HIGHLIGHTED ARTICLE

GENETICS | PERSPECTIVES

What Do You Mean, “Epigenetic”?

Carrie Deans*,1 and Keith A. Maggert†
*Department of Entomology, and †Department of Biology, Texas A&M University, College Station, Texas 77843

ABSTRACT Interest in the field of epigenetics has increased rapidly over the last decade, with the term becoming more identifiable in
biomedical research, scientific fields outside of the molecular sciences, such as ecology and physiology, and even mainstream culture. It
has become increasingly clear, however, that different investigators ascribe different definitions to the term. Some employ epigenetics
to explain changes in gene expression, others use it to refer to transgenerational effects and/or inherited expression states. This
disagreement on a clear definition has made communication difficult, synthesis of epigenetic research across fields nearly impossible,
and has in many ways biased methodologies and interpretations. This article discusses the history behind the multitude of definitions
that have been employed since the conception of epigenetics, analyzes the components of these definitions, and offers solutions for
clarifying the field and mitigating the problems that have arisen due to these definitional ambiguities.
KEYWORDS transgenerational; maternal effects; gene expression; epigenetic inheritance

I NTEREST in epigenetics, as well as the usage of the term

epigenetic, has increased significantly since the field was
first conceived by Conrad Waddington in the early 1940s. In
entirely philosophical. In this essay, we address these is-
sues by providing a brief history of epigenetics (the term
and the scientific field) and discussing various definitions,
2006, over 2500 articles related to epigenetics were pub- as well as the important differences between them. We
lished (Bird 2007), and in 2010, over 13,000 (Haig 2012). will also address the challenges that exist, and will con-
In 2013, however, this number rose to over 17,000, a striking tinue to exist, if these ambiguities are not addressed, and
45 new publications every day, in addition to increases in offer potential solutions for dealing with these challenges.
scientific meetings and grant directives dedicated to the sub-
ject. Today, epigenetic concepts have spread into fields that
do not routinely address genetics (at least explicitly), such History of the Term “Epigenetic”
as ecology (Bossdorf et al. 2008; Zucchi et al. 2013; Burris
To understand the meaning of the term epigenetics, one must
and Baccarelli 2014), physiology (Ho and Burggren 2010),
understand the context in which it was derived. Conrad
and psychology (Ngun and Vilain 2014; Zhou et al. 2014).
Waddington, who first defined the field in 1942(a), worked
Despite its apparent popularity, the unfortunate fact is that
as an embryologist and developmental biologist. In 1947, he
the increased use of the term epigenetics is likely due more to
founded and led the first genetics department at the Insti-
inconsistencies in its definition than to a consensus of in-
tute of Edinburgh and would later found the Epigenetics
terest among scientists or a paradigm shift in the rules of
Research Group in 1965 (Van Speybroeck 2002). Waddington
inheritance. The term has taken on multiple meanings, de-
had a strong appreciation for genetics and was an important
scribing vastly different phenomena. As a result, its usage
advocate for uniting genetic principles with other fields of bi-
oftentimes implies mechanistic connections between unre-
ology, such as cytology, embryology, and evolutionary biology;
lated cases. The lack of a clear definition has led to confusion
however, he was particularly interested in embryology and de-
and misuse of the term, while also making research within
velopmental genetics, specifically the mechanisms that con-
the field of epigenetics difficult to synthesize and reconcile.
trolled cellular differentiation. At the time, there were two
There are many reasons why the etymology of epigenetics
prevailing views on development, both of which were derived
is so ambiguous, many of which relate to the scientific
from the 17th century: preformation, which asserted that all
atmosphere in which the term was conceived; others are
adult characters were present in the embryo and needed simply
to grow or unfold, and epigenesis, which posited that new
Copyright © 2015 by the Genetics Society of America tissues were created from successive interactions between the
doi: 10.1534/genetics.114.173492
1
Corresponding author: 507a Minnie Belle Heep Bldg., Texas A&M University, College
constituents of the embryo (Waddington 1956; Van Speybroeck
Station, TX 77843-2475. E-mail: cadeans@tamu.edu 2002). Waddington believed that both preformation and

Genetics, Vol. 199, 887–896 April 2015 887

epigenesis could be complementary, with preformation repre- genes (Nanney 1958a; Haig 2004, 2012). Most importantly,
senting the static nature of the gene and epigenesis represent- in addition to discussing variability in expression patterns,
ing the dynamic nature of gene expression (Waddington Nanney (1958a) emphasized the fact that expression states could
1956; Van Speybroeck 2002). It is through the combination persist through cell division. Although some have claimed that
of these concepts that he coined the term epigenetics, which Nanney’s usage of the term epigenetic was developed indepen-
he referred to as, “the branch of biology that studies the dently of Waddington’s definition (he initially used the term
causal interactions between genes and their products which paragenetic) (Haig 2004), considerable overlap can be found in
bring the phenotype into being” (Waddington 1942a; Dupont their contemporary writings on genotype–phenotype relation-
et al. 2009). ships (Nanney et al. 1955, 1958a,b; Waddington 1939, 1942a,b),
It is important to note that genetics was still a young field gene expression (Nanney et al. 1955, 1958a,b; Waddington
at this time, centered on Mendel’s work on trait inheritance, 1939, 1942a.b), and the respective roles of the nucleus and
with the gene being accepted as the unit of inheritance the cytoplasm in gene regulation (Nanney 1953, 1957, 1958a;
(Johannsen 1909); but, little was known about the biochem- Waddington 1939, 1956). It is clear, however, that Nanney’s
ical nature of the gene or how it functioned. It wasn’t until contemplation of the stability of cellular expression states was
Beadle and Tatum (1941) published their work affirming an important addition to Waddington’s ideas, which had sig-
the one-gene, one-enzyme concept that an understanding nificant impacts on the future direction of epigenetics. For
of gene function took discrete shape, and subsequent work a more detailed treatment of this history please refer to Haig
on molecular biology defined gene structure. This gene-centric (2004, 2012) and Holliday (1994).
atmosphere, coupled with the emerging effort to understand
gene regulation and expression, had a strong influence on the
Definitions of Epigenetics
creation of epigenetics, both as a concept and a field of study
(Jablonka and Lamb 2002). It was largely through a shared interest in development and
At that time, many, including Waddington, were interested cellular differentiation that Waddington, Nanney, and others
in the process of gene control and expression. Experimental came to use the term epigenetic; however, the focus of those
embryologists, such as Wilhelm Roux (1888), Hans Spemann within the field did vary, with some, such as Waddington,
(1967), Viktor Hamburger (1960), and the developmental being more concerned with gene regulation and genotype–
geneticist Ernst Hadorn (1955) studied mutations by induc- phenotype interactions, and others, such as Nanney and
ing changes in development through experimentation with Lederberg, being more interested in the stability of expression
chemicals or excision. Waddington, on the other hand, was states and cellular inheritance. As stated by Haig (2004), in-
more interested in the cellular processes that brought about terest in these different aspects of epigenetics led to a division
these changes, rather than the stimuli that created them. One within the field that can be directly linked to the definitional
of Waddington’s most important contributions was his ac- identity crisis that exists today.
knowledgment of, and emphasis on, the flexible relationship Throughout the 1980s and 1990s, the definition of epige-
between genotype and phenotype (Waddington 1942a,b, netic moved farther away from developmental processes and
1957), and this was an idea that many of his contemporaries, became more generalized. For example, one definition from
such as Nanney (1958a), Huxley (1956), Ephrussi (1953, 1982 describes epigenetics as “pertaining to the interaction
1958), and Lederberg (1958) (see below), were also inter- of genetic factors and the developmental processes through
ested in. Today, Waddington’s views on epigenetics are most which the genotype is expressed in the phenotype” (Lincoln
closely associated with phenotypic plasticity, which is the abil- et al. 1982). This definition does include the term developmen-
ity of a gene to produce multiple phenotypes, but he also tal, but its meaning seems to relate more to the development of
coined the term canalization to refer to the inherent stability the phenotype than to an ontological meaning. Although only
of certain phenoytpes (particularly developmental traits) across slightly different from Waddington’s original definition, this
different genotypes and environments (Waddington 1942b; definition and others during this time broadened the meaning
Siegal and Bergman 2002). Together, his concepts of plasticity of epigenetics in important ways. It made the term more avail-
and canalization suggest a general decoupling of genotype and able and applicable to other fields by emphasizing the impor-
phenotype and imply that regulatory processes must exist be- tance of genetic and nongenetic factors in controlling gene
tween the two. This realization was fundamental to Wadding- expression, while downplaying (although not ignoring) the
ton’s concept of epigenetics. connection to development (Medawar and Medawar 1983;
In 1958, 16 years after Waddington first coined the term, Hall 1992; Jablonka and Lamb 2002).
David Nanney published a paper in which he used the term Concurrently, research being done in the 1970s and 1980s
epigenetics to distinguish between different types of cellular on the relationship between DNA methylation, cellular differ-
control systems. He proposed that genetic components were entiation, and gene expression (Holliday and Pugh 1975;
responsible for maintaining and perpetuating a library of Riggs 1975; Jones and Taylor 1980; Bird et al. 1985) became
genes, expressed and unexpressed, through a template repli- more closely associated with epigenetics. The work of Robin
cating mechanism. He then deemed epigenetic components as Holliday and others, on cellular memory and DNA methyla-
auxiliary mechanisms that controlled the expression of specific tion, particularly the finding that DNA methylation had strong

888 C. Deans and K. A. Maggert

effects on gene expression and that these effects persisted acknowledge that the lack of a universal definition has pro-
through mitosis, corresponded to Nanney’s (1958a,b) writings duced significant ambiguity across biological fields. As pre-
on the stability of expression states. This prompted Holliday to viously acknowledged by Haig (2004) and others (Bird
redefine epigenetics in a way that was more specific and 2007; Haig 2012; Mann 2014), what we have today is
squarely focused on the inheritance of expression states (while a pronounced dichotomy within the field of epigenetics.
Nanney discussed epigenetic inheritance, his definition of epi- Waddington’s epigenetics describes the interplay of genetic
genetics did not include a specific component on heritabililty). and cytoplasmic elements that produce emergent phenotypes
Holliday (1994) offered two definitions of epigenetics, both of (Van Speybroeck 2002; Jamniczky et al. 2010), and those in
which were admittedly insufficient when taken separately but the biological sciences interested in gene-by-environment
comprehensive in covering all currently acknowledged epige- interactions and phenotypic plasticity use the term in this
netic processes when taken together. The first definition posed sense. As a result, Waddington’s definition is largely used to
that epigenetics was “the study of the changes in gene expres- describe the expression of environmentally mediated phe-
sion, which occur in organisms with differentiated cells, and notypes, particularly in the fields of ecology (Rollo 1994;
the mitotic inheritance of given patterns of gene expression.” Pigliucci 2007; Bossdorf et al. 2008) and physiology
The second stated that epigenetics was “nuclear inheritance, (Jablonka 2004; Aguilera et al. 2010; Ho and Burggren
which is not based on differences in DNA sequence.” Wu and 2010). Those in the field of genetics concerned with DNA
Morris (2001) streamlined Holliday’s definition to state “the methylation, chromatin activity states, chromosomal imprint-
study of changes in gene function that are mitotically and/or ing, centromere function, etc., predominantly use Holliday’s
meiotically heritable and that do not entail change in DNA notion of epigenetics. They are interested in how expression
sequence.” patterns persist across different cells (mitosis) and genera-
The addition of heritability to Waddington’s original defini- tions (meiosis). The phenomena being described by these
tion by Holliday was a significant change. While Waddington’s two groups, and more importantly the mechanisms underly-
definition does not preclude the inheritance of expression states ing them, are vastly different, yet they both use the same
[indeed Waddington (1942a) did briefly discuss heritability in term: epigenetic.
his paper “The Epigenotype”], this aspect was not a fundamen- This ambiguity has made even the simple task of identi-
tal part of his concept of epigenetics. Despite the more thor- fying epigenetic phenomena difficult and also constrains
ough discussion of heritable expression states by Nanney and more advanced pursuits to determine how epigenetic pro-
others, this was the first definition to make heritability a neces- cesses occur. After all, how can scientists effectively study
sary part of epigenetics. a process when they cannot even agree on how to define it?
The implications of Holliday’s redefinition were significant. With the usage of the term epigenetic increasing exponentially
The field soon became a residence for perplexing phenomena across scientific and mainstream literature, one must wonder:
that didn’t fit squarely into other genetic fields and, in many for all the interest and attention epigenetics is receiving, why
regards, the inability to explain these phenomena by simple don’t we have a clearer understanding of it?
genetic explanations became a defining element of epige- The primary challenge is reconciling Waddington’s epige-
netics. Prior to understanding RNA-based regulatory me- netics with Holliday’s epigenetics, because while both exist,
chanisms, and still in early stages of understanding DNA they may not necessarily be related to each other. Is there
methylation and histone modifications, the decoupling of room within one field to entertain both definitions? More-
genotype and phenotype exemplified by epigenetics provided over, do the phenomena underlying each have any business
an attractive refuge because it offered metaphorical language being categorized together, particularly when their connec-
to describe the disconnect between a gene and its phenotypic tion is based more on history and semantics than delibera-
properties. This included occasions where the expression of tion? Answering these questions is important for streamlining
a gene varied depending on its location (such as position the field, facilitating more effective interchanges between
effect variegation in Drosophila or yeast), history (imprint- researchers, and developing clearer research objectives.
ing), or other circumstances (e.g., the establishment of cen- The second challenge lies in addressing the methodolog-
tromeres, telomere healing prior to sequence addition). The ical problems that have accumulated within the field of
thrill and charisma of a “new” genetics initiated a virtually epigenetics over time, due to the absence of a clear def-
unparalleled wave of interest in epigenetics over a very short inition. The principles that provide the foundation of any
amount of time (Cold Spring Harbor Symposium on Quanti- biological field exist to direct research and achieve objec-
tative Biology 2004; Haig 2012). tives within that field; however, without this clear founda-
tion, our desire to understand epigenetics has dictated our
experimental approaches, colored our mechanistic interpre-
The Problem
tations, and allowed us to gloss over inadequacies. Rather
It is not difficult to find articles in the current scientific than building from clear first principles, the field of epi-
literature that use epigenetic to mean any one of the defini- genetics continues to be a catchall for puzzling genetic
tions above, or others entirely. It is futile to argue over the phenomena from which categorizations and justifications
correctness of any one definition; however, it is important to were developed a posteriori. Working backward to reevaluate

Perspectives 889
the first principles of epigenetics will help put the field on surprising, given that these are the portions of DNA responsible
a stronger track and will hopefully allow research to for producing the majority of proteins vital to cell survival and
flourish. function. Repetitive sequences, including those found in the
heterochromatin, are often viewed as less important and
commonly referred to as junk DNA (Ohno 1972; Brosius and
Ruminations on Important Terms: Dependence, DNA Gould 1992; Kapranov and Laurent 2012; Graur et al.
Sequence, and Heritability 2013). The ambivalence toward repetitive sequences likely
Understanding why some genes are turned on or off is certainly stems from the fact that their function is poorly understood,
less mysterious now than when the field of epigenetics was and that the tools for investigating them are undeveloped.
born, largely because of the identification of regulatory gene– The bias toward protein-coding regions and the diffi-
gene and gene–protein interactions. These findings go a long culty in working with repetitive sequences has shaped,
way to explain the changes in gene expression that Wadding- and perhaps limited, our understanding of the role gene
ton termed epigenetics, but the real difficulty is in satisfying sequence plays in gene expression; however, there is
Holliday’s addendum of heritability. These regulatory compo- evidence that other aspects of DNA, aside from the base
nents are all encoded by DNA; however, Holliday’s concep- pair sequence within gene regions, are important for
tualization of epigenetics requires that the status of gene gene expression.
expression, not just the components needed for gene ex- One example is that the expression of a gene can be
pression, be heritable. Also, this phenomenon requires an dependent on other sequences lying outside of the coding
additional mode of inheritance that is not dependent on region (cis- and trans-regulatory elements or repetitive sequen-
DNA sequence. To fully comprehend Holliday’s definition, ces). This makes it difficult to understand, and therefore reject,
we must first make sure that all of the elements are accu- a relationship between gene expression and primary sequence
rately defined. This requires not only taking a critical look because the expression of one gene may be dependent on the
at how Holliday’s description defines the terms depen- primary sequence of another section of DNA (see Figure 1).
dence, DNA sequence, and heritability, but also the range These problems are solved by expanding the definition of
of possible meanings. a gene to include regulatory elements and a rigorous require-
ment to map the genetic locus of regulatory changes. The
Dependence
former is easily accomplished (but often suffers from ambi-
The term dependence carries several potential meanings. In guity and difficulties in precisely determining the boundaries
a strict sense, any molecule that cannot exist in the absence of a gene), while the latter is rarely pursued in epigenetics
of DNA could be considered to be dependent on DNA. literature.
Therefore, any molecule or process that relies on DNA for A second, often overlooked characteristic of DNA se-
its creation, perpetuation, and/or activation is dependent, quence is location, which can impact gene expression in
and this would include any molecule that requires DNA as both coding and noncoding regions. Position-effect variega-
a substrate. From this perspective, anything from DNA meth- tion (PEV) demonstrates that moving a gene sequence to a
yltransferases (DMNTs), which are expressed by specific different location within the genome can affect its expres-
DMNT genes, to histones, which use DNA as a substrate dur- sion (Gowen and Gay 1934; Spofford 1976; Karpen 1994),
ing modification, would be considered dependent on DNA. and in these cases nondependence is still upheld by most
It is likely, however, that Holliday and others would argue epigeneticists as long as no changes occur in the transposed
that this is not the meaning they had in mind when they sequence. But why is the location of a gene sequence viewed
made this distinction. Instead, they refer to dependence in as unimportant? To those who use transgenesis, a common
a stricter sense as the relationship between the location of practice in biology, it is abundantly clear that the location of
a particular chromosomal locus, the specific base pair DNA an inserted transgene has significant effects on its expres-
sequence within that locus, and a reliable expression state sion (Al-Shawi et al. 1990; Wilson et al. 1990). In fact,
(Holliday 1994). For example, Holliday’s argument is that Waddington explicitly promoted the idea of incorporating
the ability of the same DNA sequence to produce different gene position and arrangement as an element of the geno-
expression profiles without a base pair change shows a lack type due to its important effects on expression (Waddington
of dependence on the primary sequence because something 1939).
outside of the sequence must be controlling expression. This A third salient characteristic of DNA sequence is the copy
then requires that we understand what exactly is meant by number of nearby sequences. Studies have shown that
DNA sequence. repeat regions can play important regulatory roles (Lemos
et al. 2008; Zhou et al. 2012) and that the proximity of
DNA sequence
coding regions to repeats (Dorer and Henikoff 1997), as
Many characteristics of DNA sequence are often overlooked well as the size of the repeating regions (Howe et al.
and underappreciated. Most geneticists are primarily con- 1995; Paredes et al. 2011; Sentmanat and Elgin 2012),
cerned with euchromatic regions containing sequences can have unique effects on gene expression and chromatin
that make up genes and encode proteins. This isn’t too structure. This also means that changes in repeat regions,

890 C. Deans and K. A. Maggert

 Figure 1 Imagine the expression of gene A
is dependent on the expression of gene B (a
transcription factor or si/piRNA perhaps). If
we see variable expression in A, but no
change in the sequence of gene A, we
may conclude that this provides evidence
for the expression of A being sequence in-
dependent and a product of epigenetics, as
shown below. However, it is possible that
sequence changes have occurred in gene
B, producing transcriptional changes in A.
This would make the expression of A de-
pendent on the primary sequence of gene
B but not the sequence of A itself. This
makes the task of proving sequence inde-
pendence difficult because you cannot sim-
ply look for sequence changes in the coding
region of the gene in question, but must
also be sure expressional changes aren’t
due to mutations elsewhere on the chromo-
some or other places in the genome.

which are notoriously difficult to detect, must also be ruled to discern between changes in gene expression due to the
out to accurately show sequence independence. inheritance of an expression state and those due to a real-time
reaction to a stimulus. To show that an expression state is
Heritability
inherited, you first need to have a clear understanding of
Perhaps the most important and definitive element found the cause (i.e., stimulus). Knowing the relationship between
among definitions of epigenetics, is the heritability of expres- a given stimulus and its expressional effect(s) is paramount to
sion states. With this addition one could argue that the def- creating a timeline and conclusively showing that a barrier
inition of epigenetics was simultaneously expanded and exists between the two for which inheritance in necessary.
constricted. On the one hand, incorporating heritability into For example, this would entail that a parent cell or organism
the discussion forces us to consider epigenetics on a more experienced a stimulus that caused a specific expression pat-
conceptual level by thinking about the role of time and the tern and then that a similar expression pattern was also evi-
relationship between the stimulus that causes an expres- dent in the offspring without the offspring having ever
sional change and the lasting or fleeting effects of that change. experienced the initial stimulus.
On the other hand, requiring that expressional changes persist While these connections are easy enough to conceptualize,
through mitosis and/or meiosis in order for a phenotype to be they can be difficult to prove empirically, not only because
considered epigenetic drastically reduces the number of obser- gene expression can be capricious, but because in many cases
vations that qualify. For these reasons, this aspect of Holliday’s the stimuli impacting a parent also may impact the germ cells
definition is the most controversial, particularly since it requires residing in the parent, germ cells which will ultimately go on
the acknowledgment of a new mode of inheritance. to produce daughter cells and/or offspring. If the germ cells
From a semantics perspective, the inclusion of heritabil- respond to a stimulus experienced by the parent, no barrier
ity also expands the meaning of the term itself, which has exists between the stimulus and offspring because expression
traditionally related to the transfer of only DNA. Using her- in the primordial cells of the future offspring are also directly
itability to describe the transfer of non-DNA molecules, whether affected. For example, in mammals, any stimuli impacting
they are methyl groups, histones, or cytoplasmic compounds, a pregnant female carrying daughters may impact the mother,
broadens the concept of inheritance in an intriguing way. the fetus, and the germ cells of the fetus, which will go on to
However, Holliday’s definition doesn’t actually delineate the produce offspring (Youngson and Whitelaw 2008; Daxinger
difference between the inheritance of molecules and the trans- and Whitelaw 2012; Dias and Ressler 2014). This means that
fer of molecules, nor does it state what kind of molecules can any stimulus experienced by the mother may also result in
and cannot be inherited. Without this distinction it is very dif- direct exposure to two additional generations of potential
ficult to separate epigenetic phenomena from nonepigenetic offspring. In this scenario, one would have to show a similarity
phenomena, and also to investigate how such modes of inher- in expression between the mother and her great granddaugh-
itance may function. ter to verify a possible epigenetic connection (Skinner 2007;
Holliday’s concept of heritability also produces several Skinner et al. 2013). However, if the expression pattern of the
complications in practice. First, it can be surprisingly difficult original germ cell were apparent in the offspring, it would still

Perspectives 891
satisfy Holliday’s definition, as persistence through mitosis lack the detail to be functionally useful in directing the testing
would have had to occur (Holliday 1994). This has led to some of specific hypotheses, particularly as it relates to the location
clarifications in the identification of epigenetic phenomena, but or site (cytoplasm or nucleus) of epigenetic phenomena. To
those attempts have yet to clearly delineate Waddington’s and mitigate these shortcomings, we advocate defining epige-
Holliday’s views (Youngson and Whitelaw 2008; Berger et al. netics as “the study of phenomena and mechanisms that cause
2009; Grossniklaus et al. 2013; Dias and Ressler 2014). chromosome-bound, heritable changes to gene expression
The primary difficulty lies in identifying the mechanism that are not dependent on changes to DNA sequence.”
of inheritance. Do the compounds responsible for perpetu- We feel that this definition makes a strong distinction
ating an expression pattern have to be closely associated between gene regulation (Waddington’s definition) and epi-
with DNA, as in methylation and chromatin modification, or genetic inheritance (Holliday’s definition), and also empha-
do cytoplasmic compounds qualify? If so, should the transfer sizes that epigenetic phenomena must deal exclusively with
of cytoplasmic compounds really be considered inheritance? chromosome-bound changes. By making these distinctions, we
Waddington stressed the importance of cytoplasmic com- have efficiently separated expressional changes caused by cyto-
pounds and their effect on gene expression (Waddington plasmic compounds, which are more closely tied to gene regu-
1935), yet maternal or transgenerational effects mediated lation, from those which occur on, or in close association to, the
by cytoplasmic transfer from mother to offspring would not chromosome. Doing so makes the focus of the field much clearer
be considered epigenetic under Holliday’s definition because and identifies epigenetic mechanisms more explicitly.
the expression pattern of the offspring is not independent We feel that this definition touches on several important
and simply results from the transfer of cytoplasmic com- elements not encompassed by other definitions, yet commonly
pounds, such as RNA, transcription factors, prions, etc. implied in most uses. To further explain the reasoning behind
(Ptashne 2008; Jarosz et al. 2014). These issues make the our definition, as well as its utility for improving epigenetic
contrast between Waddington’s epigenetics and Holliday’s research, we would like to offer a clarification and a test.
epigenetics much more evident.
The Clarification
In the battle between Waddington and Holliday’s definitions,
Possible Solutions
we have clearly chosen Holliday’s conceptualization, and this
The ambiguity surrounding the field of epigenetics, as well has occurred for two reasons. First, although the usage of
as the historical basis for this definitional confusion, has Waddington’s general definition has increased within nonge-
been discussed by many over the last 15 years (Holliday netic fields, particularly ecology and physiology, to describe
2002, 2006; Jablonka and Lamb 2002; Haig 2004; Bird environmentally mediated phenotypes and trait plasticity, we
2007; Berger et al. 2009; Mann 2014). This has led to the feel that these topics fall more clearly under the heading of
development of several new definitions and terms to help gene regulation. Second, the phenomena that pose the most
clarify the issue. Bird (2007) proposed that epigenetics serious challenges to traditional genetic theory, which dictates
could be redefined as “the structural adaptation of chromo- that identical sequences should behave identically, are ge-
somal regions so as to register, signal or perpetuate altered nomic imprinting, X inactivation in mammals, centromere/
activity states,” a definition that he feels unified Holliday’s telomere establishment and stability (McClintock 1939; Ahmad
requirement for heritability with Waddington’s more gen- and Golic 1998; Barry et al. 2000; Maggert and Karpen 2001;
eral definition. Mann (2014) also advocated keeping a broad Blasco 2007; Black and Cleveland 2011; Mendiburo et al.
notion of epigenetics, but offered the term “memigenetic” to 2011), and perhaps others. Most of the work on these issues
denote expression states that are heritable. Despite these has and continues to occur in the field of genetics, and we
suggestions, a strong working definition for epigenetics believe that the epigenetics fits most appropriately within
has yet to be adopted, and we believe that this largely re- the realm of genetics, given this strong precedent of research.
sults from (1) attempting to combine Waddington’s and That being said, we do want to clarify some points regarding
Holliday’s definitions into one comprehensive term and (2) Holliday’s definition and the current state of the field of
the absence of specific terms within the available definitions epigenetics.
that identify the mechanistic components underlying epige- Holliday’s addendum on heritable expression states arose
netic phenomenon. as a hypothesis to explain the phenomena listed above; how-
We don’t feel that it is possible to reconcile Waddington’s ever, rather than this hypothesis being thoroughly tested, it
focus on gene regulation with Holliday’s more specific crite- quickly perpetuated several new ideas regarding potential
ria within one field and still maintain the level of clarity mechanisms for inheritance (methylation, histone modifica-
needed to produce a useful definition. The efforts to pre- tions, etc.) without strong empirical proof for the necessity
serve a relationship between these two conceptualizations of such mechanisms. Although Holliday’s ideas on the perpet-
have been impaired by the fact that there are just too many uation of expression states and cell memory are innovative
phenomena, with too few mechanistic connections, to cate- and may very well prove to be accurate, we feel an important
gorize into one field. Also, among the definitions that do step in the process of developing these ideas has been over-
maintain the requirement of heritability, we feel that many looked. This is particularly true when the attempts to validate

892 C. Deans and K. A. Maggert

these hypotheses have, as of yet, proved inconclusive. What concerned with the possibility of efficient inducible changes
can it mean to say that DNA methylation is repressive when masquerading as “epigenetic” cases, e.g., mating type switching
activation of a gene removes methylation (e.g., Bird 2002; in yeasts (Haber 1998), VDJ recombination (Blackwell and Alt
Nagae et al. 2011; Hackett et al. 2012; Qian et al. 2012; 1989), repeat-sequence instability (Hawley and Marcus 1989),
Gan et al. 2013; Xie et al. 2013; Bestor et al. 2014)? The and induced mutation (McClintock 1983; Piacentini et al.
search for the mechanism of semiconservative histone mod- 2014); after all, they do bear all of the hallmarks of epigenetic
ifications continues (Deal et al. 2010; Xu et al. 2010; Nakano changes save one: we happen to know their mechanism. For
et al. 2011; Tran et al. 2012; Whitehouse and Smith 2013) that reason, it is critical to refrain from negative claims (that is,
despite evidence that the modifications respond to expression assertions of “no difference”) as implied in “genetically identi-
state rather than control it (Kilpinen et al. 2013; Ptashne cal chromosomes,” when chromosomes have not been se-
2014; Teves et al. 2014). It’s not that histone modification quenced. Ideally, one should be able to make strong positive
and DNA methylation are not correlated with gene expression statements to conclude epigenetic gene regulation is at play.
differences—they are—but the possibility that they may be One can experimentally test for sequence independence
responsive rather than causal has not been disproved (Henikoff using a genetic approach. If we regard an expression state as
2005; Ptashne 2013). We include causation in our definition to a phenotype (and indeed Holliday’s, and Wu and Morris’s
reflect these shortcomings, in acknowledgment of the inade- definitions clearly make mRNA production a phenotype),
quacies in sequencing repeat regions and the conceptualization then it is a simple matter to map a phenotype to the location
of important terms (DNA sequence and heritability) discussed on the chromosome it stems from. In the example of A and B
earlier, and as an attempt to spur research that focuses on these in Figure 1, if the stable expression state of A maps to the
fundamental issues. physical location of A on the chromosome, then we can have
The definition of epigenetics proposed above contains the confidence that the expression state is a consequence of
necessarily vague “gene expression” so as to not exclude some feature (perhaps epigenetic) of A. Subsequent work
a priori any units of inheritance, including protein-encioding showing lack of sequence dependency would confirm epige-
genes, telomeres, centromeres, functional RNA gene prod- netic regulation. If however, the status of A maps to the B locus,
ucts (such as the rRNA, miRNAs, pi/siRNAs, etc), origins of or to the heterochromatin, or even to the nucleoplasm, then
replication, G-quartets, genome instabilities, or anything there is no reason (and in fact no justification) to claim that A’s
else that can manifest a phenotype. Our explicit addition expression state is epigenetic. It is likely instead controlled,
of “chromosome bound” encompasses the already- implied pop- through well-understood mechanisms, e.g., by the presence
ular use of the term epigenetic, where local changes in gene of another factor (Ptashne 2013; Serra et al. 2014; Struhl
expression are induced and inherited at the specific gene being 2014). In these cases, there is nothing meaningfully “depen-
regulated. This explicit statement added to Holliday’s (1994) dent” about the “sequence” of A in terms of its regulation.
definitions, later merged by Wu and Morris (2001), assures At an ideal extreme, identical reporter sequences should
two things. First, that epigenetics is not inferred from cyto- be placed in the same nucleus (through transgenesis or mating).
plasmic or nucleoplasmic factors, e.g. perdurance of a protein- If a regulatory change is epigenetic, then those sequences
aceous transcription factor (Ptashne 2013). Second, that should (or could) behave differently, each independently
heritable memory (rather than “inheritance”) is an explicit maintaining a memory of their states. This idea is the in-
property of epigenetic gene regulation. The most heavily cited tellectual foundation of the search for heritable histone
examples of epigenetic phenomena (e.g., genomic imprinting) modifications, DNA methylation, etc., yet is rarely directly
fulfill these criteria, and other cases that are more dubious tested. Strikingly, and underscoring our concern, in a few
(e.g., stress-sensitivity in offspring of stressed pregnant mam- cases where data have been presented, the idea of allele-
mal mothers) are excluded until better understood. specific memory is either not tested or is directly refuted
(Anway et al. 2005; Pembrey et al. 2006; Greer et al. 2011;
The Test(s)
Crews et al. 2012; Stern et al. 2012; Voutounou et al. 2012;
To make the strong claim of sequence independence, one Buescher et al. 2013; Padmanabhan et al. 2013; Wan et al.
must assure that there are no changes to any sequence in cis 2013; Gapp et al. 2014).
or in trans to the gene whose expression is being monitored. These conditions—nonsimilar behavior of identical se-
Ideally, one would sequence the entire genome, yet this is quences, mapping of the epigenetic state—are implied by
impractical on many grounds, not least of which are the most uses of the term epigenetic. Importantly, they are
large blocks of repetitive heterochromatin on most chromo- taken to imply a great deal about how gene expression
somes, which modern molecular biology cannot assemble works, suggesting that there is an entire layer of gene reg-
(and thus modern molecular biologists tend to ignore). In- ulation that we are only now becoming aware of. Or is
stead, careful (and laborious) work, such as that done by there? Before we rewrite the textbooks, divert funding ini-
some (Brink 1956; Clark and Carbon 1985; Steiner and tiatives, refocus our disease intervention strategies, or alter
Clarke 1994; De Vanssay et al. 2012) showing frequent our view of neo-Darwinian biology, it is our obligation to
switching, should be considered strong evidence in the place attempt these simple tests to assure ourselves that we are not
of exhaustive sequencing. We must, however, always be chasing a ghost.

Perspectives 893
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