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Exercise-associated DNA methylation change in skeletal muscle and the importance of imprinted genes: A bioinformatics meta-analysis

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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.
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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 alterationproducing heritable
characteristics. For example, DNA methylation modies
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 proling analyses).
Design Gene ontology (ie, gene product elucidation)/
meta-analysis.
Data sources 26 skeletal muscle and 86 imprinted
genes were subjected to g:Proler ontology analysis.
Meta-analysis assessed exercise-associated DNA
methylation change.
Data extraction g:Proler found four muscle gene
networks with imprinted loci. Meta-analysis identied 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 ve 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 identied in
skeletal muscle gene networks and exercise-associated
DNA methylation change. Exercise-associated DNA
methylation modication could rewind the epigenetic
clockas we age.
Trial registration number CRD42014009800.
INTRODUCTION
British developmental biologist Sir Conrad H
Waddington introduced the term epigeneticsas a
science of development from genotype to pheno-
type.
1
However, the term epigeneticshad an inde-
pendent origin and meaning, which led to a
conation of terms.
2
Recall Waddingtons use of
the term epigeneticsto 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-
eticin 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,aboveor outer. Examples of such changes
are DNA methylation and histone modications
(gure 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 dened as genotype-independ-
ent parent-of-origin gene expression. Specically, 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 modications 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.
813
Furthermore, epigenetic
dysregulation in imprinted geneswhich 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 gure 1a type of silencer at a
genes promoterplaces 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 inuences on the epigenome has led to a
strong hypothesis that exercise can also affect DNA
Brown WM. Br J Sports Med 2015;0:113. doi:10.1136/bjsports-2014-094073 1
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methylation and have long-term health outcomes. Specically,
we propose that the same mechanism that controls genomic
imprinting in mammals (ie, DNA methylation) allows for
phenotypic modication 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 modication, 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-specic and age-
specic manner during development. Where DNA methylation
occurs can be critical for its effect. DNA methylation (ie, adding
methyl groups to a cytosine base, gure 1) at a genes 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
gure 2). DNA methylation has been implicated in cancer, neu-
rodevelopmental disorders and autoimmune diseases.
15
Thus, if
exercise can inuence 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
prole). Specically, 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 tagDNA 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 genes promoter. Histones: Histones are proteins around which DNA can wind for compaction and histone modication can
regulate gene activity. Both epigenetic processes (ie, DNA methylation and histone modications) affect health resulting in cancer, autoimmune
disease, neurological disorders or diabetes. Image modied from the National Institutes of Health, Benjamin I. Laufer and Forluvoft.
2 Brown WM. Br J Sports Med 2015;0:113. doi:10.1136/bjsports-2014-094073
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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 Immunodeciency
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 proles, key developmental regulatory and
immune function genes show loci-specic 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
reect 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 cells 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;0:113. 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).
Haigs model
19
proposes that imprinting evolved as a result
of opposing tness 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
tness of the mother and future offspring. Haigs 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 tness.
IGF2an imprinted gene and paternally expressed in
humansregulates 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 efcacy.
20
IGF2s 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) bre size
variation of both type 1 and 2 bres, (B) type 2 bre atrophy, (C)
increased numbers of type 2C bres and (D) decreased numbers
of type 2B bre. This nding 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 inuence muscle hyper-
trophyextremely muscled hindquartersin 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 broblasts 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 barrierwill
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
transcriptomethat 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 specication of cell
identity, maintaining homoeostasis and responding to environ-
mental conditions. These epigenetic mechanisms encompass
DNA methylation, post-translational histone modications and
microRNA (gure 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 proinammatory 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 inuence 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:Proler
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 proler
32 33
(gure 4). To test the hypothesis that imprinted genes are impli-
cated in skeletal muscle gene networks, g:Proler
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 (gure 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 nalised by
the author (gure 5). Both PRISMA (see online supplementary
le 1) and MOOSE (see online supplementary le 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;0:113. 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 signicant. 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 nger 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 identied. Notably,
INS-IGF2, a read-through gene, aligns to the INS gene,
whereby INS is at the 5region and IGF2an extremely
well-studied growth regulatory imprinted geneis 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 rst 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 modication 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. Modied from Kelvin Song.
Brown WM. Br J Sports Med 2015;0:113. doi:10.1136/bjsports-2014-094073 5
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Figure 4 Enrichment prole for
selected human skeletal muscle genes
(dened 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;0:113. 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. UBE3Asprotein
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 signicantly 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 12 (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 ve 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 ow diagram for meta-analysis component of paper. Search terms used were DNA methylation exerciseor DNA methylation
physical activity. Human in vivo studies only (May 2014).
Brown WM. Br J Sports Med 2015;0:113. 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 peoplesee online supplementary table
S2)is large (mean Cohens d=1.20±1.20; 95% CI of the
mean 1.10 to 1.31) and signicantly 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 signicantly greater for people
over 40 years of age (Cohens d=2.89±1.97) compared with
those under 40 years of age (Cohens 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
designslarger 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 specicity (larger effect
sizes in tissue with more cell types, p<0.001). The fact that
sample size and effect size are signicantly 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. Specically, among older people
(people older than 40 years of age), the effect size was signi-
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. Specically, among
younger people (people less than 40 years of age), the effect
size was signicantly 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:Proler
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-519bs microRNA network.
Among younger individuals, a microRNA-regulated gene
network involved in stem cell activity was implicated.
Specically, 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 signicant (all p values
<0.04) gene networks were uncovered for the genes that
decreased in DNA methylation after exercise among younger
people. Specically, 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 signicant: F(4,471)=137.03,
p<0.001, partial η
2
=0.54 (gure 6A). As seen in gure 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 ve 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;0:113. 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 signicant: F(2,453)=126.11, p<0.001, partial η
2
=0.36
(gure 6B). As seen in gure 6B, DNA methylation change was
signicantly greater among those engaged in Tai Chi and
walking compared with those engaged in cycling. Walking and
Tai Chi were not signicantly different from one another
(p=0.25). Regardless of the specic 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 myober number loss and myosin heavy chain IIB
gene expression.
36
GRB10 was another imprinted gene missing
from g:Prolers 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
Gareld 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-specic 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 ndings
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;0:113. 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
difcult 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, le drawer
artefacts are a potential source of bias. Future work will need to
investigate cultural and geographical effects, which could bias the
ndings. 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 genesa DNA methylation-based
transgenerational epigenetic phenomenonare implicated in
skeletal muscle gene networks and responsive to exercise expos-
ure. Specically, 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. Specically, 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 nding. 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 ndings 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 organismsepigenetic ageby
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 benecial at younger ages and
costly later in life. In the context of growth effects, older indivi-
dualstumour 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;0:113. 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. Specically, 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 samplemay 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 pupsPgc-1a.
43
The bene-
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 GRB10s
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. Specically, maternal exercise during
gestation could produce dose-dependent epigenetic responses in
offspring.
44
Despite using two different methods (ie, g-Proler 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 modied by exercise. Once again, given
the relevance of imprinted genes for human cancers, one long-
standing conundrum in medicine could be resolved. Specically,
why does exercise treatment appear to reduce the incidence of
cancers? One answer is that tumour suppressor genes are reacti-
vatedat promoters on long-term exercise treatment and there is
corresponding reduction in DNA methylation.
29
Given these
possible medical benets, 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
. Specically,
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
IlluminasInnium 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 ramications 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
2629 4940
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 genesbecause 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;0:113. 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 5890 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 modies
the human epigenome. Specically, DNA methylationan
important regulator of gene expression and correlate of diverse
disease statesis altered by physical activity. No systematic
review has been conducted to elucidate these effects and
associations.
What this study adds
This study isolates imprinted genesknown to be important for
health and diseaseas important for muscle growth and
clinical targets of exercise. Further, older people received
signicant benets 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. WMBs research time was
supported by the European Ofce of Aerospace Research & Development
FA8655-10-1-3037.
Competing interests None.
Provenance and peer review Not commissioned; externally peer reviewed.
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Review
... Several studies have investigated the impact of resistance exercise on miRNA expression in skeletal muscle. [153][154][155][156][157] The miRNA expression of 800 targets was investigated in the skeletal muscle of young men who had undergone lower-body resistance training. 153 A single bout of resistance exercise, at the start of the training intervention, was determined to alter the expression of 85 miRNA; whereas, after the completion of resistance training the expression of 102 miRNAs was differently expressed from baseline. ...
... The response of young and aged skeletal muscle has been directly investigated in acute resistance exercise studies. 155,156 The first study to investigate the impact of age selected three miRNA previously associated with muscle phenotypes and investigated the response after acute resistance exercise. 155 After exercise and protein consumption, miRNA-1 was significantly lower in young compared to old individuals; however, there was no change in miRNA-133a or miRNA-206 expression. ...
... 155 Ageassociated exercise-induced miRNA expression was also reported in a second study which identified 21 miRNAs that were altered in skeletal muscle from young individuals 6 h after exercise; whereas, miRNA expression was unchanged in older individuals. 156 The lack of miRNA regulation in older skeletal muscle was associated with a blunted mRNA response in old compared to young skeletal muscle after resistance exercise. Pathway analysis identified exercise-induced alteration of miRNAs with targets associated with cellular growth and proliferation processes. ...
Chapter
This chapter introduces the epigenetic processes that govern how exercise affects the aging processes. We begin with an introduction to the molecular changes that occur with aging including methylation and histone and noncoding RNA modifications. We then present the evidence for changes in these processes by exercise and physical activity, Lastly, we present evidence for and against a role for exercise on changes in telomere length and aging.
... Studies most often focus on the acute effects of exercise to determine the epigenetic transient changes that influence muscle adaptation while others look at long-term changes in response to exercise. A few studies have shown differential changes between the sexes with females exhibiting a more dynamic and robust response in methylation changes due to exercise; however, a deeper dive into specific sex differences in methylation and subsequent downstream effects is sorely needed (Lindholm et al., 2014b;Brown, 2015). ...
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As the fields of kinesiology, exercise science, and human movement developed, the majority of the research focused on male physiology and extrapolated findings to females. In the medical sphere, basing practice on data developed in only males resulted in the removal of drugs from the market in the late 1990s due to severe side effects (some life-threatening) in females that were not observed in males. In response to substantial evidence demonstrating exercise-induced health benefits, exercise is often promoted as a key modality in disease prevention, management, and rehabilitation. However, much like the early days of drug development, a historical literature knowledge base of predominantly male studies may leave the exercise field vulnerable to overlooking potentially key biological differences in males and females that may be important to consider in prescribing exercise (e.g., how exercise responses may differ between sexes and whether there are optimal approaches to consider for females that differ from conventional approaches that are based on male physiology). Thus, this review will discuss anatomical, physiological, and skeletal muscle molecular differences that may contribute to sex differences in exercise responses, as well as clinical considerations based on this knowledge in athletic and general populations over the continuum of age. Finally, this review summarizes the current gaps in knowledge, highlights the areas ripe for future research, and considerations for sex-cognizant research in exercise fields.
... The so-called epigenetic clock delivers the most accurate measure of biological aging [49], and might be a suitable alternative for the lack of reliable aging biomarkers. Moreover, the epigenetic clock is sensitive to the most common modifiers that alter aging trajectories, such as obesity, exercise, and nutritional interventions [50,51]. ...
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Background Aging is characterized by a progressive loss of capacities linked to fundamental alterations/damage in multiple cellular and molecular pathways. It is the most significant risk factor for all non-communicable diseases (NCDs). Another contributing factor to the rise in NCDs is obesity. It has been suggested that obesity not only accelerates the onset of metabolic imbalances but also decreases lifespan and impacts cellular and molecular processes in a manner similar to aging. Obesity might accelerate the pace of aging. Guided by a lifecourse approach, we will explore how exposure to obesity in critical developmental stages disrupt homeostatic resilience mechanisms that preserve physiological integrity, inducing an early expression of aging phenotypes. Also, we will determine whether exposure to early psychosocial adversity influences vulnerability to obesity as a risk factor for accelerated aging. Methods Multiple events case–control study embedded in a prospective cohort of Chileans at 30-31y, 50% females, of low- to-middle socioeconomic status, who participated in nutrition research since birth. At 23y, 25% had obesity and cardiometabolic risk was high. We will use a multi-layer approach including: anthropometric assessment; DXA scan for body composition; abdominal ultrasound of the liver; stool samples collection and sequencing of the ribosomal RNA 16S gene to characterize the gut microbiome; determination of age-related pro-inflammatory cytokynes and anti-inflammatory miokynes. For the first time in Chile, we will address age-related epigenetic changes using the Horvath´s epigenetic clock. In a subset we will conduct a controlled physical challenge to characterize physical resilience (autophagy). Discussion ObAGE is in an excellent position to: approach aging as a process whose expression involves multiple factors from the early stages of a person's life; understand how longitudinal changes in health trajectories impact the biological mechanisms of aging; identify potential resilience mechanisms that help prevent unhealthy aging. Because SLS participants are still young, our research setting combined with advanced scientific techniques may identify individuals or groups at risk of early onset health issues. Results from ObAGE may pave the way to address the contribution of obesity to aging through lifespan from cells to systems and might be instrumental to developing interventions to improve health span in the Chilean population. Trial registration The proposed study does not consider any health care intervention on human participants.
... The current findings agree in principle with a recent metaanalysis that examined 16 research studies and concluded that nuclear DNA methylation generally decreases with exercise in older adults. 42 Additionally, our data agree in F I G U R E 3 Mitochondrial transcript and marker adaptations with training in older males. These data represent mitochondrial transcript levels (panel A), TFAM mRNA levels (panel B), TFAM protein levels (panel C), citrate synthase (CS) activity levels (panel E), and protein levels of mitochondrial complexes I-V (panel F). ...
Article
Resistance training (RT) dynamically alters the skeletal muscle nuclear DNA methylome. However, no study has examined if RT affects the mitochondrial DNA (mtDNA) methylome. Herein, ten older, Caucasian untrained males (65 ± 7 y.o.) performed six weeks of full‐body RT (twice weekly). Body composition and knee extensor torque were assessed prior to and 72 h following the last RT session. Vastus lateralis (VL) biopsies were also obtained. VL DNA was subjected to reduced representation bisulfite sequencing providing excellent coverage across the ~16‐kilobase mtDNA methylome (254 CpG sites). Biochemical assays were also performed, and older male data were compared to younger trained males (22 ± 2 y.o., n = 7, n = 6 Caucasian & n = 1 African American). RT increased whole‐body lean tissue mass (p = .017), VL thickness (p = .012), and knee extensor torque (p = .029) in older males. RT also affected the mtDNA methylome, as 63% (159/254) of the CpG sites demonstrated reduced methylation (p < .05). Several mtDNA sites presented a more “youthful” signature in older males after RT in comparison to younger males. The 1.12 kilobase mtDNA D‐loop/control region, which regulates replication and transcription, possessed enriched hypomethylation in older males following RT. Enhanced expression of mitochondrial H‐ and L‐strand genes and complex III/IV protein levels were also observed (p < .05). While limited to a shorter‐term intervention, this is the first evidence showing that RT alters the mtDNA methylome in skeletal muscle. Observed methylome alterations may enhance mitochondrial transcription, and RT evokes mitochondrial methylome profiles to mimic younger men. The significance of these findings relative to broader RT‐induced epigenetic changes needs to be elucidated.
... Since sex was dealt with as a confounder, no direct statistical comparison between methylation changes in men and women in response to exercise was performed. Also, a meta-analysis study stated that larger effect sizes in DNA methylation were observed in women than men following exercise, proposing sex differences in the epigenetic response to exercise [81]. However, specific differences between sexes were not examined because it was not the aim of the study. ...
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BACKGROUND CONTEXT Chronic low back pain (LBP) is a multifactorial disorder with complex underlying mechanisms, including associations with intervertebral disc (IVD) degeneration in some individuals. It has been demonstrated that epigenetic processes are involved in the pathology of IVD degeneration. Epigenetics refers to several mechanisms, including DNA methylation, that have the ability to change gene expression without inducing any change in the underlying DNA sequence. DNA methylation can alter the entire state of a tissue for an extended period of time and thus could potentially be harnessed for long-term pain relief. Lifestyle factors, such as physical activity, have a strong influence on epigenetic regulation. Exercise is a commonly prescribed treatment for chronic LBP, and sex-specific epigenetic adaptations in response to endurance exercise have been reported. However, whether exercise interventions that attenuate LBP are associated with epigenetic alterations in degenerating IVDs has not been evaluated. PURPOSE We hypothesize that the therapeutic efficacy of physical activity is mediated, at least in part, at the epigenetic level. The purpose of this study was to use the SPARC-null mouse model of LBP associated with IVD degeneration to clarify (1) if IVD degeneration is associated with altered expression of epigenetic regulatory genes in the IVDs, (2) if epigenetic regulatory machinery is sensitive to therapeutic environmental intervention, and (3) if there are sex-specific differences in (1) and/or (2). STUDY DESIGN Eight-month-old male and female SPARC-null and age-matched control (WT) mice (n=108) were assigned to exercise (n=56) or sedentary (n=52) groups. Deletion of SPARC is associated with progressive IVD degeneration and behavioral signs of LBP. The exercise group received a circular plastic home cage running wheel on which they could run freely. The sedentary group received an identical wheel secured in place to prevent rotation. After 6 months, the results obtained in each group were compared. METHODS After 6 months of exercise, LBP-related behavioral indices were determined, and global DNA methylation (5-methylcytosine) and epigenetic regulatory gene mRNA expression in IVDs were assessed. This project was supported by the Canadian Institutes for Health Research. The authors have no conflicts of interest. RESULTS Lumbar IVDs from WT sedentary and SPARC-null sedentary mice had similar levels of global DNA methylation (%5-mC) and comparable mRNA expression of epigenetic regulatory genes (Dnmt1,3a,b, Mecp2, Mbd2a,b, Tet1-3) in both sexes. Exercise attenuated LBP-related behaviors, decreased global DNA methylation in both WT (p < 0.05) and SPARC-null mice (p < 0.01) and reduced mRNA expression of Mecp2 in SPARC-null mice (p < 0.05). Sex-specific effects of exercise on expression of mRNA were also observed. CONCLUSIONS Exercise alleviates LBP in a mouse model. This may be mediated, in part, to changes in the epigenetic regulatory machinery in degenerating IVDs. Epigenetic alterations due to a lifestyle change could have a long-lasting therapeutic impact by changing tissue homeostasis in IVDs. CLINICAL SIGNIFICANCE This study confirmed the therapeutic benefits of exercise on LBP and suggests that exercise results in sex-specific alterations in epigenetic regulation in IVDs. Elucidating the effects of exercise on epigenetic regulation may enable the discovery of novel gene targets or new strategies to improve the treatment of chronic LBP.
Article
Aging causes degenerative changes such as epigenetic changes and mitochondrial dysfunction in skeletal muscle. Exercise can upregulate muscle mitochondrial homeostasis and enhance antioxidant capacity and represents an effective treatment to prevent muscle aging. Epigenetic changes such as DNA methylation, histone posttranslational modifications, and microRNA expression are involved in the regulation of exercise-induced adaptive changes in muscle mitochondria. Reactive oxygen species (ROS) play an important role in signaling molecules in exercise-induced muscle mitochondrial health benefits, and strong evidence emphasizes that exercise-induced ROS can regulate gene expression via epigenetic mechanisms. The majority of mitochondrial proteins are imported into mitochondria from the cytosol, so mitochondrial homeostasis is regulated by nuclear epigenetic mechanisms. Exercise can reverse aging-induced changes in myokine expression by modulating epigenetic mechanisms. In this review, we provide an overview of the role of exercise-generated ROS in the regulation of mitochondrial homeostasis mediated by epigenetic mechanisms. In addition, the potential epigenetic mechanisms involved in exercise-induced myokine expression are reviewed.
Chapter
Neuroinflammation is a neuroprotective mechanism that orchestrated by microglial cells and astrocytes in response to neural trauma or infection. Neuroinflammation not only results in elevation in levels of proinflammatory cytokines, chemokines, lipid mediators, proteases, and oxidants, but also supports removal of dead cells and restoration of normal structure and function of the brain. A prolonged chronic inflammatory state produces detrimental effects on health and predisposes humans to a number of chronic diseases. Chronic inflammation is also a robust predictor of both disability and mortality even in the absence of clinical disease. Therefore, it is thought that the inflammatory pathway is a potential therapeutic target for interventions to reduce risk of disease and disability. Lifestyle behavioral interventions, including changes in food/dietary intake and physical activity, may have clinically significant benefits for improving neuroinflammation over the long term.
Chapter
Many different types of exercise and physical activity induce mechanical, hormonal, and metabolic stimuli that lead to adaptions in almost every tissue and cell type. Recent research has shown that both acute exercise and chronic exercise are potential epigenetic modifiers (affecting DNA methylation, posttranslational histone modifications, and the expression of microRNAs) that change the functional genome in gametes, muscle, blood, and fat cells in brain tissue as well as in cells of the cardiovascular system. The results of these studies reveal that exercise-induced epigenetic modifications may counteract pathophysiological alterations of the epigenome in diseased states (e.g., cancer, neurodegenerative disorders, cardiovascular diseases). It is hypothesized that the beneficial preventative and rehabilitative effects of exercise are at least partly driven by epigenetic modifications, which contribute to reduced systemic inflammation and local adaptions, such as the demethylation of tumor suppressor genes. Furthermore, data show that responders to certain exercise programs (e.g., resistance training) have different epigenetic patterns compared to non-responders. Additionally, different types and intensities of exercise exhibit specific epigenetic responses that can be directly measured in muscle tissue and the circulation. Therefore, epigenetic adaptions may be used in clinical trials or in the field as markers for exercise conducting and controlling in the future. However, more research is needed to generate meaningful knowledge about exercise-induced epigenetic modifications in particular tissues as well as the local and systemic consequences of these modifications. As exercise seems to provoke different responses throughout the life span—and considering the popular belief that exercise can rewind the “epigenetic clock”—age should always be considered as a covariate in further studies.
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Using a mouse model of conditional and inducible in vivo fluorescent myonuclear labeling (HSA-GFP), sorting purification of nuclei, low-input reduced representation bisulfite sequencing (RRBS), and a translatable and reversible model of exercise (progressive weighted wheel running, PoWeR), we provide the first nucleus type-specific epigenetic information on skeletal muscle adaptation and detraining. Adult (>4 month) HSA-GFP mice performed PoWeR for 8 weeks then detrained for 12 weeks; age-matched untrained mice were used to control for the long duration of the study. Myonuclei and interstitial nuclei from plantaris muscles were isolated for RRBS. Relative to untrained, PoWeR caused similar myonuclear CpG hypo- and hypermethylation of promoter regions and substantial hypomethylation in interstitial nuclear promoters. Over-representation analysis of promoters revealed a larger number of hyper- versus hypomethylated pathways in both nuclear populations after training and evidence for reciprocal regulation of methylation between nucleus types, with hypomethylation of promoter regions in Wnt signaling-related genes in myonuclei and hypermethylation in interstitial nuclei. After 12 weeks of detraining, promoter CpGs in documented muscle remodeling-associated genes and pathways that were differentially methylated immediately after PoWeR were persistently differentially methylated in myonuclei, along with long-term promoter hypomethylation in interstitial nuclei. No enduring gene expression changes in muscle tissue were observed using RNA-sequencing. Upon 4 weeks of retraining, mice that trained previously grew more at the whole muscle and fiber type-specific cellular level than training naïve mice, with no difference in myonuclear number. Muscle nuclei have a methylation epi-memory of prior training that may augment muscle adaptability to retraining.
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Exertional rhabdomyolysis may occur when an individual is subjected to strenuous physical exercise. It is occasionally associated with myoglobinuria (i.e. "cola-colored" urine) alongside muscle pain and weakness. The pathophysiology of exertional rhabdomyolysis involves striated muscle damage and the release of cellular components into extracellular fluid and bloodstream. This can cause acute renal failure, electrolyte abnormalities, arrhythmias and potentially death. Exertional rhabdomyolysis is observed in high-performance athletes who are subjected to intense, repetitive and/or prolonged exercise but is also observed in untrained individuals and highly trained or elite groups of military personnel. Several risk factors have been reported to increase the likelihood of the condition in athletes, including: viral infection, drug and alcohol abuse, exercise in intensely hot and humid environments, genetic polymorphisms (e.g. sickle cell trait and McArdle disease) and epigenetic modifications. This article reviews several of these risk factors and proposes screening protocols to identify individual susceptibility to exertional rhabdomyolysis as well as the relevance of proteomics for the evaluation of potential biomarkers of muscle damage.
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Abnormal conditions during early development adversely impact later health. We investigated whether maternal exercise could protect the offspring from adverse effects of maternal HFD with a focus on the metabolic outcomes and epigenetic regulation of the metabolic master regulator, peroxisome proliferator activated receptor γ co-activator-1α (Pgc-1α). Female C57BL/6 mice were exposed to normal chow, HFD, or HFD with voluntary wheel exercise for 6 weeks prior to and throughout pregnancy. Methylation of the Pgc-1α promoter at CpG site -260 and Pgc-1α mRNA expression were assessed in skeletal muscle from neonatal and 12 month-old offspring, and glucose and insulin tolerance tests (GTT and ITT, respectively) were performed in the female offspring at 6, 9 and 12 months. Hypermethylation of the Pgc-1α promoter caused by maternal HFD was detected at birth, which was maintained to 12 month of age with a trend of reduced Pgc-1α mRNA (P = 0.065) and its target genes. Maternal exercise prevented maternal HFD-induced Pgc-1α hypermethylation and enhanced Pgc-1α and its target gene expression concurrent with amelioration of age-associated metabolic dysfunction at 9 months of age in the offspring. Therefore, maternal exercise is a powerful lifestyle intervention in preventing maternal HFD-induced epigenetic and metabolic dysregulation in the offspring.
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Epigenetic mechanisms are implicated in gene regulation and the development of different diseases. The epigenome differs between cell types and has until now only been characterized for a few human tissues. Environmental factors potentially alter the epigenome. Here we describe the genome-wide pattern of DNA methylation in human adipose tissue from 23 healthy men, with a previous low level of physical activity, before and after a six months exercise intervention. We also investigate the differences in adipose tissue DNA methylation between 31 individuals with or without a family history of type 2 diabetes. DNA methylation was analyzed using Infinium HumanMethylation450 BeadChip, an array containing 485,577 probes covering 99% RefSeq genes. Global DNA methylation changed and 17,975 individual CpG sites in 7,663 unique genes showed altered levels of DNA methylation after the exercise intervention (q<0.05). Differential mRNA expression was present in 1/3 of gene regions with altered DNA methylation, including RALBP1, HDAC4 and NCOR2 (q<0.05). Using a luciferase assay, we could show that increased DNA methylation in vitro of the RALBP1 promoter suppressed the transcriptional activity (p = 0.03). Moreover, 18 obesity and 21 type 2 diabetes candidate genes had CpG sites with differences in adipose tissue DNA methylation in response to exercise (q<0.05), including TCF7L2 (6 CpG sites) and KCNQ1 (10 CpG sites). A simultaneous change in mRNA expression was seen for 6 of those genes. To understand if genes that exhibit differential DNA methylation and mRNA expression in human adipose tissue in vivo affect adipocyte metabolism, we silenced Hdac4 and Ncor2 respectively in 3T3-L1 adipocytes, which resulted in increased lipogenesis both in the basal and insulin stimulated state. In conclusion, exercise induces genome-wide changes in DNA methylation in human adipose tissue, potentially affecting adipocyte metabolism.
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Background: DNA methylation patterns are heritable but can change over time and in response to exposures. Lower global DNA methylation, which may result in increased genomic and chromosomal instability, has been associated with increased cancer risk. Physical activity is a modifiable factor that has been inversely related to the risk of cancer. Changes in DNA methylation may be a mechanism by which lifestyle and environment factors influence disease. We investigated the relationship between DNA methylation and physical activity in a sample of women enroled in The Sister Study, a large United States (U.S.) cohort study of women aged 35-74 years with a family history of breast cancer. Methods: Global DNA methylation was measured using bisulphite-converted DNA and pyrosequencing of a LINE-1 repetitive sequence in the peripheral blood of 647 non-Hispanic white women. Physical activity (average hours per week) was retrospectively assessed for three time periods: childhood (ages 5-12), teenage years (ages 13-19) and the previous 12 months. Findings: Compared with women with physical activity levels below the median for all three time periods, those at or above the median physical activity for one (β = 0.20, 95% confidence interval (CI): -0.10, 0.49), two (β = 0.22, 95% CI: -0.08, 0.52) or all three (β = 0.33, 95% CI: 0.01, 0.66) time periods had increased global methylation. Interpretation: Maintaining higher levels of physical activity over these three time periods was associated with increased global DNA methylation, consistent with reported associations between exercise and decreased cancer risk.
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Genome-wide association studies have identified many genetic variants associated with complex traits. However, at only a minority of loci have the molecular mechanisms mediating these associations been characterized. In parallel, whereas cis regulatory patterns of gene expression have been extensively explored, the identification of trans regulatory effects in humans has attracted less attention. Here we show that the type 2 diabetes and high-density lipoprotein cholesterol-associated cis-acting expression quantitative trait locus (eQTL) of the maternally expressed transcription factor KLF14 acts as a master trans regulator of adipose gene expression. Expression levels of genes regulated by this trans-eQTL are highly correlated with concurrently measured metabolic traits, and a subset of the trans-regulated genes harbor variants directly associated with metabolic phenotypes. This trans-eQTL network provides a mechanistic understanding of the effect of the KLF14 locus on metabolic disease risk and offers a potential model for other complex traits.
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As part of a systematic screen for novel imprinted genes of human chromosome 7 we have investigated GRB10, which belongs to a small family of adapter proteins, known to interact with a number of receptor tyrosine kinases and signalling molecules. Upon allele-specific transcription analysis involving multiple distinct splice variants in various fetal tissues, we found that human GRB10 is imprinted in a highly isoform- and tissue-specific manner. In fetal brains, most variants are transcribed exclusively from the paternal allele. Imprinted expression in this tissue is not accompanied by allele-specific methylation of the most 5' CpG island. In skeletal muscle, one GRB10 isoform, yl, is expressed from the maternal allele alone, whereas in numerous other fetal tissues, all GRB10 splice variants are transcribed from both parental alleles. A remarkable finding is paternal-specific expression of GRB10 in the human fetal brain, since, in the mouse, this gene is transcribed exclusively from the maternal allele. To our knowledge, this is the first example of a gene that is oppositely imprinted in mouse and human.
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Imprinted genes are defined by their parent-of-origin-specific monoallelic expression. Although the epigenetic mechanisms regulating imprinted gene expression have been widely studied, their functional importance is still unclear. Imprinted genes are associated with a number of physiologies, including placental function and foetal growth, energy homeostasis, and brain and behaviour. This review focuses on genomic imprinting in the brain and on two imprinted genes in particular, Nesp and paternal Grb10, which, when manipulated in animals, have been shown to influence adult behaviour. These two genes are of particular interest as they are expressed in discrete and overlapping neural regions, recognised as key "imprinting hot spots" in the brain. Furthermore, these two genes do not appear to influence placental function and/or maternal provisioning of offspring. Consequently, by understanding their behavioural function we may begin to shed light on the evolutionary significance of imprinted genes in the adult brain, independent of the recognised role in maternal care. In addition, we discuss the potential future directions of research investigating the function of these two genes and the behavioural role of imprinted genes more generally.