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Exercise-associated DNA methylation change in
skeletal muscle and the importance of imprinted
genes: a bioinformatics meta-analysis
William M Brown
▸Additional material is
published online only. To view
please visit the journal online
(http://dx.doi.org/10.1136/
bjsports-2014-094073).
Correspondence to
Dr William M Brown,
Department of Sport Science
and Physical Activity, Faculty of
Education and Sport, Institute
of Sport and Physical Activity
Research, University of
Bedfordshire, Polhill Avenue,
Bedford, Bedfordshire,
MK41 9EA, UK;
william.brown@beds.ac.uk
Accepted 18 February 2015
To cite: Brown WM. Br J
Sports Med Published Online
First: [please include Day
Month Year] doi:10.1136/
bjsports-2014-094073
ABSTRACT
Background Epigenetics is the study of processes—
beyond DNA sequence alteration—producing heritable
characteristics. For example, DNA methylation modifies
gene expression without altering the nucleotide
sequence. A well-studied DNA methylation-based
phenomenon is genomic imprinting (ie, genotype-
independent parent-of-origin effects).
Objective We aimed to elucidate: (1) the effect of
exercise on DNA methylation and (2) the role of
imprinted genes in skeletal muscle gene networks
(ie, gene group functional profiling analyses).
Design Gene ontology (ie, gene product elucidation)/
meta-analysis.
Data sources 26 skeletal muscle and 86 imprinted
genes were subjected to g:Profiler ontology analysis.
Meta-analysis assessed exercise-associated DNA
methylation change.
Data extraction g:Profiler found four muscle gene
networks with imprinted loci. Meta-analysis identified 16
articles (387 genes/1580 individuals) associated with
exercise. Age, method, sample size, sex and tissue
variation could elevate effect size bias.
Data synthesis Only skeletal muscle gene networks
including imprinted genes were reported. Exercise-
associated effect sizes were calculated by gene. Age,
method, sample size, sex and tissue variation were
moderators.
Results Six imprinted loci (RB1,MEG3,UBE3A,
PLAGL1,SGCE,INS) were important for muscle gene
networks, while meta-analysis uncovered five exercise-
associated imprinted loci (KCNQ1,MEG3,GRB10,
L3MBTL1,PLAGL1). DNA methylation decreased with
exercise (60% of loci). Exercise-associated DNA
methylation change was stronger among older people
(ie, age accounted for 30% of the variation). Among
older people, genes exhibiting DNA methylation
decreases were part of a microRNA-regulated gene
network functioning to suppress cancer.
Conclusions Imprinted genes were identified in
skeletal muscle gene networks and exercise-associated
DNA methylation change. Exercise-associated DNA
methylation modification could rewind the ‘epigenetic
clock’as we age.
Trial registration number CRD42014009800.
INTRODUCTION
British developmental biologist Sir Conrad H
Waddington introduced the term ‘epigenetics’as a
science of development from genotype to pheno-
type.
1
However, the term ‘epigenetics’had an inde-
pendent origin and meaning, which led to a
conflation of terms.
2
Recall Waddington’s use of
the term ‘epigenetics’to refer to the causal pro-
cesses of development, with an emphasis on inter-
actions among genes and between genes and the
environment. In contrast, Nanney
3
used ‘epigen-
etic’in 1958 to describe a system of cellular hered-
ity that was not based on DNA sequence.
Most molecular biologists use the term epigenet-
ics to mean the study of heritable changes in gene
expression or cellular phenotype caused by
mechanisms other than changes in the underlying
DNA sequence. The epi in epigenetic is Greek for
‘over’,‘above’or ‘outer’. Examples of such changes
are DNA methylation and histone modifications
(figure 1), both of which can regulate gene expres-
sion without altering the underlying DNA
sequence. One well-studied epigenetic phenomenon
based on DNA methylation in mammals (and
notably humans) is genomic imprinting.
4
One
purpose of this review is to introduce this term to
the sports and exercise medicine/physiotherapy
community and explain its importance.
Genomic imprinting
Genomic imprinting is defined as genotype-independ-
ent parent-of-origin gene expression. Specifically, for
most genes we inherit two working parental copies.
However, in the case of imprinted genes, an epigen-
etic tag (via DNA methylation) is placed on either the
maternal or paternal copy rendering the other
inactive. Such parent-of-origin gene expression is
mediated by epigenetic modifications which differ
between the two parentally derived chromosomes.
5
Approximately 1% of the human genome is
imprinted.
67
Despite their rarity, imprinted genes are
of great medical importance. Studies of metabolic
growth and neurodevelopmental disorders have
shown that imprinted genes are absolutely essential
for healthy development.
8–13
Furthermore, epigenetic
dysregulation in imprinted genes—which often have
growth-enhancing and tumour suppressor functions
—predict disease and cancer outcomes.
Once epigenetic mechanisms emerged during
mammalian evolution (eg, genomic imprinting), a
source of environmental information (ie,
parent-of-origin of a gene) was transmitted transge-
nerationally. Imprinting machinery (eg, DNA
methylation, see figure 1—a type of silencer at a
gene’s promoter—places marks on a gene when the
gametes are produced) allowed for biased gene
expression in the subsequent generation (ie, mono-
allelic gene expression).
The realisation that the environment has pro-
found influences on the epigenome has led to a
strong hypothesis that exercise can also affect DNA
Brown WM. Br J Sports Med 2015;0:1–13. doi:10.1136/bjsports-2014-094073 1
Review
methylation and have long-term health outcomes. Specifically,
we propose that the same mechanism that controls genomic
imprinting in mammals (ie, DNA methylation) allows for
phenotypic modification and the possibility of multiple sources
of environmental information (eg, exercise, nutrition) to be
transmitted to the next generation. Muscle and nerve cells share
the property of responding to electrochemical/environmental
stimuli and thus are ideal epigenetic interfaces for transgenera-
tional phenotypic modification, which may explain in part why
imprinted genes are particularly involved in neural
development.
DNA methylation
DNA methylation refers to the adding of a methyl group on a
cytosine base. It occurs primarily in the context of CpG dinu-
cleotides, which cluster in regions called CpG islands.
14
‘CpG’
refers to regions of DNA where a cytosine nucleotide appears
next to guanine nucleotide interconnected by phosphate. When
cytosines in CpG dinucleotides are methylated to form
5-methylcytosine, a gene can be turned off. CpG dinucleotides
are rare in mammals (∼1%), but ∼50% of gene promoters are
linked to CpG islands which are often unmethylated in healthy
cells. Cells become methylated in a tissue-specific and age-
specific manner during development. Where DNA methylation
occurs can be critical for its effect. DNA methylation (ie, adding
methyl groups to a cytosine base, figure 1) at a gene’s promoter
is linked to silencing (ie, less gene expression); in contrast, DNA
methylation outside the promoter region (eg, gene body) is
sometimes associated with increased gene expression. In the
case of genomic imprinting, hypermethylation of one of the
two parental alleles leads to monoallelic expression (conceptu-
ally similar to gene-dosage reduction in X-inactivation, see
figure 2). DNA methylation has been implicated in cancer, neu-
rodevelopmental disorders and autoimmune diseases.
15
Thus, if
exercise can influence DNA methylation, it may be the mechan-
ism that underpins the lower cancer rate in those who are phys-
ically active.
DNA methylation, health and diverse disease states
Cancer cells are characterised by a global loss of DNA methyla-
tion among growth enhancers; and the coordinated acquisition
of hypermethylation at the CpG islands of tumour suppressor
genes. Global hypomethylation occurs primarily at parasitic
DNA regions of the genome. For example, the LINE family
member L1 is hypomethylated in a variety of cancers, such as
those of the breast and colon.
16
Neurological disorders are also associated with epigenetic dys-
regulation (ie, reversed patterns of a normal DNA methylation
profile). Specifically, dysregulation of DNA methylation occurs
in several neurological diseases, giving rise to hypermethylated
and hypomethylated CpG sites. FMR1 promoter hypermethyla-
tion occurs among individuals diagnosed with Fragile X
Figure 1 DNA methylation: DNA methylation occurs when methyl groups ‘tag’DNA and activate or repress gene expression. DNA methyltransferase
is a catalyst, which transfers methyl groups to DNA. Silencing of a gene activity can occur if the hydrogen (H) molecule of cytosine (C) is replaced by a
methyl (Me) group at a gene’s promoter. Histones: Histones are proteins around which DNA can wind for compaction and histone modification can
regulate gene activity. Both epigenetic processes (ie, DNA methylation and histone modifications) affect health resulting in cancer, autoimmune
disease, neurological disorders or diabetes. Image modified from the National Institutes of Health, Benjamin I. Laufer and Forluvoft.
2 Brown WM. Br J Sports Med 2015;0:1–13. doi:10.1136/bjsports-2014-094073
Review
syndrome. Rett syndrome, an X-linked neurological disease, is
caused by point mutations in MECP2, which encodes a methyl
binding protein and is proposed to be a gene silencer.
17
DNA methylation dysregulation is associated with auto-
immune disease. For example, the Immunodeficiency
Centromeric Instability and Facial Anomalies (ICFA) syndrome
is caused by heterozygous mutations in DNMT3B. Individuals
with ICFA show DNA hypomethylation among Alu repeats.
Interestingly, despite patients with ICFA having normal global
DNA methylation profiles, key developmental regulatory and
immune function genes show loci-specific epigenetic
dysregulation.
15
Whether it is cancers, neurological disorders or autoimmune
disease, DNA methylation and imprinted genes are emerging
causal factors. Some imprinted genes are involved in multiple
phenotypes,
18
suggesting that imprinting performs a regulatory
function during ontogeny (eg, regulation of other genes).
The current paper argues that imprinted genes are important
for skeletal muscle development and their phenotypic effects
reflect an underlying ancestral tug of war between parental
genomes over offspring growth and developmental trajectories.
Imprinted genes and skeletal muscle gene networks:
growth suppression and enhancement
Imprinted genes are genes whereby an epigenetic mark is laid
down during gametogenesis, indicating a key environmental
source of information, the parental origin of a particular gene.
There are few imprinted genes in the human genome, but they
are often associated with growth, neural functioning and behav-
iour. Parental antagonism theory
19
is currently the best theory
Figure 2 (A) Genomic imprinting: Genomic imprinting is parent-of-origin (but genotype independent) gene expression. When males and females
produce gametes (ie, sperm or eggs) an epigenetic mark (eg, DNA methylation, which silences one of the parental alleles) is placed on the DNA to
indicate parent-of-origin. Regardless of sex of offspring imprinted genes affect growth and neural development differentially by parent-of-origin.
Once the child produces its own gametes the imprints are erased and new parent-of-origin marks are established. Imprinted genes are rare but have
profound effects on growth and neurodevelopment. (B) X-inactivation: X-inactivation is a process by which one parental copy of the X chromosome
in women is randomly deactivated. X-inactivation prevents females from having twice as much X chromosome gene production as males (which
only have one copy of the X chromosome). Once the X chromosome is deactivated, it remains silent throughout the cell’s lifetime. Compared with
transcriptionally active X chromosome, the inactive X has higher levels of DNA methylation, which is associated with gene silencing. One difference
between imprinting and X-inactivation is the former is not a random process with respect to which parental allele is epigenetically silenced.
Brown WM. Br J Sports Med 2015;0:1–13. doi:10.1136/bjsports-2014-094073 3
Review
for the phenotypic effects of growth regulators (eg, the paternal
genome within offspring fosters growth at a cost to the maternal
genome, while the maternal genome attempts to minimise these
costs by suppressing growth).
Haig’s model
19
proposes that imprinting evolved as a result
of opposing fitness interests of parental genomes. For example,
in polygamous species, patrigenes (genes expressed within off-
spring inherited from the father) favour fetal growth at the
expense of depleting maternal resources and disadvantaging
future offspring. Meanwhile, matrigenes (genes expressed
within offspring inherited from the mother) will oppose the
paternal effect and conserve resources to optimise inclusive
fitness of the mother and future offspring. Haig’s theory
19
pre-
dicts that paternally expressed imprinted genes will often
promote growth, while maternally expressed genes will have
opposite effects to reduce costs on matrilineal inclusive fitness.
IGF2—an imprinted gene and paternally expressed in
humans—regulates muscle development. IGF2 is upregulated
early in MyoD-induced in myocyte differentiation and IGF2
inhibition leads to reduced expression of MyoD target genes,
which suggests that IGF2 is essential for amplifying and main-
taining MyoD efficacy.
20
IGF2’s role as a paternally derived skel-
etal muscle growth enhancer is consistent with the theoretical
orientation of this paper.
Germane to sports medicine, some imprinting disorders affect
muscle growth. For example, Angelman (AS) and Prader-Willi
syndromes (PWS) are imprinting disorders affecting muscle
development and health. PWS is caused by an overexpression of
maternal genes on chromosome 15, while AS is due to an overex-
pression of paternal genes. Muscle biopsies of 11 PWS children
have been investigated using histochemical and morphometric
methods.
21
The phenotypic abnormalities included (A) fibre size
variation of both type 1 and 2 fibres, (B) type 2 fibre atrophy, (C)
increased numbers of type 2C fibres and (D) decreased numbers
of type 2B fibre. This finding is consistent with the overexpres-
sion of maternal genes suppressing skeletal muscle growth. In
addition to their low muscle tone, PWS individuals experience
chronic hunger, potentially leading to overeating and obesity.
DNA methylation and imprinted loci influence muscle hyper-
trophy—extremely muscled hindquarters—in callipyge sheep.
22
Hypomethylation of Clpg1 causes muscle hypertrophy, in part due
to the overexpression of Dlk1,
22
a paternally expressed imprinted
gene associated with muscle precursor cell (myoblast) differenti-
ation.
23
DNA demethylation promotes skeletal myotube matur-
ation.
24
Early experiments in the 1970s showed that DNA
methyltransferase inhibitors (eg, 5-azacytidine) induced transdif-
ferentiation of fibroblasts into myoblasts.
25
More recently, in
C2C12 culture, Hupkes et al
24
noticed that on treatment with the
methylation inhibitor (ie, 5-azacytidine), myotubes spontaneously
acquired repetitive membrane activity, intracellular calcium transi-
ents and contractility. Hupkes et al
24
suggested that DNA methyla-
tion may pose an epigenetic barrier to C2C12 myotubes reaching
maturity. However, when imprinted genes are involved in skeletal
muscle development, the so-called ‘DNA methylation barrier’will
likely be parent-of-origin dependent. Beyond the distinct possibil-
ity that imprinted genes coordinate mammalian skeletal muscle
development (eg, regulating skeletal muscle gene networks), it
remains to be investigated whether the DNA methylation of
imprinted loci are responsive to human exercise.
Exercise epigenetics and DNA methylation
Traditionally, exercise biologists envision biological systems
changing by the regulation of protein synthesis (eg, alteration of
receptor expression or intracellular signalling). Since
transcription precedes translation, it is often at the level of the
‘transcriptome’that adaptations can be tracked at the molecular
level. Subtle changes in gene transcription occur through epi-
genetic regulatory machinery. A variety of epigenetic mechan-
isms allow for transcriptional activation and specification of cell
identity, maintaining homoeostasis and responding to environ-
mental conditions. These epigenetic mechanisms encompass
DNA methylation, post-translational histone modifications and
microRNA (figure 3). Much of the previous research on envir-
onmental epigenomics involves nutrition; however, exercise
physiology is coming to the forefront. There is evidence that
DNA methylation can change due to short bouts of exercise (eg,
exercising to exhaustion
26
) and longer, more sustained exercise
regimens (eg, 6 months of controlled walking
27
). For example, a
high-intensity interval walking regimen increased DNA methyla-
tion of the proinflammatory gene ASC (apoptosis-associated
speck-like protein containing a caspase recruitment domain)
among older adults nearly to the levels of healthy younger
adults.
28
Among breast cancer sufferers, DNA methylation
changes in L3MBTL1 (an imprinted and possible tumour sup-
pressor gene) due to exercise (eg, brisk walking on treadmill)
has been demonstrated.
29
Exercise epigenetics: research questions
What is the average effect of exercise on changes in DNA
methylation and does age, research design, sample size, sex or
tissue heterogeneity influence the size of the effect? Are
imprinted genes implicated in skeletal muscle gene networks
and exercise-associated DNA methylation changes in humans?
Since imprinted genes regulate adiposity, energy expenditure
and glucose homoeostasis, it was hypothesised that imprinted
genes will be involved in human skeletal muscle gene networks
and targets of exercise-associated DNA methylation change. To
test these hypotheses, gene ontology and meta-analytic method-
ologies were utilised.
METHODS
A human skeletal muscle gene network: testing the
importance of imprinted genes
It is hypothesised that human skeletal muscle growth is regu-
lated by imprinted genes. A gene ontology networking web
server called g:Profiler
30 31
was used to assess the functional
involvement of imprinted genes for skeletal muscle gene net-
works. First, human skeletal muscle genes were determined
using the Xavier laboratory gene enrichment profiler
32 33
(figure 4). To test the hypothesis that imprinted genes are impli-
cated in skeletal muscle gene networks, g:Profiler
30 31
was used.
Human imprinted genes (29 maternally expressed and 57 pater-
nally expressed) were selected from http://www.geneimprint.
com. Imprinted genes were combined with 25 skeletal muscle
genes (figure 4).
Meta-analysis on exercise-associated DNA methylation
change
This meta-analysis was limited to English as no foreign language
results were found. Only published papers measuring DNA
methylation and exercise in humans were used. The following
search strings were entered into PubMed (1968 to 2 May 2014):
(1) ‘DNA methylation and exercise’; and (4) ‘DNA methylation
and physical activity (human-only)’. Search results finalised by
the author (figure 5). Both PRISMA (see online supplementary
file 1) and MOOSE (see online supplementary file 2) guidelines
followed. One author did not respond to a direction of effect
query. Reference lists did not yield additional articles.
4 Brown WM. Br J Sports Med 2015;0:1–13. doi:10.1136/bjsports-2014-094073
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RESULTS
Imprinted genes and skeletal muscle gene networks
Table 1 shows the imprinted genes associated with skeletal
muscle gene networks. Six imprinted genes (ie, three maternally
expressed genes RB1,MEG3 and UBE3A and three paternally
expressed genes INS,PLAGL1 and SGCE) were revealed to be
part of the gene networks of highly enriched skeletal muscle
loci. Considering the rarity of imprinted genes in the human,
this is biologically significant. Below is a description of each
imprinted gene involved.
Paternally expressed genes linked to muscle-related
phenotypes
1. Pleomorphic adenoma gene-like 1 (PLAGL1): As seen in
table 1,PLAGL1 is part of two gene ontology networks:
‘muscle organ development’(GO: 0007517) and ‘skeletal
muscle tissue development’(GO: 0007519). PLAGL1
encodes a zinc finger protein with transactivational and
DNA-binding functions. PLAGL1 has antiproliferative prop-
erties making it a candidate for functioning as a tumour sup-
pressor gene. Overexpression of this gene during fetal
development underlies transient neonatal diabetes mellitus
(TNDM). In most tissues (eg, skeletal muscle), PLAGL1
appears to be expressed from the paternal allele.
34
2. Sarcoglycan, epsilon (SGCE): As seen in table 1,SGCE is
part of a gene ontology network called ‘muscle organ devel-
opment’(GO: 0007517) and a human phenotype gene
network called ‘abnormality of the musculature of the neck’
(HP: 0011006). SGCE encodes the epsilon member of the
sarcoglycan family (ie, transmembrane proteins which are
part of the dystrophin-glycoprotein complex linking the
actin cytoskeleton to the extracellular matrix). Epsilon sarco-
glycan is more broadly expressed (ie, not just restricted to
striated muscle). Mutations in this gene are associated with
the myoclonus-dystonia syndrome and it is imprinted ( pref-
erentially expressed from the paternal copy).
34
3. Insulin (INS): As seen in table 1,INS is part of a human
phenotype gene network called ‘motor delay’(HP: 001270).
The INS gene encodes for proinsulin (a prohormone precur-
sor to insulin), which is post-translationally cleaved into
three peptides. Binding of insulin to the insulin receptor
(INSR) stimulates glucose uptake. A multitude of mutant
alleles with phenotypic effects have been identified. Notably,
INS-IGF2, a read-through gene, aligns to the INS gene,
whereby INS is at the 5’region and IGF2—an extremely
well-studied growth regulatory imprinted gene—is at the 3’
region.
34
Maternally expressed genes linked to muscle-related
phenotypes
1. Retinoblastoma 1 (RB1): As seen in table 1,RB1 is part of
two gene ontology networks: ‘muscle organ development’
(GO:0007517) and ‘skeletal muscle tissue development’
(GO:0007519). RB1 encodes a protein that negative regu-
lates cell cycle and was the first tumour suppressor gene dis-
covered. The encoded protein maintains overall chromatin
structure. Defects in RB1 cause childhood retinoblastoma
(RB), bladder cancer and osteogenic sarcoma.
34
2. Maternally expressed 3 non-protein coding (MEG3): As seen
in table 1,MEG3 is part of two gene ontology networks:
‘muscle organ development’(GO: 0007517) and ‘skeletal
muscle tissue development’(GO: 0007519). MEG3 is
expressed in many healthy tissues, but expression is lost in
multiple cancer cell lines of various tissue types. Notably,
MEG3 suppresses tumour cell proliferation in vitro and
interacts with tumour suppressor p53. Deleting MEG3
enhances angiogenesis in vivo. Many studies show that
MEG3 is a long non-coding RNA tumour suppressor.
34
Figure 3 MicroRNAs (miRNAs): A miRNA is a small (ie, approximately 19 to 25 nucleotides in length) non-coding RNA molecule functioning to
silence RNA and involved in the post-transcriptional modification of gene expression. miRNA should not be confused with messenger RNA. miRNAs
are another important molecular epigenetic regulator. miRNAs can result in small, but important reductions in physiologically relevant gene
expression by blocking translation. Modified from Kelvin Song.
Brown WM. Br J Sports Med 2015;0:1–13. doi:10.1136/bjsports-2014-094073 5
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Figure 4 Enrichment profile for
selected human skeletal muscle genes
(defined as genes that have high
enrichment scores within tissue and
between tissues). Embryonic stem cells,
t cells, b cells and myeloid gene
loadings have been removed from heat
map.
32 33
6 Brown WM. Br J Sports Med 2015;0:1–13. doi:10.1136/bjsports-2014-094073
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3. Ubiquitin protein ligase E3A (UBE3A): As seen in table 1,
UBE3A is part of a human phenotype gene network called
‘motor delay’(HP:001270). UBE3A encodes an E3
ubiquitin-protein ligase. This imprinted gene is maternally
expressed in the brain and most likely biallelically expressed
in skeletal muscle. Maternally inherited deletion of this gene
causes a neurodevelopmental disorder AS which is charac-
terised by severe motor and intellectual impairments, ataxia,
hypotonia, epilepsy and absence of speech. UBE3A’sprotein
in part causes ubiquitination and proteolysis of tumour
protein p53.
34
Meta-analysis of exercise-associated DNA methylation
change
To determine if there is a directional bias in exercise-associated
methylation change, a binomial signed test was conducted.
Two-hundred and eighty-seven genetic elements (out of 478)
showed significantly decreased DNA methylation after exercise
(binomial test p<0.001). Online supplementary table S2 reports
the 478 genetic elements showing exercise-associated DNA
methylation change, 5 of which are imprinted genes (maternally
expressed (GRB10,KCNQ1,MEG3) and paternally expressed
(PLAGL1,L3MBTL)). Despite appearing like a small percentage
of imprinted loci, this is much higher than the expected number
of 1–2 (ie, assuming there are ∼90 imprinted genes in a human
genome containing ∼22 300 genes): binomial test p<0.03. All
imprinted genes showed a decrease in DNA methylation after
exercise, except for GRB10 and KCNQ1 (adipose tissue only).
Table 2 provides location, ontology and growth-related effects
for the imprinted genes showing exercise-associated DNA
methylation change. Unfortunately, it was not possible to deter-
mine if exercise-associated DNA methylation change in the
imprinted loci was near differentially methylated regions
(DMRs) as each imprinted gene in table 2 has clinically relevant
single-nucleotide polymorphisms. Among the five imprinted
genes revealed by the meta-analytic search in table 2, DNA was
extracted from diverse tissues (ie, skeletal muscle, adipose and
blood).
Figure 5 PRISMA flow diagram for meta-analysis component of paper. Search terms used were ‘DNA methylation exercise’or ‘DNA methylation
physical activity’. Human in vivo studies only (May 2014).
Brown WM. Br J Sports Med 2015;0:1–13. doi:10.1136/bjsports-2014-094073 7
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Degree of exercise-associated DNA methylation change:
effect of age and confounded factors
The effect size of exercise on DNA methylation for the 478
genetic elements (387 were unique genes) across 16 different
publications and 1580 people—see online supplementary table
S2)—is large (mean Cohen’s d=1.20±1.20; 95% CI of the
mean 1.10 to 1.31) and significantly different from a test value
of zero (ie, no effect of exercise on DNA methylation): one-
sample t(477)=22.77, p<0.001. Analysis of covariance
(ANCOVA) revealed that the effect size of exercise-associated
DNA methylation change was significantly greater for people
over 40 years of age (Cohen’s d=2.89±1.97) compared with
those under 40 years of age (Cohen’s d=0.90±.51): (F(1,471)
=197.26, p<0.001, partial η
2
=0.30). In this model, the effect
of age was independent of research design (experimental
designs’larger effect size, p<0.001), sample size (smaller studies’
larger effect sizes, p<0.001), sex (larger effect size among
females than males, p<0.03) and tissue specificity (larger effect
sizes in tissue with more cell types, p<0.001). The fact that
sample size and effect size are significantly and negatively corre-
lated suggests the presence of publication bias: r (477)=−0.11;
p<0.02. Sample size has been included as a covariate in analyses.
Online supplementary table S2 shows that most of the genes
decreased in DNA methylation percentage after exercise (238/
387 different genes). ANCOVA (sample size controlled) found a
direction of change by age interaction: F(1,381)=20.14,
p<0.001, partial η
2
=0.05. Specifically, among older people
(people older than 40 years of age), the effect size was signifi-
cantly larger (p<0.05) when DNA methylation increased with
exercise (3.85; 95% CI of the mean 3.32 to 4.38) compared
with when DNA methylation decreased with exercise (3.04,
95% CI of the mean 2.45 to 3.63). However, the reverse was
true for people under 40 years of age. Specifically, among
younger people (people less than 40 years of age), the effect
size was significantly smaller ( p<0.05) when DNA methylation
increased with exercise (0.90; 95% CI of the mean 0.83 to
0.97) compared with when DNA methylation decreased with
exercise (1.00, 95% CI of the mean 0.95 to 1.05).
To elucidate the possible function of this interaction among
older people, the genes showing increases and decreases after
exercise were exposed to g:Profiler
30 31
for ontology analysis by
age. As seen in table 3, among older people the genes that
increased in DNA methylation after exercise were associated
with growth regulation (GO:0022603), and the genes that
become less methylated after exercise are targets of a putative
tumour suppressing microRNA miR-519B. Two imprinted genes
(L3MBTL1,PLAGL1)—both of which are tumour suppressors
—are associated with miR-519b’s microRNA network.
Among younger individuals, a microRNA-regulated gene
network involved in stem cell activity was implicated.
Specifically, as seen in table 4, genes that increased in DNA
methylation among younger people were part of a
microRNA-regulated (hsa-miR-130b*) gene network that sup-
presses stem cell activity. Statistically significant (all p values
<0.04) gene networks were uncovered for the genes that
decreased in DNA methylation after exercise among younger
people. Specifically, these gene networks are important for the
biological processes of the extracellular matrix, skeletal muscle
and cartilage development (table 4).
Tissue heterogeneity
Tissue type is a moderator of the degree of exercise-associated
DNA methylation change. To further elucidate this apparent
moderator, a one-way ANCOVA (controlling for sample size)
was conducted and found significant: F(4,471)=137.03,
p<0.001, partial η
2
=0.54 (figure 6A). As seen in figure 6A,
exercise-associated DNA methylation change was greater in
blood samples compared with all tissue types (ie, buccal and
salvia, breast and adipose, skeletal muscle and gastric tumour).
For three out of five tissue types, the effect sizes are large. For
buccal cells and saliva and gastric tumours, the effect sizes were
medium.
Table 1 Imprinted genes networked with human skeletal muscle genes and network function
Function ID p Value Genes PAT MAT
Muscle organ development GO: 0007517 0.0005 PLAGL1, SGCE, RB1, MEG3, MYL1, NEB, ACTA1,
TTN, MYLPF, RYR1, CASQ1
PLAGL1, SGCE RB1, MEG3
Skeletal muscle tissue development GO: 0007519 0.05 RB1, MEG3, PLAGL1, ACTA1, MYLPF, RYR1, CASQ1 PLAGL1 RB1, MEG3
Motor delay HP: 001270 0.03 UBE3A, INS, NDN, NEB, ACTA1, TTN, TNNT1, TPM3, RYR1 INS UBE3A
Abnormality of the musculature of the neck HP: 0011006 0.05 SGCE, NEB, ACTA1, TTN, TPM3 SGCE –
Networks determined by g:Profiler.
30 31
Static url: http://tinyurl.com/imprinting-skm.
GO, gene ontology; HP, human phenotype; MAT, maternally expressed gene; PAT, paternally expressed gene.
Table 2 Imprinted genes that showed DNA methylation changes associated with exercise
Gene Ontology Expression Chromosome Start End Growth
PLAGL1 Cell differentiation skeletal muscle; apoptosis Paternal 6 144 328 445 144 328 885 Enhancer
GRB10 Insulin receptor pathway (negative regulation) Maternal skeletal muscle γ
splice variant
7 50 850 662 50 851 107 Suppressor
KCNQ1 Cardiovascular system development; negative regulation of insulin
secretion; gene silencing
Maternal 11 2 465 914 2 870 339 Suppressor
MEG3 Negative regulation of angiogenesis; cell proliferation, positive
regulation of skeletal muscle fibre development
Maternal 14 101 293 947 101 294 390 Suppressor
L3MBTL Regulation of megakaryocyte differentiation Paternal 20 42 142 508 42 142 820 Enhancer
Function, parent-of-origin, differentially methylated region (DMR) location and hypothesised growth effects. Location of DMRs courtesy of Randy Jirtle. For KCNQ1, the DMR
48
resides in
intron 10. Genome Reference Consortium Human Build 37 (GRCh37).
8 Brown WM. Br J Sports Med 2015;0:1–13. doi:10.1136/bjsports-2014-094073
Review
Exercise type
Exercise type is a potential moderator of the degree of DNA
methylation change. To further elucidate the effect of exercise
type on the degree of DNA methylation change, a one-way
ANCOVA (controlling for sample size) was conducted and
found significant: F(2,453)=126.11, p<0.001, partial η
2
=0.36
(figure 6B). As seen in figure 6B, DNA methylation change was
significantly greater among those engaged in Tai Chi and
walking compared with those engaged in cycling. Walking and
Tai Chi were not significantly different from one another
(p=0.25). Regardless of the specific type of exercise, effect sizes
were large.
DISCUSSION
Imprinted genes and skeletal muscle gene networks
As predicted, imprinted genes were implicated in skeletal muscle
gene networks. Table 1 is consistent with the hypothesis that
parental genomes act simultaneously to suppress and promote
skeletal muscle growth. The argument is that for skeletal muscle
the maternal genes in table 1 (RB1,MEG3 and UBE3A) suppress
growth, while paternal genes (INS,PLAGL1 and SGCE)
perform antagonistic functions (ie, growth enhancement).
Some imprinted genes expected based on non-human animal
research were conspicuously absent from the human skeletal
muscle gene networks. For example, in sheep, imprinted gene
H19 regulates muscle development.
35
H19 is also known as
ASM for ‘adult skeletal muscle’.H19 is a negative regulator of
prenatal growth and bovine muscle development.
35
H19 is
maternally expressed at high levels in embryonic and fetal skel-
etal muscle and is located closely downstream of paternally
expressed IGF2 performing antagonistic functions (ie, growth
enhancement).
Beyond H19, other imprinted genes studied in non-human
animals were absent from the human skeletal muscle gene net-
works, such as DLK1, which is a well-known imprinted gene
and paternally expressed muscle growth enhancer. In mouse
skeletal muscle cultures, the genetic ablation of Delta-like
1homolog (Dlk1) causes reductions in skeletal muscle mass, in
part due to myofiber number loss and myosin heavy chain IIB
gene expression.
36
GRB10 was another imprinted gene missing
from g:Profiler’s human skeletal muscle gene networks. In mice,
Grb10 has a tissue-dependent imprinting status (ie, paternally
expressed in the brain and maternally expressed in muscle, see
Garfield et al
37
for its links to behaviour). Holt et al
38
have
found evidence that when Grb10 is deleted, hypermuscularity
overgrowth occurs, suggesting that maternal gene expression
functions to suppress muscle growth. The same pattern occurs
in human skeletal muscle.
39
Considering the rarity of imprinted genes, it is remarkable
that six imprinted genes were discovered as part of skeletal
muscle gene networks. Imprinted genes most likely repress,
maintain and induce muscle-specific transcription during myo-
genesis. Future studies should investigate epigenomic antagon-
isms between paternally and maternally derived genes during
myogenesis, as opposed to assuming that decreased methylation
invariably leads to growth. Owing to the importance of
imprinted genes for skeletal muscle development (table 1), it
was hypothesised that imprinted genes would be implicated in
exercise-associated DNA methylation changes. These findings
are discussed below.
Table 3 Genes that showed exercise-associated increases (http://tinyurl.com/methincreased) and decreases (http://tinyurl.com/methdecreased) in
DNA methylation among older people
Change Function ID p Value Genes
Increased DNA methylation after
exercise
Regulation of anatomical structure
morphogenesis
GO:0022603 0.018 CXCL10,DCC, PPP2R3A, RASA1,SULF1,TMEM100,
WNT7A
Decreased DNA methylation after
exercise
microRNA 519 inhibits cell proliferation
and decreases tumour growth
MI:hsa-miR-519b-3p 0.016 GAB1,L3MBTL1, PLAGL1, WNK3,BCL2L11,CACNA2D3
Gene networks courtesy g:Profiler.
30 31
GO, gene ontology; MI, computationally predicted microRNA target sites from the MicroCosm database (formerly miRBase).
Table 4 Genes that showed exercise-associated increases and decreases (http://tinyurl.com/methdecreases-young) in DNA methylation among
younger people
Change Function ID p Value Genes
Increased DNA methylation
after exercise
Regulation of stem cell activity MI: hsa-miR-130b* 0.008 SNCG,NCOA6, MRPS26, SPINT4,HDACC3,ESR2,TSTD1,RGS6,FHL1,
ANO2
Increased DNA methylation
after exercise
Negative regulation of cell cycle GO:0045786 <0.02 FHL1,RB1,RPTOr,ZFHX3,CAB39,CDK9,HDAC3,MED25, PSMC5,
BRCA1, RUNX3
Decreased DNA methylation
after exercise
Extracellular structure
organisation; extracellular matrix
organisation
GO:0043062,
GO:0030198
<0.02 LTBP4, COL15A1, COL18A1,COL4A1,LAMA2,NID1, NRXN1,
OLFML2A,PTK2,SFRP2,SULF2,COMP,FBLN2
Decreased DNA methylation
after exercise
System development; skeletal
system development; cartilage
development
GO:0048731,
GO:0001501,
GO:0051216
<0.04 PRKG1,ALDH1A2, ANK3,ANKS1A, BATF,CAMK2B, CASP8,CD74,
CENPF,CHL1, COL15A1, COL18A1,COL4A1,DKK3,EHD1,EYA1,FLG,
HDAC9,HYAL2,IL7,IPMK,KCNQ1, KERA,LAMA2, LFNG,LILRB1, LMO4,
LY6D, MBNL1,MEF2A, MEPE,MITF,NFIB, ND1,NNMT, NPR2,NRXN1,
PLXND1, PTK2,RUNX1,SCIN,SERPINI1,SFRP2,SIX6,SLC35D1, SULF2,
TRPV4, TTLL7,CHRDL2, COMP,PAX6, RUNX3,SIM1
Gene networks courtesy g:Profiler.
30 31
GO, gene ontology; MI, computationally predicted microRNA target sites from the MicroCosm database (formerly miRBase).
Brown WM. Br J Sports Med 2015;0:1–13. doi:10.1136/bjsports-2014-094073 9
Review
Exercise-associated DNA methylation change
Human exercise has medium to large effect sizes on the DNA
methylation of genes extracted from different tissues across sex
and lifespan. The effect sizes are strong among older people
above and beyond the independent effects of research design,
sample size, sex and tissue type. Publication bias is possible and
difficult to rule out. However, the correlation between effect size
and sample size was small (r=−0.11) and driven in part by two
studies, both of which were tightly controlled molecular exercise
physiology experiments with small sample sizes and large effect
size. The large effect size in these studies could be due to meth-
odological rigour. Nonetheless, sample size and other moderat-
ing factors were included in the analyses, suggesting that exercise
has substantial effects on DNA methylation. Since this work was
limited to published studies from western cultures, file drawer
artefacts are a potential source of bias. Future work will need to
investigate cultural and geographical effects, which could bias the
findings. Given the plasticity of human development and popula-
tion genetic variation, we may expect regional variation in the
size of exercise-associated DNA methylation change.
As expected, imprinted genes—a DNA methylation-based
transgenerational epigenetic phenomenon—are implicated in
skeletal muscle gene networks and responsive to exercise expos-
ure. Specifically, skeletal muscle gene network analyses revealed
both maternally expressed (RB1,MEG3,UBE3A) and paternally
expressed (PLAGL1,SGCE,INS) imprinted genes. These
imprinted genes play important growth regulatory functions.
Likewise, the meta-analysis imprinted genes showed changes in
DNA methylation associated with exercise. Specifically, mater-
nally expressed (GRB10,KCNQ1,MEG3) and paternally
expressed (L3MBTL1,PLAGL1) genes were represented in the
meta-analysis. Sixty per cent of the 478 genetic elements uncov-
ered in the meta-analysis showed decreased DNA methylation
after physical exercise. Among older people, the genes that
increased in DNA methylation were involved in growth, while
the genes that decreased in DNA methylation were part of the
cancer-suppressing microRNA gene network. This strongly sug-
gests that exercise may have a protective function among older
people, perhaps shielding them to a degree from the age-related
diseases and decline.
It is notable that two of the six genes that decreased in DNA
methylation among older people after exercise (ie, L3MBTL1,
PLAGL1) are imprinted targets of tumour suppressor
microRNA miR-519b. Considering that imprinted genes (eg,
hypermethylation of tumour suppressors) are often associated
with diverse forms of cancer, this is both a biologically and
medically important finding. Exercise-associated decreases in
L3MBTL1 DNA methylation are associated with decreased mor-
tality among patients with breast cancer.
29
Exercise-associated
decreases in CACNA2D3 (also included in the microRNA
network reported here) DNA methylation may help reduce
gastric tumorigenesis.
40
These findings suggest that, at least for
older people, exercise could have a protective effect against a
variety of cancers in both sexes. More experimental approaches
(ie, a mouse transfection model) likewise suggest that miR-519b
suppresses breast cancer.
41
The ability of the miR-519b network
to inhibit cell proliferation and decrease tumour growth
42
makes it potentially an epigenetically labile network for clinical
researchers interested in exercise. In contrast, among younger
people (less than 40 years of age), a microRNA-regulated
(hsa-miR-130b*) gene network, which functions to suppress
stem cell proliferation, increased in DNA methylation after
exercise.
The fact that exercise-associated DNA methylation change
was stronger in older compared with younger people indicates
that exercise could alter an organism’s‘epigenetic age’by
warding off senescence. Why would exercise have more pro-
found effects on older compared with younger epigenomes?
One possibility is that as organisms age, epigenetic errors accu-
mulate, and because there are more to correct or reset, older
people experience greater (and more positive) DNA methylation
change compared with younger people (who have experienced
fewer epigenetic errors). Since physically active grandparents
were probably a characteristic of the majority of human
evolution, the pronounced age effect could also be an example
of an age-dependent adaptive epigenetic response to antagonis-
tic pleiotropy. Antagonistic pleiotropy is the hypothesis that
genes with multiple effects can be beneficial at younger ages and
costly later in life. In the context of growth effects, older indivi-
duals’tumour suppressor genes were becoming demethylated
Figure 6 (A/B) Effect of tissue and exercise variation on exercise-associated DNA methylation change. Error bars represent Bonferroni-corrected
95% CIs.
10 Brown WM. Br J Sports Med 2015;0:1–13. doi:10.1136/bjsports-2014-094073
Review
(possibly more expressed), but growth-related loci were becom-
ing methylated (presumably less expressed). Thus, rebuilding
new tissue would become increasingly costly for older people
relative to younger people. To reiterate, stem cells and tissue
regeneration adaptive gene complexes in younger people may
well be costly among older age groups due to cancer prolifer-
ation and premiums on cancer suppression as we age. Please
note that reverse antagonistic pleiotropic effect may be operat-
ing here, that is, growth suppression among younger individuals
may be particularly costly (eg, inability to regenerate or develop
costly secondary sexual characteristics) relative to older postre-
productive individuals. The effect of age was independent of
tissue type effects. Specifically, it was found that
exercise-associated DNA methylation changes are greater in
blood compared with breast, adipose and skeletal muscle tissues.
This latter result is consistent with the hypothesis that epigenetic
dysregulation in more heterogeneous samples (eg, blood)—com-
pared with a single tissue sample—may be a better proxy for
the accumulation of environmental stress as we age. Beyond
repeated exposure of physical exercise across the lifespan being
important for a healthy epigenome, in utero maternal epigenetic
effects are likely to be important.
There is recent work in mice showing that maternal exercise
during pregnancy can reverse the deleterious epigenetic effects
of poor maternal diet on newborn pups’Pgc-1a.
43
The benefi-
cial effects of maternal exercise during pregnancy on Pgc-1a
DNA methylation levels in the next generation suggest that a
transgenerational mechanism exists for long-lasting epigenetic
changes and is consistent with a fetal origins of disease
approach.
44
There is no evidence of transgenerational DNA
methylation effects of exercise in humans.
44
However, it is note-
worthy from the meta-analysis that GRB10 (γisoform)—mater-
nally expressed in human fetal skeletal muscle
39
—showed
greater exercise-associated DNA methylation change among
those without a type 2 diabetes family history. Relaxation of the
growth-inhibiting effects of maternal genes could be dependent
on genetic, ecological conditions or fetal exposure to maternal
exercise. For example, individuals from families exhibiting more
sedentary behaviour (ie, characterised by a history of type 2 dia-
betes) have less silencing of maternally expressed GRB10 in skel-
etal muscle. Such differentially epigenetic responses of GRB10’s
γisoform depending on a family history of type 2 diabetes
would be extremely interesting if reliable. A powerful interface
between family history and offspring epigenetics could be sig-
nalled during gestation. Specifically, maternal exercise during
gestation could produce dose-dependent epigenetic responses in
offspring.
44
Despite using two different methods (ie, g-Profiler gene ontol-
ogy network analysis vs meta-analysis), multiple imprinted loci
appear to be missing. This raises the distinct possibility that add-
itional imprinted genes will be found to be associated with
muscle adaptation and exercise adaptation in humans. For
example, in newborns with transient neonatal diabetes, the loss
of an epigenetic mark at the TNDM locus on chromosome
6q24 in the mesodermal lineage causes abdominal muscle hypo-
plasia, the so-called prune belly sequence.
45 46
When Laborie
et al
47
investigated a family with prune belly that included one
discordant set of MZ twins, the twin with prune belly (relative
to the normal co-twin) had extensive loss of methylation at the
TNDM locus, as well as at the following imprinted loci IGF2R,
DIRAS3 and PEG1. Future work in humans and other animals
should be able to develop a more comprehensive list of
imprinted genes regulating skeletal muscle and associated with
exercise. One reason that some imprinted genes may be missed
from these analyses could be due to the fact that imprinted
genes are often involved in neural systems,
18
which, unlike skel-
etal muscle, cannot be extracted from healthy human
participants.
Future work needs to be conducted to test whether or not
imprinted DMRs
48
are modified by exercise. Once again, given
the relevance of imprinted genes for human cancers, one long-
standing conundrum in medicine could be resolved. Specifically,
why does exercise treatment appear to reduce the incidence of
cancers? One answer is that tumour suppressor genes are ‘reacti-
vated’at promoters on long-term exercise treatment and there is
corresponding reduction in DNA methylation.
29
Given these
possible medical benefits, future research should look at the
relationship between exercise stress and regulation of imprinted
genes in order to understand more fully the underlying
mechanisms.
It is worth noting that exercise-associated DNA methylation
changes for imprinted genes occurred only in studies where par-
ticipants were exposed to longer term exercise (ie, 6 months) as
opposed to short bouts of exercise. This interpretation should
be taken with caution as it is biased by the fact that fewer genes
were studied in the acute study by Barrès et al
26
. Specifically,
Barrès et al
26
selected genes from a previous study of DNA
methylation in patients with type 2 diabetes, while the long-
term exercise studies revealing the imprinted genes (see online
supplementary table 2) screened many more genes using
Illumina’sInfinium HumanMethylation450 BeadChip (San
Diego, California, USA).
CONCLUSIONS
Modern epigenomics helps to end nature-nurture debates over
health and disease. The genome is sensitive to the environment
and environmental information is encoded into the epigenome
transgenerationally (eg, imprinted genes). Rather than argue
which is more important, nature or nurture, we can now
measure the interface between the two directly. Measuring the
interface between genes and the environment (eg, DNA methy-
lation) will have ramifications for health and human disease due
to an ageing and an increasingly physically inactive population.
Given the increasing amount of research from multiple inde-
pendent laboratories
26–29 49–40
indicating that human exercise
has varied associations and effects on DNA methylation, it is a
reasonable hypothesis that long-term exercise throughout the
lifespan (or exposure during sensitive periods of in utero devel-
opment) could have profound effects on the epigenome.
44
Future work should determine the optimal exercise types,
timing and duration for ameliorating epigenetic-based disease
outcomes. The strength of exercise-associated DNA methylation
change could be an overestimate due to publication bias (eg,
unpublished studies not included). Genetic background could
affect the associations reported here and was not ruled out.
Future work should sample from monozygotic twins reared
together and apart to elucidate the importance of genetic back-
ground. To rule out publication bias, a collection of unpublished
exercise epigenetics papers will need to be collected and
analysed.
Uncovering epigenetic biomarkers is likely to be more clinic-
ally relevant than looking for ‘disease genes’because epigenetic
changes can be reversed and also since disease variants are
expected to be at low frequency due to the power of natural
selection to remove deleterious genetic variants from a popula-
tion. Techniques are being developed to remove epigenetic
marks (ie, DNA demethylation), which can radically change
disease phenotypes (eg, tumour progression). Exercise medicine
Brown WM. Br J Sports Med 2015;0:1–13. doi:10.1136/bjsports-2014-094073 11
Review
should work alongside clinical epigenetics to investigate how
exercise shapes the human epigenome. An applied goal would
be to adaptively decouple chronological and epigenetic age by
using exercise interventions. The analyses presented here
suggest that exercise-associated DNA methylation change
reduces epigenetic age (eg, cancer reduction
29 40
). Controlled
exercise interventions could help the ageing epigenome, espe-
cially among older hospitalised patients. For example, one study
of older people (aged 58–90 years) with cerebrovascular disease
suggests that physical function improvements during hospitalisa-
tion covary with subtelomeric methylation of long telomeres.
59
If exercise alters the epigenome to reduce age-related disease
outcomes, it could be a relatively inexpensive treatment option
within hospital environments. In conclusion, human studies in
exercise epigenetics are required not only because of the impact
on health of ageing populations, but also because key epigenetic
elements (ie, imprinted genes) responsible for regulating adipos-
ity, energy expenditure, glucose homoeostasis and hunger are dif-
ferentially imprinted (or read differently) between mice and man.
What is already known on this subject
Recent empirical studies suggest that physical exercise modifies
the human epigenome. Specifically, DNA methylation—an
important regulator of gene expression and correlate of diverse
disease states—is altered by physical activity. No systematic
review has been conducted to elucidate these effects and
associations.
What this study adds
This study isolates imprinted genes—known to be important for
health and disease—as important for muscle growth and
clinical targets of exercise. Further, older people received
significant benefits from exercise in terms of the adaptive
epigenetic regulation of tumour suppressor genes.
Correction notice This paper has been amended since it was published Online
First. The correspondence address has been updated and the acknowledgements
section has been revised.
Twitter Follow William Brown at @coevolve
Acknowledgements Thanks to John Brewer, Thomas Chalk and Javaid Kashani
for discussions of some of the ideas presented here. WMB’s research time was
supported by the European Office of Aerospace Research & Development
FA8655-10-1-3037.
Competing interests None.
Provenance and peer review Not commissioned; externally peer reviewed.
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