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The Obstetrician & Gynaecologist 10.1576/toag.12.1.037.27556 http://onlinetog.org 2010;12:37–42 SAC review

SAC review DNA methylation:

a form of epigenetic control of
gene expression
Authors Derek H K Lim / Eamonn R Maher

Key content:
• Epigenetic factors such as DNA methylation play an important role in regulating
gene expression.
• Aberrant DNA methylation is a feature of a number of important human diseases.
• Epigenetic changes are common in human cancer cells.

Learning objectives:
• To appreciate the role of DNA methylation as a regulator of gene expression.
• To understand the role of DNA methylation in normal gene function.
• To illustrate how DNA methylation is implicated in the regulation of genomic
imprinting.
• To draw attention to how altered DNA methylation can result in human diseases
such as imprinting disorders and cancer.

Ethical issues:
• What are the implications for assisted reproductive technologies?
• There are possible difficulties in interpreting the clinical significance of alterations
in DNA methylation.
Keywords Beckwith–Wiedemann syndrome / cancer / genomic imprinting /
Russell–Silver syndrome / tumour suppressor genes
Please cite this article as: Lim DHK, Maher ER. DNA methylation: a form of epigenetic control of gene expression. The Obstetrician & Gynaecologist 2010;12:37–42.

Author details
Derek H K Lim DCH MRCPCH Eamonn R Maher BSc MD FRCP FMedSci
Clinical Research Fellow and Specialist Professor of Medical Genetics
Registrar in Clinical Genetics Department of Medical and Molecular
Department of Medical and Molecular Genetics, University of Birmingham,
Genetics, Institute of Biomedical Research, Birmingham, UK; and
University of Birmingham, Edgbaston, West Midlands Regional Genetics Service,
Birmingham B15 2TT, UK; and Birmingham, UK
West Midlands Regional Genetics Service, Email: [email protected]
Birmingham Women’s Hospital, Edgbaston, (corresponding author)
Birmingham B15 2TG, UK

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This article was commissioned

Introduction gene expression. In general, but not always, DNA
The term epigenetics refers to the study of heritable methylation is associated with loss of gene
by the Scientific Advisory
Committee (SAC) changes in phenotype or expression of genes that expression.7 One theory on the evolution of DNA
are not due to changes in the sequence of DNA.1 methylation is that it evolved as a host defence
Research into epigenetics has demonstrated that mechanism to silence foreign DNA such as viral
epigenetic regulation of gene expression has a sequences, replicated transposable elements and
critical role in normal development and cell other repetitive sequences.9
functions, including imprinting, X-inactivation
and tissue-specific gene expression.2–4 In addition, How does DNA methylation
disordered epigenetic gene regulation is a feature of affect gene expression?
a number of important human diseases, including For gene transcription to occur, the gene promoter
cancer.5,6 A number of processes have been should be readily accessible to transcription factors
implicated in epigenetic gene regulation including and other regulatory units (e.g. enhancers).10 DNA
DNA methylation; chromatin structure and methylation can directly prevent transcription
modification; and untranslated RNAs. This review factor binding and lead to changes in chromatin
aims to introduce the reader to the concept of DNA structure that restrict access of transcription factors
methylation as a regulator of gene expression, with to the gene promoter (Figure 1). For example,
examples of its involvement in genomic imprinting methylated CpGs attract methyl-CpG-binding
and cancer. domain proteins that recruit ‘repressor complexes’,
resulting in histone modification. Histones are the
DNA methylation: what is it? key protein components of chromatin and they act
DNA methylation refers to the addition of a methyl as spools for the wrapping of DNA which can be
group (CH3) covalently to the base cytosine (C) altered. The modification of histones by the
in the dinucleotide 5´-CpG-3´ (methylated cytosine recruitment of repressor complexes leads to a more
residues are sometimes referred to as the fifth condensed chromatin structure (heterochromatin)
nucleotide). The term CpG refers to the base as opposed to an open and active chromatin
cytosine (C) linked by a phosphate bond to the base structure (euchromatin) required for
guanine (G) in the DNA nucleotide sequence. Most transcription.11–13
CpG dinucleotides in the human genome are
methylated. However, unmethylated CpGs are not
randomly distributed, but are usually clustered Maintenance and resetting
together in ‘CpG islands’, which are in the promoter of methylation marks
region of many genes (the region that facilitates The epigenetic marking of the human genome by
transcription of a particular gene).7,8 The DNA methylation is heritable (from one cell to
observation that the CpG islands in the promoters another during cell division), stable and allows a
of important genes that are expressed in most cells form of epigenetic ‘memory’. DNA methylation is
(often called ‘housekeeping genes’) are mainly catalysed by the DNA methyltransferases, including
unmethylated, and that methylation of CpG islands those which establish methylation (DNMT3a and
in cancer cells often leads to silencing of gene DNMT3b) and maintain methylation (DNMT1).
expression, led to the hypothesis that DNA This allows control of expression of developmental
methylation plays an important role in regulating genes at specific times of embryonic development

Figure 1
DNA methylation regulating gene
expression. (A) The CpG island
promoter is unmethylated and
allows binding of transcription
factors, which is required for
transcription initiation. (B) The
CpG island promoter methylation
prevents binding of transcription
factors and results in gene
silencing.

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in specific tissues and also ensures maintenance of syndrome and BWS demonstrate unusual patterns
the correct marking of imprinted genes.14–16 of inheritance, with parent-of-origin effects on
disease phenotype. For example, in familial BWS,
The role of DNA methylation full-blown disease is only seen when the disease is
inherited from the mother. Whilst some cases of
in genomic imprinting and Angelman syndrome or BWS can be caused by
imprinting disorders mutations in specific genes (UBE3A and CDKN1C,
Imprinted genes are genes that are selectively respectively), in other cases there is no genetic
expressed depending on the parental chromosome of alteration in a specific gene but another mechanism
origin. Therefore only one allele (copy) of the gene is (e.g. uniparental disomy, chromosomal deletions or
expressed; for example, for insulin-like growth factor duplications and epimutations affecting the
2 (IGF2), only the copy from the father is active methylation status of imprint control region: Box 1)
whereas the opposite is true for the closely linked H19 causes abnormal expression for several imprinted
gene (Figure 2). Only a minority (about 80) of human genes. In this review we focus on the alteration of
genes are imprinted but imprinted genes often play a methylation status of the key imprinting control
critical role in fetal and neurodevelopment. Thus, two region resulting in a change in gene expression.19,21
copies of the paternal genome (and no maternal
contribution) result in a complete molar pregnancy Beckwith–Wiedemann syndrome
despite the correct number of genes being present.17 This syndrome is characterised by prenatal and/or
postnatal overgrowth, macroglossia and anterior
Important information on the role of imprinted abdominal wall defects (which range from mild
genes in human development has been derived umbilical hernia to exomphalos in severe cases).
from studies of imprinting disorders such as Additional but variable features include facial
Beckwith–Wiedemann syndrome (BWS) and naevus flammeus, hemihypertrophy, neonatal
Russell–Silver, Prader–Willi and Angelman hypoglycaemia and childhood embyronal tumours
syndromes.18–20 The majority of imprinting (especially Wilms tumours).18 Increased expression
syndromes occur sporadically; however, familial of the paternally expressed fetal growth promoter
cases of imprinting disorders such as Angelman IGF2 is implicated in some cases of BWS. The

Figure 2
Genomic imprinting of chromosome
11p15.5. Diagram showing the
methylation status of the two
imprinting centres (IC1 and IC2) and
genes that are selectively expressed
from either parental allele. There is
balance between the paternally
expressed growth promoter IGF2
and the maternally expressed growth
suppressor CDKN1C. Imprinting
centre 1 (IC1): the H19 DMR is
methylated on the paternal allele,
allowing expression of IGF2 from the
paternal chromosome. On the
maternal allele (Mat), the H19 DMR is
unmethylated, allowing expression of
H19 from the maternal chromosome.
Imprinting centre 2 (IC2): KvDMR1 is
methylated on the maternal allele,
allowing the expression of CDKN1C
from the maternal chromosome. On
the paternal allele (Pat), KvDMR1 is
unmethylated, resulting in silencing
of CDKN1C.

Box 1
Mechanism Definition/explanation
Examples of mechanisms leading
Uniparental disomy Both chromosomes in a cell are derived from a single parent (and there is no chromosomal material to imprinting disorders
from the other parent). Uniparental disomy may affect the whole chromosome (complete) or part of a
chromosome. If the chromosomal region contains an imprinted gene then uniparental disomy will be
associated with alterations in imprinted gene expression.
Deletion/duplication Deletions involving imprinted regions can affect imprinting by either:
• deletion of an expressed gene on that allele (so abolishing gene expression); or
• deletion of the imprinting control centre, which results in loss of regulatory control of imprinting.
Duplications involving the imprinted regions can double the expression of imprinted genes expressed
from that allele.
Mutation Loss of function mutations in a gene on the expressed allele will impair gene function whereas a
mutation in the silenced allele will have no apparent effect. Hence mutations in imprinted genes are
associated with parent-of-origin effects on clinical phenotype.
Epimutation An epimutation is a specific loss of methylation (hypomethylation) or gain of methylation
(hypermethylation) at an imprinting control centre without any change in DNA sequence. This alters
expression of imprinted genes on the same allele.

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imprinting status of IGF2 (and H19) is controlled how alterations in DNA methylation can affect gene
by the methylation status of an imprinting control expression and cause specific human diseases. In
region (IC1), which maps between IGF2 and H19. most cases of BWS the cause of altered DNA
On the paternal chromosome IC1 is methylated, methylation is unknown but there is a higher
IGF2 is expressed and H19 expression is silenced frequency of children born by assisted reproductive
(the H19 promoter is methylated). However, on the technologies in children with BWS who have lost
maternally inherited chromosome IC1 is genomic methylation at a second imprinting centre
unmethylated, IGF2 is silenced and H19 is (IC2) between the CDKN1C and IGF2 genes
expressed (the H19 promoter is unmethylated) (Figure 3B).22,23 Further studies are ongoing to
(Figure 2). In 5–10% of BWS cases, both maternal determine whether the reportedly higher frequency
and paternal IC1 regions are methylated (the of growth restriction and congenital abnormalities
maternal chromosome IC1 has gained after assisted reproductive technologies may, in
methylation) and this is associated with expression some cases, be related to epigenetic changes.
of IGF2 and silencing of H19 on both
chromosomes (Figure 3A). This pattern would DNA methylation and cancer
be predicted to double IGF2 expression and so Epigenetic changes are common in human cancer
cause fetal overgrowth and embryonal tumour cells (Figure 4). Initially, most attention was paid
susceptibility (increased expression of IGF2 is a to the DNA hypomethylation that is common in
feature of Wilms tumour).21 cancer cells.24 This may promote tumourigenesis
by transcriptional activation of proto-oncogenes
Russell–Silver syndrome (which promote oncogenic cell growth), loss of
Another imprinting disorder has also been linked imprinting or genomic instability (by allowing
to altered IGF2/H19 expression and IC1 activation of repetitive elements which are
methylation. Russell–Silver syndrome (RSS) is normally repressed).25,26 Gain of CpG methylation
characterised by prenatal and postnatal growth can also be a feature of cancer cells and as
restriction, body asymmetry, a triangular face and methylated cytosines are highly unstable bases this
café-au-lait patches of the skin. Hence BWS and will predispose to gene mutation as the methylated
RSS have opposite growth phenotypes and ~40% cytosines are often deaminated and converted to
of children with RSS have an abnormal IC1 thymine. Replacement of a cytosine by a thymine
methylation pattern which is opposite to that seen can lead to inactivation of tumour suppressor genes
in BWS. Thus in RSS there is loss of paternal allele (TSGs) (e.g. CGA, which encodes an arginine
IC1 methylation (so that the paternal epigenotype residue, is changed to TGA, which specifies a stop
is the same as that on the maternal allele), resulting codon resulting in a prematurely truncated protein
in reduced IGF2 expression and fetal growth on translation).27 In addition, recent research has
retardation.20 highlighted that a major consequence of epigenetic
alterations in cancer cells is acquired promoter
The genetics of BWS and RSS are complex and methylation, causing silencing of expression of key
other molecular mechanisms can result in a BWS or TSGs.5,6 These TSGs (e.g. p53, p16) are required for
RSS phenotype. However, these disorders illustrate regulation of normal growth and differentiation.

Figure 3
Example of two epigenetic errors
on chromosome 11p15.5, which
results in BWS. (A) Epimutation
with gain of methylation at the
H19DMR on the maternal allele
(Mat), allowing biallelic expression
of the growth promoter IGF2. This is
seen in 5% of sporadic BWS cases.
(B) Epimutation with loss of
methylation or hypomethylation at
the maternal allele KvDMR1,
resulting in silencing of CDKN1C of
both parental alleles. This is seen in
50% of sporadic BWS cases but is
the predominant defect seen in
BWS cases conceived by assisted
reproductive technologies.

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Figure 4
DNA methylation and cancer.
Examples of three mechanisms
leading to tumourigenesis.
TSG  tumour suppresor gene

Inactivation of both alleles of TSGs is a critical chemotherapy such as temozolomide.33 In addition,

event in the pathogenesis of most human cancers the discovery of novel epigenetically inactivated
(which TSGs are involved depends on the specific TSGs can provide insights into pathways of
cancer type). Initially, TSG inactivation was usually tumourigenesis and provide a basis for further
attributed to the acquisition of genetic mutations research to develop new therapeutic agents or
and chromosome deletions (allele loss). However, targeted therapy such as demethylating agents.
it is now clear that for some important TSGs (e.g.
RASSF1A) promoter methylation is a more
frequent cause of inactivation than mutations. Conclusion
Whereas genetic mutations and promoter This short review has covered the basic concepts of
methylation may have equivalent effects and both DNA methylation and its relevance to normal
inactivate a TSG, epigenetic silencing by promoter development and regulation of gene expression.
methylation is a potentially reversible event. Thus, Defects in DNA methylation can result in disorders
in the laboratory, treatment of cancer cells with affecting embryogenesis, genomic imprinting and
demethylating agents can reactivate TSG cancer. Epigenetics is a rapidly developing area of
expression and drugs that modify histone status are human genetics. In the same way that sequencing
being used in clinical trials for leukaemias.28 the human genome has accelerated research into
inherited diseases and cancer, it is anticipated that
In ovarian cancer cells, a number of cancer genes initiatives to define the normal human epigenome
have been identified as having aberrant promoter will enhance progress towards better understanding
hypermethylation, including OPCML, BRCA1, of the role of epigenetics in human disease.
p16 and TMS1.29,30 Tumour suppressor gene
methylation specific to cancer cells provides Recommended reading
opportunities for novel, noninvasive early detection 1. Torst J. Epigenetics. Norfolk: Caister Academic
strategies. For example, detection of methylated Press; 2008.
TSGs in sputum may be used to detect lung cancer 2. Allis CD, Jenuwein T, Reinberg D, Caparros M,
and in urine, bladder cancer.31,32 Furthermore, editors. Epigenetics. New York: Cold Spring
specific patterns of TSG methylation may also be Harbour Laboratory Press; 2007.
useful as prognostic indicators or predictors of
treatment response. For example, aberrant References
1 Egger G, Liang G, Aparicio A, Jones PA. Epigenetics in human disease
hypermethylation of MGMT has been shown to be a and prospects for epigenetic therapy. Nature 2004;429:457–63.
biomarker for a good response to alkylating agent doi:10.1038/nature02625

© 2010 Royal College of Obstetricians and Gynaecologists 41

TOG12_1_37-42_Maher.qxd 1/5/10 3:58 PM Page 42

SAC review 2010;12:37–42 The Obstetrician & Gynaecologist

2 Jones PA, Takai D. The role of DNA methylation in mammalian 18 Elliott M, Maher ER. Beckwith–Wiedemann syndrome. J Med Genet
epigenetics. Science 2001;293:1068–70. doi:10.1126/science.1063852 1994;31:560–4. doi:10.1136/jmg.31.7.560
3 Kaneda M, Okano M, Hata K, Sado T, Tsujimoto N, Li E, et al. Essential 19 Nicholls RD, Knepper JL. Genome organization, function, and imprinting
role for de novo DNA methyltransferase Dnmt3a in paternal and in Prader–Willi and Angelman syndromes. Annu Rev Genomics Hum
maternal imprinting. Nature 2004;429:900–3. Genet 2001;2:153–75. doi:10.1146/annurev.genom.2.1.153
doi:10.1038/nature02633 20 Gicquel C, Rossignol S, Cabrol S, Houang M, Steunou V, Barbu V, et al.
4 Csankovszki G, Nagy A, Jaenisch R. Synergism of Xist RNA, DNA Epimutation of the telomeric imprinting center region on chromosome
methylation, and histone hypoacetylation in maintaining X chromosome 11p15 in Silver–Russell syndrome. Nat Genet 2005;37:1003–7.
inactivation. J Cell Biol 2001;153:773–84. doi:10.1083/jcb.153.4.773 doi:10.1038/ng1629
5 Jones PA, Laird PW. Cancer epigenetics comes of age. Nat Genet 21 Maher ER, Reik W. Beckwith–Wiedemann syndrome: imprinting in
1999;21:163–7. doi:10.1038/5947 clusters revisited. J Clin Invest 2000;105:247–52. doi:10.1172/JCI9340
6 Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. 22 Maher ER, Brueton LA, Bowdin SC, Luharia A, Cooper W, Cole TR, et al.
Nat Rev Genet 2002;3:415–28. Beckwith–Wiedemann syndrome and assisted reproduction technology
7 Bird AP. CpG-rich islands and the function of DNA methylation. Nature (ART). J Med Genet 2003;40:62–4. doi:10.1136/jmg.40.1.62
1986;321:209–13. doi:10.1038/321209a0 23 Lim D, Bowdin SC, Tee L, Kirby GA, Blair E, Fryer A, et al. Clinical and
8 Ehrlich M, Gama-Sosa MA, Huang LH, Midgett RM, Kuo KC, McCune RA, molecular genetic features of Beckwith–Wiedemann syndrome
et al. Amount and distribution of 5-methylcytosine in human DNA from associated with assisted reproductive technologies. Hum Reprod
different types of tissues of cells. Nucleic Acids Res 1982;10:2709–21. 2009;24:741–7. doi:10.1093/humrep/den406
doi:10.1093/nar/10.8.2709 24 Feinberg AP, Vogelstein B. Hypomethylation distinguishes genes of some
9 Hedges DJ, Batzer MA. From the margins of the genome: mobile human cancers from their normal counterparts. Nature 1983;301:89–92.
elements shape primate evolution. Bioessays 2005;27:785–94. doi:10.1038/301089a0
doi:10.1002/bies.20268 25 Chen RZ, Pettersson U, Beard C, Jackson-Grusby L, Jaenisch R. DNA
10 Watt F, Molloy PL. Cytosine methylation prevents binding to DNA of a hypomethylation leads to elevated mutation rates. Nature
HeLa cell transcription factor required for optimal expression of the 1998;395:89–93. doi:10.1038/25779
adenovirus major late promoter. Genes Dev 1988;2:1136–43. 26 Gonzalo S, Blasco MA. Role of Rb family in the epigenetic definition of
doi:10.1101/gad.2.9.1136 chromatin. Cell Cycle 2005;4:752–5.
11 Bird AP, Wolffe AP. Methylation-induced repression – belts, braces, and 27 Rideout WM, 3rd, Coetzee GA, Olumi AF, Jones PA. 5-Methylcytosine as
chromatin. Cell 1999;99:451–4. doi:10.1016/S0092-8674(00)81532-9 an endogenous mutagen in the human LDL receptor and p53 genes.
12 Jones PL, Veenstra GJ, Wade PA, Vermaak D, Kass SU, Landsberger N, Science 1990;249:1288–90. doi:10.1126/science.1697983
et al. Methylated DNA and MeCP2 recruit histone deacetylase to repress 28 Griffiths EA, Gore SD. DNA methyltransferase and histone deacetylase
transcription. Nat Genet 1998;19:187–91. doi:10.1038/561 inhibitors in the treatment of myelodysplastic syndromes. Semin Hematol
13 Nan X, Ng HH, Johnson CA, Laherty CD, Turner BM, Eisenman RN, et al. 2008;45:23–30. doi:10.1053/j.seminhematol.2007.11.007
Transcriptional repression by the methyl-CpG-binding protein MeCP2 29 Makarla PB, Saboorian MH, Ashfaq R, Toyooka KO, Toyooka S, Minna JD,
involves a histone deacetylase complex. Nature 1998;393:386–9. et al. Promoter hypermethylation profile of ovarian epithelial neoplasms.
doi:10.1038/30764 Clin Cancer Res 2005;11:5365–9. doi:10.1158/1078-0432.CCR-04-2455
14 BestorTH, Ingram VM. Two DNA methyltransferases from murine 30 Teodoridis JM, Hall J, Marsh S, Kannall HD, Smyth C, Curto J, et al. CpG
erythroleukemia cells: purification, sequence specificity, and mode of island methylation of DNA damage response genes in advanced ovarian
interaction with DNA. Proc Natl Acad Sci USA 1983;80:5559–63. cancer. Cancer Res 2005;65:8961–7. doi:10.1158/0008-5472.CAN-05-1187
doi:10.1073/pnas.80.18.5559 31 Carvalho AL, Jeronimo C, Kim MM, Henrique R, Zhang Z, Hoque MO,
15 Okano M, Bell DW, Haber DA, Li E. DNA methyltransferases Dnmt3a et al. Evaluation of promoter hypermethylation detection in body fluids as
and Dnmt3b are essential for de novo methylation and mammalian a screening/diagnosis tool for head and neck squamous cell carcinoma.
development. Cell 1999;99:247–57. doi:10.1016/S0092- Clin Cancer Res 2008;14:97–107. doi:10.1158/1078-0432.CCR-07-0722
8674(00)81656-6 32 Hoque MO, Begum S, Topaloglu O, Chatterjee A, Rosenbaum E, Van
16 Pradhan S, Bacolla A, Wells RD, Roberts RJ. Recombinant human DNA Criekinge W, et al. Quantitation of promoter methylation of multiple genes
(cytosine-5) methyltransferase. I. Expression, purification, and in urine DNA and bladder cancer detection. J Natl Cancer Inst
comparison of de novo and maintenance methylation. J Biol Chem 2006;98:996–1004.
1999;274:33002–10. doi:10.1074/jbc.274.46.33002 33 Hegi ME, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, Weller M, et al.
17 Kajii T, Ohama K. Androgenetic origin of hydatidiform mole. Nature MGMT gene silencing and benefit from temozolomide in glioblastoma.
1977;268:633–4. doi:10.1038/268633a0 N Engl J Med 2005;352:997–1003. doi:10.1056/NEJMoa043331

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