 9 April 2014 in Wiley Online Library

(wileyonlinelibrary.com).

REVIEW

This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial License, which permits use,
distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
© 2014 The Authors. J. Exp. Zool. (Mol. Dev. Evol.) published by Wiley Periodicals, Inc.

http://creativecommons.org/licenses/by-nc/4.0/

epigenetic landscapes are being established is just beginning to be
understood (i.e., Ziller et al., 2013). Within a multicellular organism
it is critical to distinguish between those epigenetic changes that
reflect cell‐type specific changes related to cell differentiation or
are constitutive (e.g., related to sex determination), from those
epigenetic changes induced by environmental exposures.
Onepotentiallyimportantroleof epigeneticmechanismsappearsto

be a cell’s way of remembering a past gene‐regulatory event, or
holding one in reserve until it is needed. Because of this ability to
stably “remember” a gene regulatory event across the lifespan of an
organism, and, possibly, across generations (Anway et al., 2005;
Stouder and Paoloni‐Giacobino, 2010; Greer et al., 2011; Stouder and
Paoloni‐Giacobino, 2011; Ashe et al., 2012; Manikkam et al., 2012a),
epigenetics has become vital to our understanding of biology, ecology,
and evolution.

TYPES OF EPIGENETIC MODIFICATIONS
DNA methylation involves the modification of a DNA base, most
often a cytosine in a CpG dinucleotide pair, with the addition of a

methyl group thus affecting the coiling of DNA around histones
and changing the potential binding of transcriptional factors in
part by recruiting methyl CpG binding proteins (MCBPs).
Although absolute levels of DNA methylation vary between
species and cell types (Lister et al., 2009; Feng et al., 2010a;
Zemach et al., 2010; Nanty et al., 2011), in humans there is
experimental evidence for 80–96% of the CpG residues in the
genome being methylated under various conditions (Varley
et al., 2013; Ziller et al., 2013). Much of our understanding of
the function of DNA methylation has come from imprinting in
mammals (reviewed in Abramowitz and Bartolomei, 2012) and the
study of cancer cell lines (reviewed in Laird and Jaenisch, ’96),
where DNA methylation is often aberrant, both in placement, and
in pattern (Miremadi et al., 2007; Cedar and Bergman, 2012).
Previous studies have focused on the role of DNA methylation

in generally repressing gene expression through methylation of
CpG islands near promoters of genes (Jones, 2012). DNA
methylation is found throughout genes, not just in promoter
regions, in animals and plants (Feng et al., 2010a; Zemach

Figure 1. Types of epigenetic modifications and their potential effects on gene expression and chromatin structure.

EPIGENETICS, PLASTICITY, AND EVOLUTION 209

J. Exp. Zool. (Mol. Dev. Evol.)

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J. Exp. Zool. (Mol. Dev. Evol.)

210 DUNCAN ET AL.

et al., 2010; Sarda et al., 2012). Promoter methylation appears to
have evolved in the vertebrate lineage whereas methylation of
gene bodies was likely present in the last common ancestor of
plants and animals (Feng et al., 2010a; Zemach et al., 2010). It
seems that the position of DNA methylation relative to the gene
(i.e., intron, exon, transcriptional start site, or promoter)
determines how gene transcription is affected by methylation
(Jones, 2012). For instance, gene body methylation has multiple
functions including repressing intragenic promoter activity
(Maunakea et al., 2010), alternative splicing (Lyko et al., 2010;
Shukla et al., 2011; Foret et al., 2012; Sati et al., 2012) and
controlling transcriptional elongation (Lorincz et al., 2004)
ensuring that the first and last exons are included in a transcript
(Sati et al., 2012), while DNA methylation at the 50 end of the gene
is associated with transcriptional silencing (Brenet et al., 2011).
DNA methylation is established and maintained by two families

of DNA methyltransferase enzymes: DNMT1 and DNMT3
(reviewed in Goll and Bestor, 2005). We do not understand how,
or indeed if, the DNA methyltransferase enzymes are targeted to
particular sites in the genome to provide specificity for DNA
methylation, although it appears that non‐coding RNAs may play
a central role. In plants, DNA methylation can be targeted to
specific genomic loci by RNA molecules (RNA directed DNA
methylation, RdDM) (Mahfouz, 2010; Zhang and Zhu, 2011). Some
evidence supports the role for RNA, specifically small RNAs, in
directing methylation in animals (Weinberg et al., 2006; Aravin
and Bourc’his, 2008; Holz‐Schietinger and Reich, 2012).
Demethylation, the removal of a methyl group from a cytosine

residue, had been assumed to be a passive process, via loss of
methylation marks across cell divisions, but it has now been
shown to occur independent of cell division (i.e., Mayer
et al., 2000; Oswald et al., 2000). Demethylation of DNA can
occur via DNA repair pathways mediated by Gadd45 (growth
arrest and DNA damage inducible protein 45) (Barreto et al., 2007;
Ma et al., 2009a; Niehrs, 2009; Niehrs and Schafer, 2012). Gadd45
can be induced by external stimuli (i.e., Ma et al., 2009b) and
appears to target specific genes for demethylation (Jin et al., 2008;
Engel et al., 2009; Schafer et al., 2010).
A second pathway for demethylation of DNA employs TET (ten

eleven translocation) enzymes, which convert 5‐methylcytosine
to 5‐hydroxymethyl cytosine (Tahiliani et al., 2009), which is then
processed to 5‐formylcytosine and 5‐carboxylcytosine (He
et al., 2011; Ito et al., 2011). Both these derivatives act as
substrates for a thymine‐DNA glycosylase, which results in the
regeneration of a non‐methylated cytosine (He et al., 2011; Maiti
and Drohat, 2011). The biological functions of the derivatives of 5‐
methylcytosine are unknown, but in human cells each associates
with proteins not linked to DNA repair, implying that these
derivatives may also act as epigenetic marks that recruit
transcriptional regulators (Spruijt et al., 2013). As with methyla-
tion enzymes, we do not understand how demethylation enzymes
are targeted to specific regions of the genome.

Nuclear chromatin is organized into nucleosomes; a segment of
DNA wound around eight core histone proteins. These proteins are
extensively post‐translationally modified (reviewed in Peterson
and Laniel, 2004) by a suite of enzymes (Biel et al., 2005;
Marmorstein and Trievel, 2009) that are temporally and
developmentally regulated (Lin and Dent, 2006; Heintzman
et al., 2009; Kharchenko et al., 2011; Dunham et al., 2012;
Pengelly et al., 2013). The post‐translational modification of these
histone proteins is known to regulate gene‐expression by altering
the accessibility of the underlying DNA to transcription factors
(Wu et al., ’79; Bell et al., 2010). It has been proposed that histone
modifications may act as a signal integration and storage
platform, allowing cells to record and store signaling events,
including environmental signals (Badeaux and Shi, 2013).
We are beginning to understand how histones at specific loci

may be targeted by histone modifying enzymes. Specific DNA
sequences have been identified that recruit histone modifying
enzymes (Fritsch et al., ’99; Tillib et al., ’99; Klymenko et al., 2006)
and long non‐coding RNAs have also been proposed to have a role
in targeting modification of histones associated with particular
loci (Tsai et al., 2010; Spitale et al., 2011). There is increasing
evidence of cross‐talk between DNA methylation and histone
modifications (i.e., Hashimshony et al., 2003; Bartke et al., 2010;
Hagarman et al., 2013; Spruijt et al., 2013) supporting the idea that
these mechanisms act together to regulate gene expression. It is
not known how cross‐talk between these two systems is mediated,
but data implies that, in at least some circumstances, changes to
histone modifications may be induced prior to methylation
changes that then serve as more stable epigenetic marks (Park
et al., 2008).
In addition to these “classical” epigenetic systems, small RNA

molecules, such as small interfering RNA (siRNA) and piwi‐RNA
(piRNA), have epigenetic potential. siRNAs are 21–22 nucleotides
in length and are produced from endogenous double stranded
RNA. These molecules associate with Argonaute proteins that
induce localized chromatin remodelling (Fagegaltier et al., 2009;
Burkhart et al., 2011) and may maintain genes in a “poised” state,
ready to be activated (Cernilogar et al., 2011). piRNAs are larger
(23–29 nucleotides) and are produced by a different mechanism to
siRNAs (reviewed in Castel and Martienssen, 2013). piRNAs were
initially discovered in germ‐line cells, but are now known to be
widely distributed throughout somatic tissues (Yan et al., 2011;
Ishizu et al., 2012). In the germ‐line, piRNAs mediate transposon
silencing via chromatin remodeling (Brower‐Toland et al., 2007;
Wang and Elgin, 2011).
Epigenetic marks may also mediate the way that DNA is

organized in three‐dimensional space within the nucleus of a cell.
This structure can bring enhancer and promoter elements into
contact, or can recruit genes to “transcription factories”
facilitating gene expression. It means that both genetic poly-
morphisms and epigenetic polymorphisms in regulatory regions of
the genome have the potential to act in both cis and trans to affect

J. Exp. Zool. (Mol. Dev. Evol.)

EPIGENETICS, PLASTICITY, AND EVOLUTION 211

gene transcription. Looping can also allow interactions with
insulator elements causing repression of gene expression.
Compartmentalization of the genome in three‐dimensions is
dynamic and is associated with cell type specific gene expression
patterns (Lieberman‐Aiden et al., 2009; Varley and Mitra, 2010). It
is unknown whether the association of higher order chromatin
structures with particular histone modifications are a cause or
consequence of those higher order structures (Greer and Shi, 2012).

EPIGENETICS AND PHENOTYPIC PLASTICITY
Phenotypic plasticity, the ability of an individual genome to
produce different phenotypes when exposed to environmental
cues (Pigliucci et al., 2006), is widespread amongst both plants and
animals.
Well‐known examples of phenotypic plasticity include caste

polyphenisms in social insects, seasonal polyphenisms in
butterflies and as well as the mechanisms of learning and immune
system adaptation (Fusco and Minelli, 2010). Epigenetic changes
have been associated with polyphenisms like caste development in
the honeybee (Kucharski et al., 2008) and ants (Bonasio
et al., 2012; Simola et al., 2013) and phenotypic plasticity in
mammals; specifically maternal mood in humans, affecting
methylation of the glucocorticoid receptor (Oberlander
et al., 2008), and differential methylation of genes in the umbilical
cord being associated with in utero growth (Lim et al., 2012) and
childhood adiposity (Godfrey et al., 2011).
Predictive adaptive responses (PARs) are a subclass of

phenotypic plasticity where animals in early life make predictions
about their future environment based on environmental cues
received early in development (Gluckman et al., 2005). Classical
examples of PARs include the meadow vole, which receives
environmental cues in utero, via maternal melatonin levels, about
the season it is gestating toward and the infant is born with a
thicker coat in autumn than it is in spring (Lee and Zucker, ’88).
PARs are believed to be established via epigenetic marks
established by a triggering cue in early development that becomes
a proxy for predicting a subsequent environment. In turn these
epigenetic marks affect the physiological trajectory that is
followed as the organism develops—establishing effects on
metabolism or morphology that may have positive effects on
fitness as the organism survives to reproduce (Varley et al., 2009).
Yet these marks are carried beyond peak reproduction through that
organism’s life and this, together with the probabilistic nature of
early prediction of later‐life environments, may have consequen-
ces that have been extensively discussed in relation to non‐
communicable disease in humans.
Predictions that an animal makes about its future environment

may not always be correct, and the idea of mismatch underpins the
“Developmental origins of Human Disease” paradigm (Gluckman
and Hanson, 2006). This describes how the environment
influences gene expression, possibly via epigenetic mechanisms,
in the fetus and infant that are then stable throughout an

individual’s life. It is proposed these predictions evolved to
enhance survival to reproduction, even if they can become
disadvantageous in later life (Bateson et al., 2004; Gluckman and
Hanson, 2004a, 2004b, 2005). Evidence is building in clinical
studies that epigenetics underpins, or is at least associated with,
this concept of fetal programming, hypothesized to be partly
responsible for the current burden of non‐communicable diseases,
such as metabolic syndrome (Hanson and Gluckman, 2008;
Gluckman et al., 2010; Hanson et al., 2011).

Evolutionary Implications
Two recent findings have radically expanded the possible role of
epigenetics in evolution and ecology. Firstly, in some situations,
environmental cues can influence epigenetic programming (i.e.,
Dolinoy et al., 2007; Sinclair et al., 2007; Kucharski et al., 2008;
Seong et al., 2011; Gertz et al., 2012; Herb et al., 2012; Wang
et al., 2012), and secondly, this information has the potential to be
passed on to the subsequent generations via the gametes (Anway
et al., 2005; reviewed in Jablonka and Raz, 2009; Stouder and
Paoloni‐Giacobino, 2010; Greer et al., 2011; Stouder and Paoloni‐
Giacobino, 2011; Ashe et al., 2012; Manikkam et al., 2012a). This
expanded view raises the possibility that these marks may be able
to produce genetic change over time periods that may be relevant
to evolution. The idea that epigenetic marks may carry gene
expression changes across generations, probably in a limited way,
is of importance in our understanding of the genetic assimilation
of acquired traits (Bateson and Gluckman, 2011).
We are coming to understand that the genome is an exquisitely

regulated structure, with various regions and domains being
accessible to transcription at any time and in any cell. This
structure is maintained by many complex mechanisms, including
epigenetic ones, but also the related three‐dimensional organiza-
tion of the genome within the nucleus. With this understanding
the genome is perhaps well explained by Waddington’s
“epigenetic landscape.” Perturbations of this landscape through
anticipated environmental changes, as in the case of a polyphen-
ism, a continuous reaction norm, or in a PAR, leads to
modifications in the landscape, and new phenotypic outcomes
becoming favoured. In the case of a more disrupting influence,
such as a heat shock in the case of genetic assimilation, the
epigenetic landscape may be modified to buffer against damage,
and some of that modification may be passed on to future
generations through the inheritance of those epigenetic marks.
Numerous authors (i.e., Bunt et al., ’88; West‐Eberhard, 2005a,

b; Pigliucci et al., 2006), have suggested that plastic changes
somehow prefigure genetic ones, that then stabilize an environ-
mentally‐induced trait in future generations. It seems likely that if
such processes exist they involve molecular epigenetic mecha-
nisms (Feinberg and Irizarry, 2010; Bateson and Gluckman, 2011).
One feature of methylated cytosine residues is that they are
susceptible to deamination, and have a higher mutation rate to
thymine than non‐methylated bases. Is it possible that this

J. Exp. Zool. (Mol. Dev. Evol.)

212 DUNCAN ET AL.

hypermutability provides a mechanism by which epigenetic
changes may lead to genetic ones? (Bateson and Gluckman, 2011).
While this is an attractive idea, it is important to note that the
epigenetic change and subsequent mutations must occur in the
germ‐line to have any importance to evolution. This is a critical
point as, if all cell types have their own epigenetic landscapes, a
change occurring in some cell type having a beneficial effect,
would also have to occur in the germ line to have a trans‐
generational or evolutionary effect.

Mechanisms for Transmission of Epigenetic Information. Indirect
epigenetic inheritance occurs when an environmental cue induces
a behavior or physiology, via epigenetic marks, that then induces
the same epigenetic mark and associated behavior in subsequent
generations. The cue is passed behaviorally or physiologically
between generations, not via trans‐meiotic passage of the
epigenetic mark. An example of this mechanism is the epigenetic
change in neuro‐hormonal pathways in the mouse by altered
maternal grooming that lead the offspring to grow up with the
same maternal behavior that again induces the same epigenetic
mark and thus behavior in the next generation (Weaver
et al., 2004).
Direct transmission of epigenetic information between gen-

erations can occur, where the environmental influence directly
affects the germline of the parent, or is mediated via interactions
between the somatic cells and germline (reviewed in Jablonka and
Raz, 2009). In both cases in order for true transgenerational
transmission of environmental information, epigenetic marks
must be stable and heritable through meiosis (Osbourne et al., in
press).
The DNA methylation landscape can be retained through

mitosis via the activity of the DNMT1 proteins, or maintenance
methyltransferases. During meiosis, and then embryonic devel-
opment, there is substantial reprogramming of DNA methylation
of both sperm and oocytes in vertebrate model species, although a
number of loci are protected (Feng et al., 2010b; Seisenberger
et al., 2012; Jiang et al., 2013; Potok et al., 2013). This provides the
potential for environmentally induced DNA methylation patterns
to be transmitted to the next generation during gametogenesis.
Consistent with this, environmental exposure to particular
chemicals is associated with altered patterns of DNA methylation,
particularly in the sperm (i.e., Guerrero‐Bosagna et al., 2010;
Manikkam et al., 2012a,b; Tracey et al., 2013).
The histone modification landscape can also be retained, fully

or partially, through cell division although we do not understand
how this occurs. It may be that the “cross talk” between DNA
methylation and histone modifications is integral for this process.
Methylation marks, transferred via maintenance methyltransfer-
ases, may be used as a “template” to establish the histone
modification landscape de novo in the new copy of the DNA.
However, animals such as Drosophila melanogaster and Caeno-
rhabditis elegans do not have appreciable levels of DNA

methylation, yet do display inheritance of histone marks, raising
the possibility that there is another mechanism for the transmis-
sion of histone modifications between generations (Greer and
Shi, 2012).
Small RNAs, such as piRNAs, can be inherited trans‐

generationally (Brennecke et al., 2008; Ashe et al., 2012). The
piRNA interacting protein PIWI has been implicated in epigenetic
regulation (Yin and Lin, 2007) and is important for suppressing
phenotypic variation. It has been hypothesized that PIWI may
have a role in canalization of traits over evolutionary time
(Gangaraju et al., 2011).
Other small RNAs can be passed through the germline to the

developing embryo. Well known examples include small non‐
coding RNAs passed through the sperm that target the Kit locus in
mice generating a white tail phenotype in the offspring
(Rassoulzadegan et al., 2006) and the miR‐1/Cdk9 paramutants
that are associated with cardiac hypertrophy in mice (Wagner
et al., 2008). Although the exact mechanism is unknown, a recent
study has shown that the methyltransferase Dnmt2, previously
thought to target only tRNAs, is required for this process (Kiani
et al., 2013).

Population and Quantitative Epigenetics. The effects of epigenetic
changes, and epialleles of genes, have not been extensively
investigated in the fields of population and quantitative genetics
(Geoghegan and Spencer, 2013a, 2013b). Recent evidence from
comparing plants in different environmental or growth conditions
has shown that genomes are capable of containing single‐
methylation‐polymorphisms as well as single‐nucleotide poly-
morphisms (Schmitz et al., 2013). These stable differences in DNA
methylation, with no underlying change in the base‐pair that is
methylated, may have a significant role in determining individual
and population fitness, particularly in response to fluctuating
environments. There may also be evolutionary consequences, as
variation in DNA methylation may act to mediate the adaptive
value of a trait. These variants may also play a role in resolution of
genomic conflicts, both with selfish genetic elements and
intersexual conflict via imprinting (Johnson and Tricker, 2010).
We are yet to see large‐scale population studies of epigenetic

change in animal genomes, so we do not yet know if they have a
significant impact on our understanding of population dynamics.
Such studies have been hindered by the cell specific nature of
epigenetic modifications and the lack of techniques that are fast
and cheap enough to probe such modifications in multiple
samples.

The Interaction Between Epigenetic and Genomic Variation. Single
nucleotide polymorphisms (SNPs) are abundant in animal
genomes. The most common polymorphism is a transition from
a C to a T nucleotide. These polymorphisms can affect numerous
CpG sites in the genome, by altering a C in a CpG dinucleotide to
another nucleotide that cannot be methylated (Shoemaker

J. Exp. Zool. (Mol. Dev. Evol.)

EPIGENETICS, PLASTICITY, AND EVOLUTION 213

et al., 2010). Studies have shown that these SNPs can influence
gene expression via effects on DNA methylation (Bell et al., 2011;
Gutierrez‐Arcelus et al., 2013). The effect of SNPs on DNA
methylation can either be direct, by changing a C (in a CpG
dinucleotide) to a non‐modifiable nucleotide, or indirect by
altering transcription factor binding, which in turn independently
affects gene expression and DNA methylation levels (Gutierrez‐
Arcelus et al., 2013).
Polymorphisms may also affect imprinting locus control

regions and thus have an influence on epigenetic changes
associated with parental imprinting (Coolen et al., 2011). This
concept of allele‐specific methylation is growing in importance
with the recognition that this phenomenon may extend well
beyond classical imprinted genes.

Tools and Pitfalls of Epigenomic Techniques
The majority of the current methods to study DNA methylation
rely on bisulphite conversion of the DNA (Laird, 2010). Treatment
of the DNA with bisulphite causes unmethylated cytosines to be
converted to uracils which, using common molecular techniques
such as PCR and sequencing, are detected as thymines. Methylated
cytosines are protected from conversion and are detected as
cytosines. DNA methylation at individual loci can be interrogated
using PCR and Sanger sequencing, high‐resolution melting
(Wojdacz and Dobrovic, 2007, 2009), MethyLight (Eads
et al., 2000) and epiTYPER (reviewed in McLean et al., 2012).
Next generation sequencing has meant that DNA methylation can
now be interrogated on a genome wide scale by shot‐gun
sequencing (Cokus et al., 2008; Lister et al., 2009) or by reduced
representation bisulfite sequencing (RRBS) (Chatterjee
et al., 2012). DNA methylation can also be interrogated by
restriction enzymes that target methylated DNA (i.e., Guo
et al., 2011) and this method can also detect 50 hydroxymethylcy-
tosine (Davis and Vaisvila, 2011). Antibodies against 50 methyl-
cytosine and 50 hydroxymethylcytosine can be used to enrich for
methylated regions of the genome (m‐DIP (Weber et al., 2005), or
hmc‐DIP (Davis and Vaisvila, 2011)) prior to next‐generation
sequencing or array analysis.
The “gold standard” method to study histone modifications

involves using antibodies to a histone modification of interest (i.e.,
H3K27me3) to affinity purify fragments of chromatin (chromatin
immunoprecipiatation or ChIP). The DNA is then eluted and
analyzed by quantitative PCR to interrogate a single locus (ChIP‐
PCR) or array hybridization (ChIP‐chip) or next‐generation
sequencing (ChIP‐seq) for genome‐wide data (reviewed in
Furey, 2012). New approaches combine bisulfite sequencing of
ChIP DNA to simultaneously detect DNA methylation associated
with particular histone modifications (Brinkman et al., 2012;
Statham et al., 2012).
Techniques for detecting higher‐order chromatin structure

within the nucleus focus on a technique known as chromatin
conformation capture (3C) and its derivatives including 4C, 5C, 6C,

Hi‐C, and ChIA‐PET (Sajan and Hawkins, 2012; Dekker
et al., 2013).

General Limitations of Epigenomic Techniques. Many of the
techniques described above require significant quantities of
starting DNA or chromatin material. Obtaining this material
may be difficult for small animals or tissues with limited cell
numbers. These techniques have been established and used
extensively in the analysis of cell cultures; their use in animal
tissues is inherently more complex. There are hundreds of different
cell types in humans (Vickaryous and Hall, 2006), all of which
have cell‐type specific gene expression and presumably epigenetic
marks. Even within a single tissue such as the liver, multiple cell
types are present. Indeed methylation profiles have been
suggested as surrogates for characterizing cell mixtures (House-
man et al., 2012) Thus any analyses of most biospecimens gives an
aggregate reading of epigenetic marks across the tissues or cell
types. When applying these techniques in vivo it is important,
where possible, to obtain a homogenous cell population. If
working with model organisms it is possible to use fluorescent cell
type specific reporters or antibodies together with fluorescence
activated cell sorting (FACS) to obtain relatively homogenous cell
populations (i.e., Berger et al., 2012; Harzer et al., 2013).
Such approaches generate large amounts of data, particularly

when coupled with next‐generation sequencing. The amount of
data, and the fact that epigenetic marks and fixed genomic
variation likely function in combinations, means that there is
complexity in terms of data analysis and statistics. There are a
number of specialized programs for analysis of whole‐genome
epigenetic data, particularly for DNA methylation and histone
modifications, but there is no standardized way to analyze this
data and each experiment may require a customized bioinformat-
ics and statistical approach. A further issue is that epigenetic data
has a number of intrinsic characteristics, some methodological,
others relating to how it is expressed, that means it often deviates
significantly from the normal distribution, and complex trans-
formations are needed that can limit use of traditional statistical
approaches (Wutz and Gribnau, 2007). The analyses described here
can also be time‐consuming and expensive, and it is important
that these experiments are performed in a standardized way. This
ensures the quality of the data, and more importantly, allows
comparison of data generated from different laboratories in the
same experimental system.
The likelihood of epigenetic mechanisms having an influence

on the evolution of phenotypic plasticity and assimilation of
acquired traits relies in part on transgenerational inheritance of
epigenetic marks. There is evidence to support the transgenera-
tional transmission of epigenetic information (Jablonka and
Raz, 2009), but in general detecting incidences of this is
complicated, particularly in species that develop in utero as the
mother, embryo and the germ cells of the embryo (the future
grandchildren) all share a common environment. This means that

J. Exp. Zool. (Mol. Dev. Evol.)

214 DUNCAN ET AL.

at least three generations are required to confirm transgenera-
tional epigenetic inheritance in females, and two in males
(Jablonka and Raz, 2009). It is also important to distinguish
between transgenerational transmission of information, parental
transmission and so called “niche reconstruction” or indirect
epigenetic inheritance, whereby similarities in the animals
experience or environment influences epigenetic programming,
causing similarities in the epigenetic marks between parents and
offspring (Weaver et al., 2004; Champagne and Curley, 2008).
While these parental effects are transmitted from generation to
generation they are not mediated by trans‐meiotic transmission of
epigenetic marks.

Future Perspectives: How Do We Move From Phenomenon to
Function?
We now have techniques that enable rapid assessment of different
sorts of epigenetic variation. We need to begin to investigate the
role and function of such variation, and of epigenetic changes in
response to plasticity, in evolution. Here we discuss some of the
major challenges that are currently hampering progress in this
research field.

Finding mechanisms that linking environmental perturbation to
epigenetic change. We have evidence to suggest that environmen-
tal cues can be transmitted to changes in the epigenetic regulation
of the genome in many animals, and that these changes may be
passed to future generations via the gamete or, at least for one to
two generations, through parental effects. But in many cases we
do not have a good understanding of how the environmental
signals are transmitted to the affected cells. In insects environ-
mental change is often linked to hormone titer, in particular
juvenile hormone and ecdysteroids (Hartfelder and Engels, ’98;
Oostra et al., 2011; Ishikawa et al., 2012). However, it is not known
if these molecules are the primary effectors of environmental
change, and if so, how they might influence the epigenetic status
of specific cells or cell types. Most work in mammals has focused
on nutritional manipulation or glucocortiocoid‐mediated effects
mimicking stress.
Systematic analysis of molecules/signaling pathways that link

environmental perturbations, such as temperature, nutrition or
stress, with plastic events will allow us to not only understand how
environmental signals are translated into epigenetic changes but
also if, and how, these changes are transmitted to the germ‐line
from distant tissues. Direct transmission of epigenetic marks
through the germ‐line would provide a direct mechanism by
which environmental perturbation might influence evolutionary
processes. Further, undertaking such analyses in phylogenetically
diverse animals will allow us to place these molecular mechanisms
in an evolutionary context: do similar mechanisms relay
environmental challenge in diverse species. Is then the transmis-
sion of environmental information to the epigenome an ancient
and conserved feature of animals?

Linking epigenetic variation to phenotype. DNA methylation and
modified histones are associated with numerous genomic features,
including transposable elements, centromeres and transcribed
genes. If we find variation in DNA methylation, or histone
modifications around specific loci, does it have any consequence
for the cell or the animal? It is possible that some, if not the
majority, of epigenetic variation, viewed in a particular region of
DNA sequence, have little or no functional consequence under
normal conditions; they do not affect gene expression and
therefore do not affect phenotype. It is also possible that these
epigenetic variants may be functionally important in a different
context, that is, under different cellular or environmental
conditions. If we are to be able to understand the importance of
epigenetic variation, we need first to understand if such changes
have any functional effect at all. It is not enough to show that two
individuals have a difference in methylation or chromatin
modification, we have to show that that has some impact on
those individual’s phenotype. The recognition of allele‐specific
methylation raises the issue of whether a particular epiallele is
acting independently of fixed genomic variation. Linking
epigenetic variation with phenotype data firstly relies on accurate
measurements of an appropriate phenotype. We do not yet have a
good understanding of how the epigenome is affected by
environmental conditions (see above), and in a complex
environment an animal receives information about innumerable
parameters that may affect the epigenome, and phenotype. But, in
many cases, we are limited in the phenotypes that we measure and
any epigenetic variant may only contribute subtly, perhaps
negligibly, to an animal’s phenotype, making it difficult to detect
epigenetic variants associated with a phenotype. However, once
variants have been identified the biggest challenge is determining
whether the epigenetic variant is causative of the phenotype or
simply correlated.

Manipulation. We need to separate causation from correlation.
That epigenetic changes are correlated with a particular phenotype
may just indicate that you are measuring the phenotype, and that
these changes are a consequence, and not a cause, of the process
you are interested in. Currently manipulation of epigenetic
mechanisms is limited to whole‐scale, broad range perturbation,
that is by treating the animal or cells with inhibitors of DNA
methylation. However, we need to be able to interrogate the
function of DNA methylation or histone modifications at a specific
site. Thus we need to be able to develop techniques to specifically
target epigenetic modifications to particular places in a genome to
determine if those changes are causative, or indeed even
functional rather than simply correlated with a phenotype.

Cellular approaches. It is important to be careful to acknowledge
the huge problem that the cell‐type specific nature of epigenetic
modifications poses for our understanding of their role in
evolution, as well as their analysis. As stated previously,

J. Exp. Zool. (Mol. Dev. Evol.)

EPIGENETICS, PLASTICITY, AND EVOLUTION 215

epigenetic marks and changes are, on the whole, cell specific. If
epigenetic changes are to be assayed in evolutionary studies, it is
vital that it is done in specific cell types. Tools, such as
fluorescence‐activated cell sorting, laser capture microscopy or
micro‐dissection exist to do this, but they are rarely employed.
Tissues with low cellular heterogeneity like buccal smears, may
also avoid the averaging effect.

Future Perspectives: Evaluating the Role of Plasticity and
Epigenetics in Evolution
The role of plasticity (West‐Eberhard, 2003; Pigliucci et al., 2006;
Crispo, 2007) and indeed epigenetics (Feinberg and Irizarry, 2010;
Bateson and Gluckman, 2011) in driving evolutionary processes is an
active area of research. Historically, it has been proposed that
plasticity may affect evolutionary processes directly, in that the
ability to alter phenotypes to accommodate a fluctuating environ-
ment means that plastic individuals within a species will be more
likely to reproduce and, assuming that plasticity is encoded
genetically, this will be heritable (Baldwin, ’02). This will, over
evolutionary time, result in stabilization of generalized plasticity
within species, but not allow a specific plastic trait to become
stabilized within a species. It is argued that, over evolutionary time,
Darwinian selection will act on existing genetic variation for that
trait to be genetically accommodated (West‐Eberhard, 2003). This
theory predicts that plasticity in species exposed to fluctuating
environments will increase over evolutionary time, and that some
plastic traits will become encoded by the genome if they are
advantageous, and if genetic variation exists in the population to
allow this. If this mechanism prevails then we might expect
epigenetics and plasticity to have a limited affect on evolutionary
processes. Recent studies indicate that there may be a more direct
route from environmental perturbation to genetic accommodation;
via transgenerational transmission of epigenetic information (An-
way et al., 2005; reviewed in Jablonka and Raz, 2009; Stouder and
Paoloni‐Giacobino, 2010; Greer et al., 2011; Stouder and Paoloni‐
Giacobino, 2011; Ashe et al., 2012; Manikkam et al., 2012a). This
mechanism may allow specific information about the environment
to be passed by an animal to its offspring, prefiguring the next
generation to be successful in a particular environment. Direct
transmission of epigenetic information between generations in-
creases the scope for evolutionary processes to be affected by
epigenetic information and plasticity. Ultimately this also requires a
mechanism for stableintegration of this information into thegenome
(such as deamination of methylated cytosine residues (Feinberg and
Irizarry, 2010; Bateson and Gluckman, 2011)). Understanding how,
and with what frequency, environmental perturbation can influence
evolutionary processes is critical to our understanding of plasticity
and the integration of plasticity into evolutionary theory.

Summary
The ubiquity of epigenetic modification of the genome, its
influence through the life‐course and transgenerationally, and its

environmental responsiveness, mean that epigenetic modifica-
tions are likely to have a significant impact in evolutionary
studies. The advent of high throughput sequencing and
biochemical techniques to measure modifications allows re-
searchers to access the epigenome, and perhaps begin to
understand the interface between epigenetic and evolution. There
are pitfalls to these approaches, and only with knowledge of these,
and the invention of techniques to manipulate epigenetic marks,
will we be able to see clearly the influence of epigenetics on
phenotype, plasticity and ultimately, evolution.

ACKNOWLEDGMENTS
This work was supported by a Gravida, National Centre for Growth
and Development grant to P.K.D. (MP04). E.J.D. is funded by
Gravida, and a Royal Society of New Zealand Marsden Grant (11‐
UOO‐124). The authors thank members of the Laboratory for
Evolution and Development for useful discussion and P.M.
Dearden for critical reading of the manuscript. P.K.D. serves on
the Editorial board for this Journal.

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220 DUNCAN ET AL.

Review
https://doi.org/10.1038/s41586-019-1411-0

Advances in epigenetics link genetics to
the environment and disease
Giacomo Cavalli1* & edith Heard2,3*

Epigenetic research has accelerated rapidly in the twenty-first century, generating justified excitement and hope, but
also a degree of hype. Here we review how the field has evolved over the last few decades and reflect on some of the
recent advances that are changing our understanding of biology. We discuss the interplay between epigenetics and DNA
sequence variation as well as the implications of epigenetics for cellular memory and plasticity. We consider the effects
of the environment and both intergenerational and transgenerational epigenetic inheritance on biology, disease and
evolution. Finally, we present some new frontiers in epigenetics with implications for human health.

Biologists have long sought to understand how a fertilized egg can form an organism composed of hundreds of specialized cell types, each
expressing a defined set of genes. Cellular identity
is now accepted to be the result of the expression of
specific combinations of genes (Fig. 1). This expression pattern must be
established and maintained—two distinct, but connected, processes. The
pluripotency of the initial cell and the establishment of cell types depend
to a large extent on the coordinated deployment of hundreds of transcrip-
tion factors that bind to specific DNA sequences to activate or repress the
transcription of cell lineage genes1. This establishment phase corresponds
most closely to what is generally cited as the first definition of epigenetics
by Conrad Waddington, namely the study of the mechanisms by which
the genotype produces the phenotype in the context of development2.
The maintenance phase often involves a plethora of non-DNA sequence
specific chromatin cofactors that set up and maintain chromatin states
through cell division and for extended periods of time—sometimes in
the absence of the initial transcription factors3. This phase is more akin
to a definition of epigenetics put forward by Nanney4, then elaborated
on by Riggs and Holliday5–7 and further modified by Bird8 and others9
to mean the inheritance of alternative chromatin states in the absence
of changes in the DNA sequence. DNA methylation was proposed early
on as a carrier of epigenetic information with subsequent work revealing
that chromatin proteins and noncoding RNAs are also important for this
process10–14. For example, histone variants and histone modifications
can influence local chromatin structure, either directly or indirectly.
Such modifications can be heritable but reversible and are governed
by a series of writers (that deposit them), readers (to interpret them)
and erasers (to remove them). Finally, higher-order 3D chromosome
folding is also thought to modulate gene expression and might contribute
to inheritance15.

Since 1942, when the word was first coined, epigenetics has been
redefined multiple times16 (Table 1). In this Review, we use epigenetics
to mean “the study of molecules and mechanisms that can perpetuate
alternative gene activity states in the context of the same DNA sequence”.
This operational definition has several implications. First, it encom-
passes transgenerational inheritance as well as mitotic inheritance and
the persistence of gene activity or chromatin states through extended
periods of time, even without cell division—for instance, in long-lived
post-mitotic cells such as adult neurons. Second, the DNA sequence to

be considered depends on the biological system. In
mitotic inheritance, one should consider the genomic
sequence of individual cells, whereas in transgener-
ational inheritance one should consider the DNA of
the whole organism (including its microbiota, if this

can contribute to inheritance). Finally, this definition explicitly extends
the usage of ‘epigenetic’ to regulatory processes that involve molecules
known to participate in epigenetic inheritance, even when not address-
ing the epigenetic memory function per se. We argue that this common
practice should be accepted, as it conveys to non-specialists the broader
field of epigenetic research. We also note that cases of inheritance that
do not involve chromosomal components have been documented14,17,18
and it will be important to study how widespread they are and whether
similar phenomena occur in humans.

Here, we review the interplay between regulatory plasticity and
stable epigenetic heritability, including cell fate and reprogramming events
that occur during development, in response to physiological stimuli,
and in disease. We discuss how noncoding RNAs, DNA methylation,
heterochromatin, Polycomb and Trithorax proteins and 3D genome
architecture (Box 1) can regulate both inheritance and gene expres-
sion plasticity, and how new technologies allow these phenomena to be
analysed in a spatiotemporal fashion, in small numbers of cells or even single
cells, and at multiple scales from the nucleotide to the chromosome
(Box 2). We discuss evidence for a hotly debated topic—epigenetic inher-
itance across generations—particularly focusing on mammalian exam-
ples because of the potential biomedical implications. We also consider
two other important new research areas: the potential influence of the
environment and the effects of epigenetic changes on genome integrity. In
closing, we highlight how epigenetic research may benefit human health.

Epigenetic inheritance versus plasticity
An appreciation of the role of chromatin as a carrier of epigenetic
information that can propagate active and silent activity states during
cell division came from the study of different biological processes and
model organisms. These include, to name but a few, heterochromatin
inheritance in yeast, X-chromosome inactivation (the process by which
one of the copies of the female X chromosome is silenced), or genomic
imprinting (the parent-of-origin-specific repression of certain genes)
in mammals; vernalization (the induction of flowering by exposure
to prolonged cold during winter) in plants; position effect variegation

1Institute of Human Genetics, CNRS and University of Montpellier, Montpellier, France. 2European Molecular Biology Laboratory (EMBL), Heidelberg, Germany. 3Collège de France, Paris, France.
*e-mail: giacomo.cavalli@igh.cnrs.fr; edith.heard@embl.org

2 5 J U L Y 2 0 1 9 | v O L 5 7 1 | N A T U R e | 4 8 9

https://doi.org/10.1038/s41586-019-1411-0

mailto:giacomo.cavalli@igh.cnrs.fr

mailto:edith.heard@embl.org

ReviewReSeARCH

(the silencing of a gene in some cells through its abnormal juxtaposition
to heterochromatin) in Drosophila. These studies demonstrated that
differentially expressed states can be transmitted across cell divisions,
once they are established and in the absence of the original signal.
Studies of cellular reprogramming in the germline and early embryo-
genesis19–22, during induced pluripotency (iPS)23,24, or upon somatic
nuclear transfer25,26 have shown that chromatin and DNA methylation
act as important ‘epigenetic barriers’ (Fig. 1) that prevent changes in
gene expression and cell identity.

Epigenetic systems (Box 1) include heterochromatin (HP1 and
H3K9me3 (trimethylation of histone 3 lysine 9)), Polycomb (PRC1
and PRC2) and Trithorax (COMPASS (complex proteins associated
with SET1)) complexes. These complexes are thought to perpetuate
functional responses by modifying histone proteins in chromatin
and by binding their own histone marks in order to convey stable
inheritance. Indeed, nucleosomes are subject to constant remodelling,
histones are exchanged and all DNA and histone marks discovered
so far are reversible, although the rates of exchange and the stability
of the marks vary in different genomic domains27. Therefore, most
regulatory signals would be rapidly lost in the absence of tight self-
reinforcing loops that maintain the memory of the chromatin state28.

Furthermore, the inheritance of epigenetic marks through cell division
requires that they survive DNA replication and mitosis (Fig. 2). This is
particularly relevant for histone modifications, because nucleosomes
do not have a DNA template-based duplication system. Deposition
of parental H3 and H4 histones occurs within few hundred base
pairs of their pre-replication position and, upon replication, they
are roughly equally distributed to the leading and the lagging strand
daughter DNA molecules, through the action of dedicated molecular
complexes29,30. Chromatin maturation factors, including DNMT1–
UHRF1, EZH2 and HP1, use the proliferating cell nuclear antigen
(PCNA; a DNA clamp that is essential for replication) or origin recog-
nition complex (ORC) proteins as tethering components31–34 (Fig. 2a).
In addition, Polycomb components utilize their DNA-anchoring fac-
tors to propagate mitotic memory. Loss of the target DNA sequence
elements results in loss of PcG proteins and of gene silencing within a
few cell divisions in Drosophila35,36, although sequence-independent
propagation of silencing can be maintained in mammalian cell cul-
ture37. Mitotic retention of regulatory components (Fig. 2c), including
transcription factors and some of the epigenetic machineries described
above38,39, has been well-documented in recent years40,41. Inheritance
through meiosis is also possible at least to some extent, as shown

Gene A

Gene B

Gene C

Neuron

Muscle

Precursors
(cells with speci�c sets
of transcription factors)

One
genome

Cells with developmentally
induced epigenomes

Altered
epigenome
in disease

Germline
reprogramming

Germline
cell

PRC1/

2

Trithorax

Heritable (somatic) states
(epigenetic barriers)

H3K4me3

H3K27me3

Promoter
Nucleosome

Transcription
factor

Fig. 1 | Epigenetic mechanisms that maintain cell identities during
development and throughout life. Starting from the zygotic genome,
stage- and cell-type-specific transcription factors initiate regulatory
cascades that induce cell differentiation. Epigenetic components (for
example, Polycomb PRC1/2 and Trithorax group proteins) maintain the
‘off ’ states of certain genes and the ‘on’ states of others, in a cell-type- and
time-specific manner (the bottom panels show three genes, depicted
schematically as chromatinized templates, in which transcription is

triggered by specific transcription factors and silent or active states are
maintained by PRC1/2 or Trithorax proteins, respectively). In doing so,
they constitute barriers against accidental reprogramming that maintain
developmental and physiological homeostasis. Altered epigenomes can
lead to changes in programmed cell differentiation or, when accidental, to
disease (bottom right). Germline reprogramming resets the majority (but
not all) of the epigenome to achieve reproduction (top right).

4 9 0 | N A T U R e | v O L 5 7 1 | 2 5 J U L Y 2 0 1 9

Review ReSeARCH

by the ability of maternally deposited H3K27me3 to control DNA
methylation-independent imprinting42,43. An additional possibility is
that only a fraction of the marks can be meiotically transmitted, but
this might be sufficient to reconstruct chromatin organization in the
subsequent generation44.

Owing to the lack of a precise ‘replication’ process for parental
nucleosomes and to the loss of many DNA-binding factors and
chromatin-associated components during mitosis and meiosis, the
inheritance of single nucleosome marks poses specific challenges28.
Mathematical modelling and biological evidence suggest that chroma-
tin heritability requires the establishment of domains of several or even
hundreds of kilobases in size45–47. Indeed, the genome is now known to
be hierarchically organized in a series of 3D structures, starting from
nucleosome clutches, to chromatin loops, to chromosomal domains
called topologically associating domains (TADs), and finally to active
or repressive compartments and chromosome territories15,46,48–50.
TADs and compartments might stabilize functional states and drive
their own inheritance. Furthermore, multiple epigenetic machineries
often act together to stabilize heritable states. For example, PRC2 col-
laborates with PRC1 complexes and DNA methylation is sustained by
heterochromatin proteins and/or small RNA pathways51. In summary,
epigenetic inheritance can involve multiple layers; and usually entails
the cooperation of partially overlapping signals, initially dependent
on DNA sequence (elicited by transcription factor binding or RNA-
mediated mechanisms). Each of these layers adds a degree of stability,
but each of them is also reversible, allowing plasticity in the presence of
regulatory cues47,52. The inheritance of chromatin states in the absence
of chromatin domains, or without self-reinforcing mechanisms, is
more challenging28. This might require retention of transcription
factors, histone variants and histone modifiers during DNA replication
and mitotic bookmarking53.

Epigenetics and DNA sequence variation
DNA sequence variation and epigenetics are inextricably linked.
Chromatin states can influence transcription factor binding54,
and DNA sequence polymorphism influences chromatin states.
Chromatin and DNA methylation display extensive variation in
humans55. Furthermore they regulate genome stability and mutability.
Transposable elements are frequent targets of epigenetic silencing that
can sometimes be environmentally influenced and can influence gene
expression as well as genome integrity.

Genetic effects on epigenetics
The genome of each individual experiences both natural and envi-
ronmentally induced mutations. While most mutations are neutral,

Table 1 | Summary of the history and definitions of epigenetics
Authors Epigenetics is the study of: References

Waddington the processes by which the genotype
brings the phenotype into being

2

Nanney the systems that regulate the expression
of the ‘library of specificities’ (that is, the
genetic material, which is meant to be the
DNA or RNA sequence)

4

Riggs, Holliday,
Martienssen, Russo

mitotically and/or meiotically heritable
changes in gene function that cannot be
explained by changes in DNA sequence

5,151

Bird structural adaptations of chromosomal
regions so as to register, signal or
perpetuate altered activity states

8

Greally, Lappalainen properties of a cell, mediated by genomic
regulators, that confer on the cell the
ability to remember a past event.

59

Nicoglou various intracellular factors that have an
effect on the stability of developmental
processes through their action on
genome potentialities

16

Box 1
Major carriers of epigenetic
information
Heterochromatin components
Pericentric heterochromatin contains a large number of proteins,
but its most distinctive feature is the presence of megabase-
sized repetitive DNA domains coated in a specific histone H3K9
trimethylation mark, which is deposited by the enzymes SUV39 and
SETDB1. This mark is bound by the chromo domain of SUV39H1,
which stimulates catalytic activity of the enzyme152. Furthermore,
the same mark is bound by the HP1 protein, which can bridge
adjacent nucleosomes153. Therefore, heterochromatin components
can both write and read the H3K9me3 mark and compact their
target chromatin. Heterochromatin factors also collaborate with
RNAi in plants, yeast and some animals to convey epigenetic
inheritance.
Polycomb proteins
Early genetic studies classified Polycomb (PcG) and Trithorax
into two antagonistic groups that maintain the memory of spatial
patterns of expression of homeotic genes throughout development.
These complexes also have key roles in the maintenance of
developmentally or environmentally programmed expression states,
such as X-chromosome inactivation or cold-induced vernalization
in plants3. PcG proteins are found in two main classes of complex—
PRC2 and PRC1—that are responsible for deposition of the
H3K27me3 and H2AK119Ub marks via EZH2 and RING1A/1B,
respectively3. PcG proteins can be recruited to specific regions of
the genome by DNA-binding proteins or noncoding RNAs3. PRC2
complexes contain a writer, the histone methyltransferase enzyme
EZH2 (or its less efficient paralogue EZH1), and a reader, the EED
subunit. Similar to HP1, CBX subunits of PRC1 complexes contain
a chromodomain that specifically recognizes H3K27me3. Finally,
another PRC1 subunit, PHC1-3, can oligomerize and induce 3D
clustering in nuclear foci in vivo3.
Noncoding RNAs
Noncoding RNAs (ncRNAs) belong to several classes, and neither
their production nor their functions can be generalized. Many
ncRNAs, such as microRNAs, regulate post-transcriptional
processes, whereas others are involved in transcriptional regulation.
Short noncoding RNAs, such as short interfering RNAs (siRNAs) and
PIWI-interacting RNAs (piRNAs), are shorter than 30 nucleotides,
whereas long noncoding RNAs (lncRNAs) vary in size (up to more
than 100 kilobases). The best characterized of these is probably
the X-inactive specific transcript (Xist)154. Many short ncRNAs act
within or outside chromatin, and some, for example siRNAs and
tRNA fragments, can diffuse extracellularly14, whereas many nuclear
lncRNAs are chromatin-associated. Enhancer RNAs can activate
genes155, but most short and long ncRNAs are repressive, act via
chromatin (H3K9me3, Polycomb) or DNA methylation154,156, and
can induce epigenetic memory by building self-enforcing loops with
heterochromatin or the RNAi machinery. They are also involved in
the regulation of higher-order chromatin architecture.
DNA methylation
The mechanisms that allow DNA methylation to be copied during
DNA replication represent one of the best-understood epigenetic
systems, and involve specific proteins that recognize CpG hemi-
methylated DNA and thereby redeposit DNA methylation on
newly replicated DNA. DNA methylation is maintained by the DNA
methyltransferase DNMT1 and its partner UHRF1 (also known
as NP95), which specifically binds hemimethylated DNA and
stimulates DNMT1 via its ubiquitin ligase activity (Fig. 2). Therefore,
as recently reviewed in detail157, a single complex contains both the
‘writer’ and the ‘reader’ of the epigenetic methyl CpG mark, and
both moieties are essential for the maintenance of DNA methylation.

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sequence polymorphisms can affect epigenomic landscapes. For
example, analysis of chromatin accessibility and ‘CCCTC-binding
factor’ (CTCF) DNA binding in parents and children from families
with different ancestry found a substantial percentage of bound sites
that were unique to each ancestry, with differential binding being
explained mainly by genetic variation56. As CTCF can affect 3D genome
architecture and gene expression, this finding suggests that rewiring
of epigenomic landscapes might frequently occur as a consequence
of mutations. On the other hand, mutations that affect histone and
DNA methyltransferases, or demethylases (TET enzymes), chromatin
remodellers and other chromatin factors including histones, are fre-
quently found in disease57 and their effects may be specifically targeted
by therapeutic interventions58. The often cryptic relationship between
DNA sequence and epigenetic changes can mean that mutations may
be overlooked or mistaken for epimutations, leading to misconcep-
tions about the driver versus passenger role for epigenetic changes59.
This issue should be partly addressed by cheaper and faster sequencing

methods, which will be able to produce genomic and epigenomic infor-
mation from the same sample.

Chromatin and DNA methylation in mutagenesis
Mutation rates vary in different parts of the genome, at different stages of
the life cycle and in diseases such as cancer, where they depend on the cell
of origin, environmental exposure and cancer type60–62. Mutation rates
can be affected by DNA methylation63 and nucleosome positioning64.
Higher-order chromosome folding also influences mutagenicity.
A large-scale survey of balanced chromosomal abnormalities in patients
with congenital anomalies revealed disruption of TADs encompassing
known syndrome-linked loci in 7.3% of cases65, and a combination of
Hi-C chromosome capture with whole-genome sequencing in multiple
myeloma showed significant enrichment of copy number variation
breakpoints at TAD boundaries66 that are frequently bound by
CTCF. Furthermore, CTCF is frequently mutated in human cancer57.
Hypermutation of the heterochromatic inactive X chromosome has
also been noted in cancer and may be due to DNA replication stress in
aberrantly proliferating cells67.

The role of the repetitive genome
Transposable elements are intimate components of genomes, with gene
regulatory potential that may lead to phenotypic diversity. Indeed,
transposable elements and their relics constitute a major fraction of
most eukaryotic genomes. McClintock proposed that transposons were
turned on or off by environmental changes or during development,
acting as ‘control elements’. We now know that transposons can influ-
ence gene activity in multiple ways, acting as regulatory elements or
interfering with transcription68. Genomes have evolved species-specific
mechanisms to limit transposon activity, for example by targeting
repressive heterochromatin machinery, either through specific RNAs
or DNA binding factors. In Drosophila, heterochromatin-dependent
mechanisms allow the expression of specific clusters of transposon
relics in order to produce PIWI-interacting RNAs (piRNAs) that, in
turn, inhibit transposition. piRNAs are maternally heritable and can be
amplified via a ping-pong system, effectively allowing the organism to
resist new invasions and adapt their genome to them69. Caenorhabditis
elegans uses heterochromatin components to prevent illegitimate
repetitive DNA transcription and genome instability70. Plants pro-
duce small RNAs derived from double-stranded precursors, which are
synthesized by dedicated polymerases and target DNA methylation
and the H3K9 methylation machinery71. Finally, numerous strategies
are deployed in mammals, the most recently characterized being the
repression of endogenous retroviruses (ERVs) by the KAP1 protein
(also known as TRIM28), which co-recruits heterochromatin proteins
such as SETDB1 by interacting with Krüppel-associated box (KRAB)
domain-containing zinc-finger proteins (KZFPs). This strategy also
enables the rapid evolution of gene regulation strategies via binding
of KZFP to ERVs near genes72, thus influencing gene expression
dynamics and levels.

Environmental epigenetics
Recently the influence of the environment in development and
physiology has been underlined. Gene × environment interactions
determine how individuals with the same or different genotypes will
respond to environmental variation. The importance of epigenetics in
environmental responses is well-established in plants, particularly in
Polycomb-based vernalization73, but similar processes appear to take
place in some animal species.

Environmental epigenetic regulation in animals
In Drosophila, environmentally induced phenotypes that depend
on epigenetic regulation involve transmission across several genera-
tions74–76. C. elegans has been shown to translate several environmental
stimuli, such as viral infection, starvation or elevated temperatures, into
modification of epigenetic components77–79. Whereas starvation and
viral infection induce inheritance via the production of small RNAs77,78,

Box 2
Novel approaches for epigenetics
The understanding of epigenetic inheritance requires the ability to
tell whether progenies retain parental phenotypes. Early studies
on mitotic inheritance were severely hampered by limitations
in describing the molecular states of individual cells. However,
modern low-cell and single-cell technologies are allowing high-
throughput quantitative measurements of molecular species
in a few or even single cells, and simultaneous measurement
of multiple molecules, including proteins and RNAs, RNAs and
DNA methylation or RNAs and chromatin accessibility158,159.
These techniques are complemented by increasingly robust and
predictive analytical tools160. Furthermore, techniques such as
single-cell Hi-C can provide information on three-dimensional
chromatin folding161–164, which can be complemented by high-
content super-resolution microscopy and molecular modelling
approaches165. Low-cell and single-cell studies allow the
investigation of individual germline cells, mature gametes, zygotes
and early stages of embryonic development166.

The study of epigenetic inheritance also requires the ability to
follow molecular changes through time and cell division. Recent
progress in lineage-tracing techniques has been instrumental in
accomplishing this goal, as such techniques allows cell pedigrees to
be established. Early tracing techniques led to the labelling of one
or a small group of cells. However, more recent approaches allow
prospective multiplex tracing by introducing barcodes of essentially
unlimited complexity into dividing cells, as well as retrospective
tracing by extensive DNA sequencing and reconstruction of the
history of acquisition of spontaneous mutations. The division tree
of large cell populations can thus be reconstructed167. Coupling
lineage tracing with single-cell ‘omic’ technologies thus promises to
make it possible to understand the gene expression histories of cell
lineages.

These descriptive techniques can be complemented by the
versatile toolbox of genome engineering technologies such
as CRISPR–Cas. It is possible to mutate the genome precisely,
reversibly and at multiple sites simultaneously168. Furthermore,
one can tether proteins of interest to selected genomic positions
in order to silence or activate genes, to induce reversible changes
in 3D chromatin architecture and to visualize loci of interest in
live imaging experiments169,170. This surge in approaches and
technologies is revolutionizing the ways in which epigenetic
processes can be studied, understood and harnessed to understand
biological and pathological processes and to develop novel
therapeutic strategies.

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apparently without involvement of chromatin, temperature-dependent
epigenetic inheritance involves the H3K9 methylation machinery
(SET-25)79, without RNA interference (RNAi), suggesting that, depend-
ing on the type of stimulus, the RNAi machinery and chromatin regu-
lators can act differently to drive inheritance.

Examples of environmental effects are by no means limited to model
organisms. Temperature is a major sex-determining factor in many
reptiles. In a turtle species in which sex is determined by tempera-
ture during egg incubation, the KDM6B H3K27me3-specific demeth-
ylase exhibits sexually dimorphic, temperature-dependent expression
that regulates the sex-determining gene Dmrt180. In Australian cen-
tral bearded dragons, chromosomal sex determination is overrid-
den by high temperatures to produce sex-reversed female offspring.
Temperature induces alternative splicing of KDM6B and of JARID2, a
PRC2-recruiting component81. It is intriguing that temperature affects
PRC2 factors in diverse animal and plant species, suggesting that tem-
perature sensing by PRC2 might be evolutionarily conserved, although
this is not the only environmental effect that can stably modify chroma-
tin. Another form of environmentally induced chromatin regulation is
found in social insects such as the carpenter ant Camponotus floridanus,
in which the balance between morphologically distinct worker castes
depends on the levels of histone acetylation, which may be influenced
by feeding behaviour82.

Metabolism and epigenetics in mammals
DNA and chromatin modifications use metabolic products. For
example, S-adenosylmethyonine (SAM) is the methyl donor in DNA
and histone methylation; folate and vitamins B6 and B12 induce
SAM production; α-ketoglutarate (αKG) is required for DNA and
histone demethylation; succinate and fumarate inhibit DNA and his-
tone demethylases; acetyl-coenzyme A is the acetyl donor for histone

acetylation; β-hydroxybutyrate inhibits class I histone deacetylases;
and the NAD+/NADH ratio regulates the sirtuins (class III histone
deacetylases). Therefore, metabolic alterations can induce global
perturbations of the epigenome and mutant metabolic components
represent potential therapeutic targets83,84. On the other hand, meta-
bolic changes can affect specific loci and induce long-lasting epigenetic
modifications, including intergenerational epigenetic inheritance85–87.
The effectors of these perturbations are DNA methylation, Polycomb
components, and transfer RNA (tRNA) fragments, which, among other
effects, repress genes associated with endogenous retroelements and
might thereby help to preserve genome integrity87–89. A protein restric-
tion diet in mice can also induce DNA methylation and repression of
a subset of ribosomal DNA (rDNA) genes90, although the inducer and
the roles of this rDNA ‘epiallele’ remain to be identified. In summary,
there are compelling examples in which the environment is linked to
epigenetic regulation. However, confounding effects, the impact of
multifactorial exposure, access to appropriate tissues and assessment
of causality for DNA sequence versus epigenetic variation remain
major challenges, particularly in humans. Most importantly, there is
an urgent need to identify direct links between environmental changes,
metabolic changes and epigenetic components. The recent discovery
that histone demethylases KDM5A and KDM6A (also known as UTX)
can sense oxygen concentrations and thereby modulate H3K4me3 and
H3K27me3 levels91,92 is a first step in this direction.

Transgenerational epigenetics
The modern evolutionary synthesis93 postulates that evolution acts
mainly via natural selection on phenotypes, ultimately affecting DNA
sequences. The discovery that non-DNA sequence information, such
as parental, ecological, behavioural and cultural information, can be
heritable94 has not broken the modern framework of evolutionary

DNA polymerase

DNMT1

PCNA

Newly replicated DNA

H3K9me3
H2AK119ub

DNA 5meC

Unmethylated C

a Replicating heterochromatin (S phase)

c Maintaining chromatin through mitosis

b Maintaining chromatin in interphase (G1, S and G2)

Mitotic bookmarking of
transcription

factors

and PolII factors

HP1

Constitutive heterochromatin

Euchromatin

Facultative heterochromatin

Other factors

H3K4me3 ATP
ADP

H3K27me3

PRC2 PRC1

Domains of
heterochromatin

PRC1/2 interplay

HMTs

UHRF1

Trithorax/
COMPASS

SWI/SNF

Fig. 2 | Maintaining chromatin states through the cell cycle. a, DNA
replication during the S phase of the cell cycle is a challenge to the
maintenance of nucleosome marks. Epigenetic components, such as
HMTs and UHRF1, interact with components of the DNA replication
machinery, such as the PCNA clamp, in order to reconstitute chromatin
domains after the passage of the fork. The case of DNA methylation
is depicted schematically. Newly replicated DNA is unmethylated
(empty lollipops; the methylated template DNA strand is not shown
here for simplicity). The UHRF1/DNMT1 complex associated with
PCNA facilitates remethylation of hemimethylated DNA after DNA
replication. b, Both constitutive (involving H3K9 methylases and

HP1) and facultative (involving PRC1 and PRC2) heterochromatin,
as well as euchromatic features (involving an interplay between PRC1,
PRC2, Trithorax/COMPASS and ATP-dependent chromatin remodelling
complexes), are stably maintained during interphase in order to prevent
genes from inappropriately switching their functional states. SWI/SNF is a
nucleosome remodelling complex. c, During mitosis, most chromosome-
associated factors are evicted during chromosome condensation, but
‘mitotic bookmarking’ of genes is achieved by the maintenance of key
components (such as certain transcription factors or RNA polymerase III)
bound to their target loci.

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synthesis. Indeed, one could postulate that complex chains of DNA-
driven events ultimately drive parental and ecological behaviours and,
therefore, DNA sequence alone would still explain these complex forms
of inheritance. A direct demonstration that other molecules, in addition
to DNA, carry substantial heritable information would represent an
important conceptual change in evolutionary biology.

When adults are exposed to a stimulus or an intervention, their
germline, as well as the germline of the fetus in pregnant females, is
exposed. We thus distinguish intergenerational inheritance, in the F1
of exposed males and up to the F2 of exposed females, from transgener-
ational inheritance, starting from the F2 of exposed males and the F3 of
exposed females44,95. There is abundant evidence for intergenerational
inheritance in plants and some animals86,95–100, suggesting that this
phenomenon might be involved in establishing early developmen-
tal patterning. What about transgenerational epigenetic inheritance
(TEI)100? In yeast, TEI has been well-documented101 and is known to
involve RNAi-dependent heterochromatin deposition, leading to Clr4-
dependent H3K9me3 marking of silent heterochromatin47,102. By con-
trast, in the absence of RNAi-dependent amplification, H3K9me3 alone
is insufficient to drive stable epigenetic memory, unless the histone
demethylase Epe1 is mutated47,102. In Tetrahymena, TEI participates
in the phenomenon called ‘programmed DNA elimination’ from the
transcriptionally active somatic nucleus and, again, it involves small
RNA-dependent formation of heterochromatin on the DNA elements
to be eliminated103. In plants, TEI has also been well-described. Plant
epialleles can be stable over many generations, and TEI is generally con-
veyed by RNA-directed DNA methylation (Fig. 3a), a mechanism that
can also promote recovery from loss of DNA methylation in a subset

of epialleles in Arabidopsis95,104. Chromatin components such as the
histone chaperone CAF-1 also modulate DNA methylation-dependent
TEI105. Future work is needed to elucidate the link between nucleosome
dynamics and inheritance of DNA methylation.

Unlike plants, the germline is separated from the soma in most sex-
ually reproducing organisms, and Weismann postulated that informa-
tion can flow only from germ cells to the soma106. Furthermore, a large
number of epigenome features are erased in germline cell chromatin
before and during meiosis. An important open question, however, is
how much of the epigenome resists erasure? Evidence for substan-
tial epigenetic inheritance of molecules other than DNA through
gametes would overturn a fundamental tenet of neo-Darwinism.
C. elegans epialleles (epigenetically modified alleles that induce specific
phenotypes and are heritable over many generations) involve hetero-
chromatin components (Fig. 3b), which, depending on the induction
paradigm, may or may not involve piRNAs79,107. In Drosophila
(Fig. 3c), heterochromatin components can induce TEI upon heat
shock or osmotic stress108, whereas piRNAs produce TEI in response
to transposable element activity69. A second mechanism that can lead
to TEI in Drosophila relies on Polycomb proteins109. Post-eclosion
dietary manipulation with a low-protein diet that resulted in elevation
of the PRC2 enzymatic subunit E(z) or inhibition of PRC2 by RNAi or
by an E(z) inhibitor induced a change in H3K27me3 and in longevity
that could be inherited for at least two generations110. Furthermore,
perturbation of chromosome architecture and of PRC2 function was
shown to induce stable but reversible TEI in Drosophila111. Exposure
of C. elegans to bisphenol A also induced alterations in the levels of
H3K9me3 and H3K27me3 through five generations112 and, in plants,

a Arabidopsis

Possible
triggers

F3

Transgene expression

At least F20 F2 to F50

Heterochromatin

H3K9me3

H3K27-
me3

b C. elegans c Drosophila

H3K4me3

• Mutations in chromatin,
DNA methylation and
RNAi components
• Altered environmental
conditions

• Viral infection
• Starvation

• KD Polycomb
• Diet change
• Toxic challenge

• piRNA
expression
• Hybrid crosses

Multiple phenotypes

Many generations

Control of
retrotransposition

Different
amounts of
Polycomb

• Reporter transgene
expression
• Longevity
• Resistance to toxic
challenges

Several
generations

• Viral
resistence
• Metabolism
• Lifespan

Stability
N generations

Phenotypic
effects

Carrier
molecular

machineries

Small
RNAs

Piwi factors

Nuclear
RNAi

factors

Hetero-
chromatin

factors

Polycomb factors

piRNA
machinery

• piRNA expression • Mutation in
chromatin
components
• Temperature

• Heat shock
• Osmotic
stress

DNA meC

Heterochromatin
vs open
chromatin

• Mutation in chromatin: lifespan
• Temperature:
transgene expression
• Heat shock, osmotic stress:
heterochromatin alterations

• Mutation in chromatin: F3
• Temperature: F14
• Heat shock, osmotic stress: F3

Fig. 3 | Transgenerational epigenetic inheritance. Hallmarks of TEI
in plants (a), C. elegans (b) and flies (c). From top to bottom, the Figure
shows the triggering mechanisms, the molecules involved in establishment
and transmission of transgenerational memory (carrier molecular

machinery), the phenotypic consequences of epigenetic changes and the
stability of TEI phenomena in terms of the number of generations (N) in
which inheritance has been reported.

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TEI of vernalization is prevented by the function of ELF6, which is a
H3K27-specific demethylase52. These data suggest that both hetero-
chromatin and Polycomb can induce TEI. Notably, the presence of a
histone binding domain that recognizes the same mark as is deposited
by the enzymatic moiety in both heterochromatin and PRC2 might
provide these systems with amplification potential28. Differential levels
of their marks might be reconstituted at each generation through dif-
ferential affinity of PRC2 or other heterochromatin complexes to chro-
matin regions endowed with differential initial densities of marked
nucleosomes (Fig. 3c).

Transgenerational inheritance in mammals
In vertebrates, DNA methylation is globally reduced twice in each gen-
eration: immediately after fertilization and in developing primordial
germ cells113. Histone marks and 3D genome organization are also

reprogrammed in the germline and after fertilization19. Furthermore, it
is difficult in mammals and virtually impossible in humans to exclude
potential confounding elements, such as maternal contribution, com-
ponents of seminal fluids, changes in utero or postnatal effects114. So,
what is the evidence for mammalian TEI? A classic example of multi-
generational inheritance, insertion of the IAP endogenous retrovirus
at the mouse agouti coat-colour locus, depends on heritable, variable
methylation of the IAP retrovirus on an alternative promoter for the
agouti gene115. A recent systematic survey of murine IAP insertions
has indicated that multigenerational inheritance is rare, however116.
Several reports have attracted attention for their suggestion that diet
or exposure to chemicals and behavioural stresses can be transmitted
to the progeny for multiple generations117–119, but some of these results
have been criticized120. In humans, epidemiological evidence from
the Överkalix population cohort established links between grandpa-
ternal food supply at the beginning of the twentieth century and the
mortality rate of subsequent generations121, although molecular
evidence is unavailable for this cohort. DNA methylation has been
suggested as a potential mechanism for these effects, and retroele-
ments and some genes involved in neurological and metabolic dis-
orders remain methylated during the wave of DNA demethylation in
human primordial germ cells122. However, a recent report in which
a high-fat diet induced insulin resistance, obesity and addictive-like
behaviours up to the third generation did not identify heritable
changes in the DNA methylome123. Other chromatin components
might also be involved. For example, transient overexpression of the
H3K4-specific KDM1A histone demethylase in mouse spermato-
genesis has been shown to induce TEI124. These studies suggest that
TEI is limited but possible in humans. Future work should address
the underlying mechanisms of TEI, and epigenome-wide association
studies should complement genome-wide association studies in order
to assess the relative contributions of DNA sequence and epigenome
alterations in disease59.

Epigenetics, health and disease
Changes in the levels of DNA methylation, histone modifications
and changes in non-coding RNA (ncRNA) function are common
in disease, as are mutations in epigenetic components57,125. The ability
to distinguish driver from passenger roles for epigenetic alterations
will make it possible to identify diseases in which epigenetics might
affect diagnosis, prognosis and therapy. Dissecting the interplay
between epigenetic components and other disease pathways
will also allow the development of combinatorial intervention
approaches.

The epigenetics of ageing
The application of machine learning to high-throughput DNA meth-
ylation data has identified indicators of chronological or biological
age. One study found that changes in CpG methylation at 353 genomic
sites produced a score that was highly correlated with age across
tissues126 (epigenetic clock; Fig. 4a). Strikingly, a comparison of dif-
ferent molecular predictors of age indicated that the epigenetic clock
is the most highly correlated to biological age127. Furthermore, epi-
genetic age is adversely affected (accelerated) by a high body mass
index, whereas it is reduced by high levels of education or physical
activity, a low body mass index and consumption of fish, poultry,
fruits and vegetables128. Many of the 353 CpGs investigated are located
close to poised promoters of bivalent genes (marked by H3K4me3 and
H3K27me3), or to active promoters126, suggesting that ageing may
correlate with reduced plasticity in the expression of some bivalent
genes, which might resolve into repressed or active states, and with
active genes changing their expression levels. More recently, integra-
tion with composite clinical measures of phenotypic age identified
a set of CpG genomic sites that better predicts lifespan as well as
healthspan129. Establishing the mechanistic links between the ageing
process and variations in CpG methylation will be critical in order to
identify the causes of ageing.

a DNA methylation in ageing: an epigenetic clock

b Epigenetic alterations and cancer

Chronological age

D
N

A
m

e
th

yl
a
ti
o

n
-b

a
se

d
a

g
e

Tumorigenic insults
Smoke, pollutants,
chemotoxic agents, ageing,
nutrition…

Mutations in epigenetic
regulators
Polycomb,Trithorax,
DNA methylation, chromatin
remodellers, CTCF…

• Altered cell signalling
• Epigenetic amplification of
altered cell states
• Maintenance of altered cell
states by TF-dependent
regulatory feedback loops

Cancer

• Loss of cell cycle control
• Loss of differentiation
• Loss of cell adhesion
• Senescence bypass
• Increase in proliferation
• Increase in cell–cell
heterogeneity

Cellular effects

Fig. 4 | Epigenetics and disease. a, The ‘epigenetic clock’ consists of
a specific set of genomic CpG sites whose levels of DNA methylation
change progressively with age, leading to an estimate of age based on
DNA methylation that correlates tightly with chronological age. Rather
than a global change in methylation levels, some of the age-related CpG
sites show increased methylation (black lollipops, red outline), whereas
others show decreased methylation (white lollipops, blue outline).
The relationship between changes in DNA methylation and chromatin
architecture in ageing remains to be investigated, as well as the cause–
consequence relationships between ageing, DNA methylation and gene
expression changes. b, The genes encoding epigenetic components such
as DNA methylases and demethylases, Polycomb, Trithorax, chromatin
remodellers, DNA methylation components and CTCF are frequently
mutated or dysregulated in cancer, often as a result of environmental
insults or physiological changes such as ageing. These mutations alter
cellular properties such as cell division, cell differentiation, adhesion and
proliferation, and increase the heterogeneity of gene expression, thereby
promoting tumorigenesis.

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Developmental epigenetics and disease
Drawing initially on epidemiological studies, Barker formulated the
hypothesis of the fetal or developmental origin of health and disease
(DOHaD)130, which suggests that exposure to environmental factors
such as chemicals, drugs, stress or infections during specific sensitive
periods of intrauterine fetal development or early childhood might
predispose an organism to diseases in adult life. Later work proposed
that epigenetic components might mediate some of these effects131,132.
Long-lasting changes to the epigenome that affect cancer susceptibility
and biology have also been documented133. Other areas of intense study
include obesity and diabetes134, neurological disorders125 and age-
related conditions such as Parkinson’s and Alzheimer’s diseases135,136.
Embryonic development and early life are two major susceptibility
windows during which epigenetic programming is sensitive to envi-
ronmental influences, such as diet, temperature, environmental
toxins, maternal behaviour or childhood abuse137. Behavioural molecular
genetics has identified a third susceptibility window, adolescence,
during which adverse life experiences affect the risk of anxiety,
depression and aggressive behaviour, associated with DNA methyl-
ation of specific genes138 or with alterations in levels of HDAC1139.
Furthermore, memory formation, a behavioural response to environ-
mental stimuli, is associated with changes in histone and DNA modifi-
cation at selected loci140,141. Future studies should establish whether any
of these alterations are in fact causal. Interestingly, one study found that
low maternal care in mice decreases DNMT3a and DNA methylation
at the L1 promoter and simultaneously induces the mobilization of L1
elements in the hippocampus, suggesting that environmental variation
can cause genetic and epigenetic changes simultaneously142.

Cancer epigenetics
Genome-wide association studies of specific types of cancer or from
the cancer genome atlas project have identified frequent mutations
in genes that encode epigenetic components57,58,143. These include
mutations in DNA methylases and demethylases, histones144 and
histone modifiers, and genes involved in chromatin remodelling
and chromosome architecture, but also metabolic genes such as IDH1
and IDH2 that affect histone and DNA methylation57 and might per-
turb 3D genome architecture145 (Fig. 4b). Repetitive DNA elements
can also contribute to cancer. For instance, in Hodgkin lymphoma,
transcription of the IRF5 transcription factor gene is induced by DNA
hypomethylation of a normally dormant endogenous retroviral long
terminal repeat located upstream of the promoter, a phenomenon
dubbed onco-exaptation146, whereas in other tumours, DNA demeth-
ylating agents can have the opposite effect147. Although epigenetic
perturbations are generally accompanied by mutations in cancer
driver genes, sporadic cases in which cancer can be induced in the

absence of obvious driver DNA mutations have also been reported in
mice148. Furthermore, analysis of pancreatic cancer metastases did not
uncover any obvious driver mutations; instead, large-scale chromatin
reprogramming was observed, with changes in the level of H3K9me3
in many chromosomal domains149. These findings suggest that
epigenetic changes can be major driver of oncogenic processes in
certain circumstances.

Concluding remarks
Epigenetic mechanisms buffer environmental variation while allow-
ing plastic responses to the most extreme environmental conditions.
In this sense, epigenetics is returning to and expanding the original
Waddington definition. A frequently held misconception about epige-
netics is that it is a carrier of freedom from a presumed DNA-encoded
destiny. The great discoveries of the second part of the twentieth
century have generated much excitement about the role of DNA in
evolution, biology and medicine, which led to the view of DNA as the
‘book of life’. The fact that the same DNA can correspond to differ-
ent heritable phenotypes has now been portrayed as proof that ‘DNA
isn’t your destiny’, a statement which merely reflects the level of hype
about epigenetics. Most organisms buffer environmental variation in
physiology and inheritance, although buffering does not erase every
bit of epigenetic information (Fig. 5). Phenotypes thus depend on
specific combinations of genome composition, epigenetic components
and environmental inputs. The advent of increasingly sophisticated
and economically feasible approaches to genomics, biochemistry and
genetics can at last clarify the extent to which epigenetic mechanisms
influence life, inheritance and evolution. This will allow us to progress
towards personalized precision medicine, as well as to investigate and
clarify the effects that lifestyle and ‘mind–body’ interventions may have
on health. It has been suggested that the extrapolation of epigenetic
findings from mice before they are confirmed in humans may lead to
‘serving epigenetics before its time’—that is, to rushed, unsupported
conclusions that can cause harm and unnecessary anxiety150. We
suggest that we are approaching ‘the right time for serving epigenet-
ics’, for several reasons. The molecular machineries and mechanisms
that enable states to be propagated are finally becoming clear; it is pos-
sible to test whether these mechanisms matter for biological processes,
ageing or disease; and epigenetic alterations are more readily reversible
than DNA mutations and can be targeted with increasing specificity.
This allows the biomedical community to test the relevance of
epigenetic components in specific diseases functionally, to exploit them
as prognostic and diagnostic markers, and to use them as actionable
targets for therapy. This path will deepen our knowledge and deliver
benefits for human health. Therefore, the field of epigenetics is finally
coming of age.

H3K27me3
PRC2

• DNA mutations/
enviromental exposure
• Alteration of epigenetic
machinery

Next generation
Metastable
epigenetic
inheritance

F1 F2

Epigenetically induced
somatic alterations

Gametes

Modification of the
germline epigenome

Transmission of the germline
epigenome modification

Fig. 5 | Interactions between genome sequence, the environment and
epigenetics in inheritance. Environmental exposure can affect both the
soma and the germline. When transient mutations or perturbations in
epigenetic components occur (for example, PRC2 Polycomb components in

Drosophila111, as shown), the germline chromatin may acquire an alternative
state that can be transmitted and produce a phenotype (here, a change in
eye colour) in subsequent generations. The degree of epigenetic inheritance
varies and depends on the molecular features of each system and species.

4 9 6 | N A T U R e | v O L 5 7 1 | 2 5 J U L Y 2 0 1 9

Review ReSeARCH

Received: 29 October 2018; Accepted: 14 June 2019;
Published online 24 July 2019.

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Acknowledgements We thank G. Papadopoulos for analysis of CpG methylation
and epigenomic data linked to ageing. We sincerely apologize to all authors
whose work could not be cited owing to space constraints. G.C. is supported
by the CNRS and the ERC (Advanced Grant ERC-2018-AdG, No. 788972
(3DEpi)). E.H. is supported by CNRS, INSERM and ERC-2015-AdG, No. 671027
(XPRESS).

Author contributions Both authors contributed ideas and figures. G.C. wrote
the draft manuscript and both authors finalized the article.

Competing interests The authors declare no competing interests.

Additional information
Correspondence and requests for materials should be addressed to G.C. and E.H.
Peer review information Nature thanks John Greally, Matthew Lawrence
and the other anonymous reviewer(s) for their contribution to the peer review of
this work.
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• Advances in epigenetics link genetics to the environment and disease

Box 1Major carriers of epigenetic information
Box 2Novel approaches for epigenetics
Epigenetic inheritance versus plasticity
Epigenetics and DNA sequence variation
Genetic effects on epigenetics
Chromatin and DNA methylation in mutagenesis
The role of the repetitive genome
Environmental epigenetics
Environmental epigenetic regulation in animals
Metabolism and epigenetics in mammals
Transgenerational epigenetics
Transgenerational inheritance in mammals
Epigenetics, health and disease
The epigenetics of ageing
Developmental epigenetics and disease
Cancer epigenetics
Concluding remarks
Acknowledgements
Fig. 1 Epigenetic mechanisms that maintain cell identities during development and throughout life.
Fig. 2 Maintaining chromatin states through the cell cycle.
Fig. 3 Transgenerational epigenetic inheritance.
Fig. 4 Epigenetics and disease.
Fig. 5 Interactions between genome sequence, the environment and epigenetics in inheritance.
Table 1 Summary of the history and definitions of epigenetics.