Analysis of repetitive element DNA methylation by MethyLight.

Abstract

Repetitive elements represent a large portion of the human genome and contain much of the CpG methylation found in normal human postnatal somatic tissues. Loss of DNA methylation in these sequences might account for most of the global hypomethylation that characterizes a large percentage of human cancers that have been studied. There is widespread interest in correlating the genomic 5-methylcytosine content with clinical outcome, dietary history, lifestyle, etc. However, a high-throughput, accurate and easily accessible technique that can be applied even to paraffin-embedded tissue DNA is not yet available. Here, we report the development of quantitative MethyLight assays to determine the levels of methylated and unmethylated repeats, namely, Alu and LINE-1 sequences and the centromeric satellite alpha (Satalpha) and juxtacentromeric satellite 2 (Sat2) DNA sequences. Methylation levels of Alu, Sat2 and LINE-1 repeats were significantly associated with global DNA methylation, as measured by high performance liquid chromatography, and the combined measurements of Alu and Sat2 methylation were highly correlative with global DNA methylation measurements. These MethyLight assays rely only on real-time PCR and provide surrogate markers for global DNA methylation analysis. We also describe a novel design strategy for the development of methylation-independent MethyLight control reactions based on Alu sequences depleted of CpG dinucleotides by evolutionary deamination on one strand. We show that one such Alu-based reaction provides a greatly improved detection of DNA for normalization in MethyLight applications and is less susceptible to normalization errors caused by cancer-associated aneuploidy and copy number changes.

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Analysis of repetitive element DNA methylation
by MethyLight
DanielJ.Weisenberger,MihaelaCampan,TiffanyI.Long,MyungjinKim,ChristianWoods1,
Emerich Fiala2, Melanie Ehrlich1and Peter W. Laird*
Department of Surgery and Department of Biochemistry and Molecular Biology, Keck School of Medicine, USC/Norris
Comprehensive Cancer Center, University of Southern California, Los Angeles, CA, USA,1Tulane Cancer Center,
Human Genetics Program and Department of Biochemistry, Tulane Medical School, New Orleans, LA, USA and
2Nelson Institute of Environmental Science, New York University School of Medicine, Tuxedo, NY, USA
Received August 19, 2005; Revised October 13, 2005; Accepted November 11, 2005
ABSTRACT
Repetitive elements represent a large portion of
the human genome and contain much of the CpG
methylationfound in normal
somatic tissues. Loss of DNA methylation in these
sequences might account for most of the global
hypomethylation that characterizes a large percent-
age of human cancers that have been studied. There
is widespread interest in correlating the genomic
5-methylcytosine content with clinical outcome, diet-
aryhistory,lifestyle,etc.However,ahigh-throughput,
accurate and easily accessible technique that can
be applied even to paraffin-embedded tissue DNA is
not yet available. Here, we report the development
of quantitative MethyLight assays to determine the
levels of methylated and unmethylated repeats,
namely, Alu and LINE-1 sequences and the centro-
meric satellite alpha (Sata) and juxtacentromeric
satellite 2 (Sat2) DNA sequences. Methylation levels
of Alu, Sat2 and LINE-1 repeats were significantly
associated with global DNA methylation, as meas-
ured by high performance liquid chromatography,
andthecombinedmeasurements
Sat2 methylation were highly correlative with global
DNA methylation measurements. These MethyLight
assays rely only on real-time PCR and provide
surrogate markers for global DNA methylation ana-
lysis. We also describe a novel design strategy
for the development of methylation-independent
MethyLightcontrolreactionsbasedonAlusequences
depleted of CpG dinucleotides by evolutionary
deamination on one strand. We show that one such
Alu-based reaction provides a greatly improved
humanpostnatal
of Aluand
detection of DNA for normalization in MethyLight
applications and is less susceptible to normalization
errors caused by cancer-associated aneuploidy and
copy number changes.
INTRODUCTION
DNA methylation in mammalian cells is required for normal
embryonic development, X-chromosome inactivation and
genomic imprinting, and involves the addition of a methyl
group to the C-5 position of cytosine, predominantly in a
50-CpG-30sequence context [reviewed in (1)]. This is accomp-
lished bytheactivitiesofoneormoreDNA methyltransferases
(DNMTs), which use S-adenosylmethionine (AdoMet) as a
cofactor. CpG dinucleotides are underrepresented in the
human genome by a factor of about 5, due to the spontaneous
deamination of 5-methylcytosine residues, resulting in C-to-T
transition mutations at CpG dinucleotides (2). However, there
are regions of the genome termed CpG islands that have
retained their expected CpG content (3,4). Most CpG islands
overlap the 50end of gene regions, including promoters, and
are typically unmethylated in normal somatic tissues (4,5).
However, only 40% of promoter regions are associated with
CpG islands (4,5). The unique and repeated sequences in the
remainder of the genome are often highly methylated at their
CpG sites in somatic tissues (6).
CpG dinucleotides are often aberrantly methylated in
humancancerstogiveregionalhypermethylationatsomeCpG
islands despite an overall reduction in 5-methylcytosine in
the DNA (global DNA hypomethylation) (7–9). The frequent
hypomethylation of repetitive elements in diverse human can-
cers is thought to largely account for the global hypomethyla-
tion commonly seen in human cancers (6).
Repetitive elements comprise ?45% of the human genome
(10,11) and consist of interspersed repeats derived from
non-autonomous or autonomous transposable elements (12–
*To whom correspondence should be addressed. Tel: +1 323 865 0650; Fax: +1 323 865 0158; Email: plaird@usc.edu
? The Author 2005. Published by Oxford University Press. All rights reserved.
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Nucleic Acids Research, 2005, Vol. 33, No. 216823–6836
doi:10.1093/nar/gki987

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14) and tandem repeats of simple sequences (satellite DNA)
or complex sequences. The most plentiful short interspersed
nucleotide element (SINE) in human DNA is the Alu repeat,
an ?282bpnon-LTR(Long Terminal Repeat)DNA sequence,
which comprises 10% of the human genome and is present
in ?1 million copies per haploid genome (12). Other abundant
non-LTR sequences are long interspersed nucleotide elements
(LINEs) of up to 6 kb that comprise ?20% of the human
genome [reviewed in (6,13)]. LINE-1 elements are present
at over 500000 copies in the human genome; however, only
3000–4000 are full length and 30–100 are active retrotrans-
posons (6,13).
LINE-1 elements are usually methylated in somatic tissues,
and LINE-1 hypomethylation is a common characteristic of
human cancers (15–18). Moreover, Alu sequences are also
normally methylated in somatic tissues (19–21) and are
thought to become hypomethylated in human cancer cells.
However, not all Alus are hypomethylated in human cancers.
Alu sequences located upstream of the CDKN2A promoter
were found to be hypermethylated in cancer cell lines (22),
and an Alu sequence located in intron 6 of TP53 showed
extensive methylation in normal and cancer cells (22,23).
While LINEs and SINEs are interspersed throughout the
genome, satellite DNA is largely confined to the centromeres
or centromere-adjacent (juxtacentromeric) heterochromatin
and to the large region of heterochromatin on the long arm
of the Y chromosome. Satellite a (Sata) repeats are composed
of 170 bp DNA sequences and represent the main DNA
component of every human centromere (24). Satellite 2 (Sat2)
DNA sequences are found predominantly in juxtacentromeric
heterochromatin of certain human chromosomes and are most
abundant in the long juxtacentromeric heterochromatin region
of chromosome (Chr) 1. Sat2 sequences are composed of
variants of two tandem repeats of ATTCCATTCG followed
by one or two copies of ATG (25). Both Chr1 Sata and Chr1
Sat2 sequences, as well as Sata repeats present throughout all
the centromeres, are highly methylated in normal postnatal
tissues, hypomethylated in sperm and often hypomethylated
in various cancers (26–29). In addition, Sat2 sequences on
Chr1 and Chr16 are also hypomethylated in the ICF (immun-
odeficiency, centromeric region instability and facial abnor-
malities) syndrome, which usually involves mutations in
DNMT3B (30,31).
Previous studies describing repetitive element DNA
methylation have been mostly based on Southern blot ana-
lyses, which require large amounts of high-molecular-weight
genomic DNA (7,27,29,32,33). Accurate global genomic
5-methylcytosinecontentisoftendeterminedbyhighperform-
ance liquid chromatography (HPLC) (7,27,29,32,33), which,
although highly quantitative and reproducible, also requires
large amounts of high-quality genomic DNA and is not suit-
able for high-throughput analyses. In a recent report, Alu
and LINE-1 methylation levels were obtained by COBRA
[COmbined Bisulfite Restriction Analysis, first described in
(34)] and pyrosequencing of bisulfite-converted DNA (18).
Althoughthese quantitative
advancements in determining repetitive element DNA methyl-
ation levels, both require post-PCR manipulation, are labor-
intensive and,therefore,may
high-throughput analyses.
methods representmajor
notbesuitable for
In this study, we advanced MethyLight assay technology, a
quantitative, TaqMan-based real-time PCR system to analyze
DNA methylation profiles (35), by extending it to the analysis
of highly repeated DNA sequences. We designed and applied
MethyLight assays to examine the methylation levels of
Alu, LINE-1, and Chr1 centromeric Sata and juxtacentro-
meric Sat2 repeat sequences. We evaluated repetitive element
MethyLight measurements on a panel of normal and tumor
DNA samples for which accurate HPLC-based global DNA
methylation measurements were available. These data suggest
that methylation of either interspersed or tandem repeats can
be used as a surrogate marker for estimating global DNA
methylation levels. The combination (mean) of Alu and Sat2
repeat methylation measurements yielded a particularly close
correlation with global genomic 5-methylcytosine content
measurements obtained by HPLC.
Additionally, we exploited the high Alu copy number
to design an Alu-based MethyLight control reaction to sens-
itively determine input DNA levels for normalization in
MethyLight assays.
MATERIALS AND METHODS
Design of the Alu-based MethyLight control reaction
TheAlu-based control reaction wasdesignedinsilicobasedon
a deaminated Alu consensus DNA sequence, in which we
assumed that all CpGs on one strand underwent evolutionary
deamination. The strategy is indicated with deamination of the
top strand in Figure 2 for simplicity; however, either the top or
the bottom strands can be chosen. In designing the control
reaction, we deaminated the CpGs on the bottom (opposite)
strand of the consensus sequence to TpGs in silico, and as a
result, CpG dinucleotides on the top strand of the consensus
sequence became CpA dinucleotides. These CpA dinuc-
leotides are converted to TpA dinucleotides upon bisulfite
conversion and PCR, thereby generating a methylation-
independent unique sequence. Using this deaminated and
bisulfite-converted DNA sequence, we selected the PCR
primer and probe sequences.
Bisulfite conversion and DNA recovery
DNA, in an 18 ml volume, was denatured at 100?C for 10 min,
then centrifuged briefly, and chilled on ice. NaOH was added
to a final concentration of 0.3 M in a 20 ml volume, and the
sample was incubated at 42?C for 20 min. Sodium bisulfite
solutions were prepared on the day of use by adding 1.9 g
sodium metabisulfite (Sigma) to 3.2 ml of 0.44 M NaOH and
heating at 50?C to dissolve the bisulfite. After addition of
hydroquinone(0.5mlofa1Msolution),120mlofthissolution
was mixed with each DNA sample. The reaction was allowed
to proceed at 50?C for 16 h in the dark.
Following bisulfite conversion, the DNA was recovered
using the Qiagen Viral RNA Mini Kit (Qiagen) according
to the manufacturer’s specifications with the following
changes: after loading the column with the supplied lysis
buffer and 100% EtOH, the filtrate was re-loaded to increase
DNA recovery. After washing with two supplied wash buffers,
the DNA was eluted in 80 ml (2 · 40 ml elutions). To desulf-
onate the sample, 50 ml of 0.2 M NaOH was then added for
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15 min, followed by neutralization with 10 ml of 1 M HCl.
The supplied lysis buffer and EtOH was added to the desulf-
onated sample, and the bisulfite-converted DNA was then
purified a second time. The eluted DNA sample was stored
at ?20?C.
M.SssI methylation assay
Peripheral blood leukocyte (PBL) DNA (Promega) was used
as a substrate for M.SssI treatment. PBL DNA (0.05 mg/ml)
was incubated with M.SssI at a concentration of 1 U/mg DNA
(0.05 U/ml) and 0.16 mM AdoMet overnight at 37?C. Then
extra AdoMet (to 0.20 mM) and M.SssI (to 0.065 U/ml) were
added followed by a second overnight incubation at 37?C. The
sample was stored at 4?C, and 18 ml (0.9 mg DNA) aliquots
were used for bisulfite conversion and recovery as described
above.
Whole-genome amplification (WGA)
To generate unmethylated human DNA as control samples
for testing the MethyLight reactions, sperm and PBL DNAs
(10 ng each) were amplified using a WGA kit (Molecular
Staging) as described by the manufacturer. The DNA was
then recovered by phenol–chloroform extraction and ethanol-
precipitation, dissolved in water and stored at ?20?C. An
aliquot (1–2 mg) was then treated with bisulfite and recovered
as described above.
MethyLight reactions
The PCR primers and probes are listed in Table 1. Probes were
either labeled with a black hole quencher (BHQ-1, Biosearch
Technologies), or a minor groove binder non-fluorescent
quencher (MGBNFQ, Applied Biosystems). MethyLight PCR
was performed in a 30 ml reaction volume with 200 mM
dNTPs, 0.3 mM forward and reverse PCR primers, 0.1 mM
probe, 3.5 mM MgCl2, 0.01% Tween-20, 0.05% gelatin and
0.1 U of Taq polymerase using the following PCR program:
95?C for 10 min, then 50 cycles of 95?C for 15 s followed by
60?C for 1 min. The samples in 96-well plates were analyzed
on an Opticon DNA Engine Continuous Fluorescence
Detector (MJ Research/Bio-Rad). A standard curve for the
Alu repeat control reaction was generated from 1:25 serial
dilutions of bisulfite-converted, M.SssI-treated DNA for the
methylated reactions and 1:25 serial dilutions of bisulfite con-
verted DNA after WGA for the unmethylated MethyLight
reactions.
Methylation calculations
The MethyLight data specific for methylated repetitive
elements were expressed as percent of methylated reference
(PMR) values and were calculated similarly to a recent report
(36), but with the following changes. DNA treated with M.SssI
served as a methylated reference, and the Alu-based control
reaction (ALU-C4 in Table 1) was used as a control reaction to
Table 1. Description of repetitive element MethyLight reaction information
Reaction IDGenBank
number
Amplicon
start
Amplicon
end
Forward primer
sequence 50to 30
Reverse primer
sequence 50to 30
Probe sequence
50to 30a
ALU-C4 Consensus
Seq., Figure 1
Y07755
198 GGTTAGGTATAGTGGTTT-
ATATTTGTAATTTTAGTA
ATTATGTTAGTTAG-
GATGGTTTCGATTTT
ATTAACTAAACTAATCTTA-
AACTCCTAACCTCA
CAATCGACCGAACGCGA
CCTACCTTAACC-
TCCC-MGBb
CCCAACACTTTAA-
AAAACCGAAATAA-
ATAAATCACGA
AAATAATCCGCCC-
GCCTCGACCT
CGCGTTTGTAATTT-
TAGTTATTCGGGA-
GGTTG
AAACGATTCTCC-
TACCTCAACCTC-
CCGAA
CAAACACACACC-
ACCACACCCAAC-
TAATTT
TCGAATATTGCG-
TTTTCGGATCG-
GTTT
CACCCTACTTCA-
ACTCATACACAA-
TACACACACCC
TATCCCGTTTC-
CAACGAA-MGBb
TTTATCCCATTT-
CCAACAAA-MGBb
CGATTCCATTCGA-
TAATTCCGTTT-MGBb
ALU-M1c
50595164
ALU-M2 Consensus
Seq., Figure 1
Consensus
Seq., Figure 1
7 95GCGCGGTGGTTTACGTTT AACCGAACTAATCT-
CGAACTCCTAAC
CCCGAATTCAAAC-
GATTCTCC
ALU-M3133 210ATTAGTCGGGCGTGGTGG
ALU-M5d
AC007256 156130156221GGTATGATGGCGTATG-
TTTGTAATTT
CGACTCACCACAACT-
TCCACC
ALU-U3 Consensus
Seq., Figure 1
91192TGGTTAATATGGTGAAA-
TTTTGTTTTTATT
TCCTACCTCAACCTCCC-
AAATAACT
LINE-1-M1X52235251 331 GGACGTATTTGGAAA-
ATCGGG
AATCTCGCGATACGCCGTT
LINE-1-U3X52235 110 210TTTATTAGGGAGTGTTA-
GATAGTGGGTG
CCTTACACTTCCCAAAT-
AAAACAATACC
SATa-M1M38468 139260TGATGGAGTATTTTTAAA-
ATATACGTTTTGTAGT
TTGATGGAGTATTTTTAAA-
ATATATGTTTTGTAGT
TCGAATGGAATTAATATT-
TAACGGAAAA
AATTCTAAAAATATTCC-
TCTTCAATTACGTAAA
AAATTCTAAAAATATT-
CCTCTTCAATTACATAAA
CCATTCGAATCCATTC-
GATAATTCT
SATa-U1M38468 138261
SAT2-M1X72623 1074 1153
dThis MethyLight reaction was designed toward an Alu sequence in the CASP8 gene.
aAll probes contain a 6FAM fluorophore and a BHQ-1 probe unless otherwise noted.
bMGB refers to a Minor Groove Binder non-fluorescent quencher in the 30terminus of the probe (MGBNFQ).
cThis MethyLight reaction was designed toward an Alu sequence in the S100A2 gene.
Nucleic Acids Research, 2005, Vol. 33, No. 216825

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measure the levels of input DNA to normalize the signal for
each methylation reaction. The levels of unmethylated repet-
itive elements were expressed as percent of unmethylated
reference (PUR) values and were calculated similarly to PMR
values except that bisulfite-converted, unmethylated human
DNA obtained by WGA, as described above, was used as
an unmethylated reference for PUR determinations of each
repetitive element.
In analyzing the panel of DNAs described in Figure 4,
each MethyLight reaction was performed 3–6 times, except
for ALU-M3, which was only analyzed in duplicate. The PMR
or PUR values represent the mean values, and the error
bars represent standard error of the mean. Standard error of
the mean values were not included for the ALU-M3 reaction
because we obtained only two PMR measurements for this
reaction. In correlating MethyLight measurements to HPLC-
based global 5-methylcytosine levels (Figure 5), each
MethyLight reaction was performed in triplicate, and the
data shown are the mean PMR or PUR values of the three
measurements. The data were plotted as PMR or PUR mean
values for each repetitive element versus HPLC-based global
5-methylcytosine measurements for each sample. The com-
posite methylation measurements of Alu and Sat2 (Figure 5H)
were determined by obtaining the mean between the triplicate
ALU-M2 and SAT2-M1 PMR values, and then plotting
the composite mean PMR value versus the HPLC-based
global 5-methylcytosine measurement for each sample.
Linear regression analyses were performed using GraphPad
InStat version 3.0a for Macintosh (GraphPad Software,
San Diego, CA).
HPLC measurements of global genomic
5-methylcytosine content
The overall DNA 5-methylcytosine content was determined
byHPLC onheat-denatured DNA digestedtonucleosides. The
global 5-methylcytosine content for each sample is listed in
Table 2 and represents the mean value of 2–3 measurements
(29). The average replicate percentage standard deviation
for replicates was 2% (37). Linear regression was used to
determine a correlation between MethyLight-based PMR
values and HPLC-based global methylation measurements
for each repetitive element. Global 5-methylcytosine content
in humans has been shown to be tissue-specific with a range of
3.43–4.26% of cytosine residues methylated in normal tissues
(32,37). DNA samples were classified as hypomethylated if
the global methylation was between 3.20 and 3.40% and sub-
stantially hypomethylated if the amount of global methylation
was <3.20%. These designations were only included as an
indication of global 5-methylcytosine content in various
tissues and in cancer cells.
Southern blot-based method of determining
Chr1 Sat2 hypomethylation
Chr1 Sat2 hypomethylation was determined by Southern blot
analysis, comparison of band patterns in autoradiograms and
quantitation of Phosphorimager results as described previ-
ously (33). The MethyLight PMR values were compared
with the hypomethylation scores for each sample using
ANOVA.
RESULTS
Development of an Alu-based, methylation-independent
MethyLight control reaction
MethyLight is a quantitative, TaqMan-based, real-time PCR
assay to measure DNA methylation profiles, using bisulfite-
converted DNA as a substrate (35,38). MethyLight is compat-
ible with DNA samples derived from fresh tissue, cell lines, as
well as formalin-fixed, paraffin-embedded tissues or bodily
fluids such as plasma or serum, where the amount of DNA
available is usually limiting. Each MethyLight-based methyla-
tion data point, expressed as a PMR value [first described in
(39,40)], involves the use of a CpG-independent, bisulfite-
specific control reaction to measure input DNA levels. The
control reaction should be highly sensitive to accurately meas-
ure small amounts of DNA and should not detectably vary in
its ability to be amplified from different human DNA samples,
including cancer tissues.
Control reactions amplifying the low- or single-copy genes
MYOD1, ACTB and COL2A1 have been routinely used in
previous reports as a measure of input bisulfite-treated DNA
levels (33,35,36,38). However, these single-copy genes may
not always be reliable in human cancers, where chromosomal
deletions, duplications and gene amplifications are frequent
events. We therefore designed an Alu-based MethyLight con-
trol reaction to evaluate input DNA levels that would be both
more sensitive in analyzing low amounts of input DNA, and at
the same time would be less subject to local cancer-associated
genetic alterations, compared with the single-copy control
genes that we have traditionally used. The high copy number
Table 2. HPLC-based measurements of global 5-methylcytosine content in
normal and cancer DNA samples
SampleLaird ID Normal/cancerGlobal
methylation:
mC/(mC + C) (%)
WT4
OvCa E
WT31
WT9
OvCa I
WT14B
WT6
WT29
OvCa L
WT7A
WT24
OvCa D
WT22
WT 27
OvCa B
Liver A
OvCa J
OvCa Q
OvCa M
Lung C
OvCa O
OvCa A
Kidney B
Spleen C
Thyroid B
Cerebellum A
6027
6020
6036
6030
6021
6031
6028
6035
6023
6029
6033
6019
6032
6034
6018
6042
6022
6026
6024
6046
6025
6017
6043
6045
6044
6041
Cancer
Cancer
Cancer
Cancer
Cancer
Cancer
Cancer
Cancer
Cancer
Cancer
Cancer
Cancer
Cancer
Cancer
Cancer
Normal
Cancer
Cancer
Cancer
Normal
Cancer
Cancer
Normal
Normal
Normal
Normal
2.88
2.94
2.94
3.09
3.11
3.18
3.20
3.25
3.27
3.28
3.28
3.31
3.39
3.46
3.48
3.55
3.56
3.57
3.61
3.68
3.73
3.76
3.77
3.77
3.77
4.26
6826 Nucleic Acids Research, 2005, Vol. 33, No. 21

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of Alu repeats, which are dispersed throughout the genome,
makes it unlikely that copy number shifts at specific genomic
loci would substantially influence their PCR product yield and
also allows for sensitive detection of minute amounts of DNA.
In addition, the presence of rare single-nucleotide polymoph-
isms(SNPs)should notinterferewith the PCRamplification of
the Alu control reaction, butmay hinderthe PCR amplification
of single- or low-copy sequences. Recently, Stroun et al. (41)
usedanAlu-basedreal-timePCR toanalyzetheamountoffree
DNA in plasma/serum of cancer patients and healthy controls.
Alu repeats are highly heterogeneous due to the depletion of
CpG dinucleotides by spontaneous evolutionary deamination,
in which C-to-T transition mutations are generated. In order to
develop a methylation-independent, Alu-based control reac-
tion, we first generated an Alu consensus sequence based on a
panel of young and old individual Alu repeats subfamilies
in which we identified all CpG dinucleotides as well as those
that became evolutionarily deaminated to TpG (or CpA on the
strand opposite of an evolutionary deamination event) in older
Alu sequences (Figure 1). However, we could not identify
a subregion in the consensus sequence that was devoid of
CpGs. We therefore took advantage of the evolutionary deam-
ination process to design a control reaction toward Alu
sequences in which all cytosines in a CpG context have
been deaminated on one of the two DNA strands. These deam-
inated Alusequences should beCpG methylationindependent.
We reasoned that such strand-specifically deaminated Alu
sequences should exist in the genome by chance, even though
they were not present among the selected Alu sequences listed
in Figure 1.
The design of this control reaction is complicated because
MethyLightreactionsarespecificforbisulfite-convertedDNA.
After bisulfite conversion, the two DNA strands are non-
complimentary (Figure 2), and the PCR primers are designed
toward either the top or the bottom DNA strands. Methylated
CpGs are refractory to bisulfite and remain as CpG on both
DNA strands, whereas an unmethylated CpG dinucleotide
is deaminated to a TpG after bisulfite conversion. However,
Figure1. AnAluconsensusDNAsequencedeterminedfromthesequencesofyoungandoldindividualAlurepeats.OldAlusequences(Alu-J,SluSp,AluSx,AluSq
andAluSc)andyoungAlusequences(AluY,AluSb2,AluYb8,AluYa5andAluYa8)werecomparedinordertogenerateanAluconsensussequenceforthepurpose
of designing an Alu-based MethyLight control reaction. Since the goal was to design a methylation-independent reaction of as many individual Alu repeats as
possible,allCpGdinucleotidesbecameapartoftheconsensussequence.OldAlusequencesarethepredominantforminhumancells,andthisisalsoreflectedinthe
Alu consensus sequence. The continuous and dashed lines underneath the consensus sequence panels indicate the MethyLight PCR amplicon locations within the
consensus sequence for the Alu control reaction (ALU-C4), two reactions toward the methylated consensus sequence (ALU-M2 and ALU-M3) and one reaction
toward the unmethylated Alu consensus sequence (ALU-U3).
Nucleic Acids Research, 2005, Vol. 33, No. 216827

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a TpG dinucleotide also results from the evolutionary
deamination of a methylated CpG. Sequences deaminated
during evolution can be distinguished from those resulting
from bisulfite conversion if the bisulfite-PCR primers are
specific for the DNA strand opposite to the evolutionarily
deaminated DNA strand (Figure 2, Methylation-Independent
Sequence). This CpA containing strand will be converted to
a distinct TpA sequence after bisulfite conversion.
Using this strategy, we simulated evolutionary deamination
of the Alu consensus sequence in silico by first replacing every
CpG dinucleotide with a CpA dinucleotide (representing the
evolutionary deamination of the opposite strand of the con-
sensus sequence), and then selecting primers and a probe for
the MethyLight control reaction specific for the bisulfite-
converted form of this DNA sequence. The locations of the
PCR amplicon and the primer/probe sequences are shown in
Table 1, and the location of the PCR amplicon within the Alu
consensus sequence is shown in Figure 1. To satisfy the
PCR melting temperature requirements for the Alu control
reaction, the probe contains a minor groove binding non-
fluorescent quencher (MGBNFQ). The use of MGB probes
in real-time PCR-based, DNA methylation analyses was
also recently reported for the purpose of improving PCR
specificity (42).
To assess whether the Alu-based MethyLight control reac-
tion could detect a high number of evolutionarily deaminated
Alu repeats, we compared the threshold cycle [C(t) value] of
this reaction to the the C(t) value of the single-copy COL2A1
control reaction using real-time PCR on 1:25 serial dilutions
of bisulfite-converted, M.SssI-treated DNA (Figure 3). The
Alu reaction fluorescence was detected ?15 cycles earlier
than the COL2A1 reaction on undiluted, bisulfite-converted
M.SssI-DNA. The Alu reaction, after a 1:15 000 dilution,
still could detect an appreciable amount of input DNA
compared with the COL2A1 reaction, which at the same
dilution failed to amplify M.SssI-DNA (Figure 3). To address
the variability of the Alu control reaction, we compared the
cycle threshold [C(t)] values of the Alu and COL2A1 control
reactions on M.SssI-treated PBL and HCT116 colon cancer
cell line DNAs. The mean C(t) values for Alu and COL2A1
reactionsonM.SssIDNAwere15.14 ± 0.21and29.71 ± 0.25,
respectively, and the mean C(t) values on HCT116 DNA were
15.57 ± 0.04 and 30.86 ± 0.25, respectively, These data dem-
onstratenotonlythe reproducibilityof the Alucontrol reaction
but also its high sensitivity. The greatly increased sensitivity
for detecting input DNA in the methylation-independent
reaction is especially useful when analyzing samples with
limited amounts of DNA. However, this Alu-based control
reaction does not increase the sensitivity of MethyLight-
based methylation detection, which is a function of the
methylation-specific MethyLight reactions. However, the
availability of a highly sensitive control reaction allows us
Figure 2. Strategy for designing an Alu-based MethyLight control reaction against the Alu consensus DNA sequence in Figure 1. Since CpGs in Alu repeats can
either be methylated (red) or unmethylated (green), one cannot distinguish if a CpG dinucleotide was subjected to evolutionary deamination of a methylated CpG
(yellow)orwasanunmethylatedCpGdeaminateddueto reactionwithbisulfite,asbotheventsresultin aTpGafterbisulfite-specificPCR.However,ifthebisulfite
PCRprimersaredesignedforamplificationofthestrandoppositetothatwhichwasevolutionarilydeaminated,aTpAsequenceresults(seeMethylation-Independent
Sequence, shaded in black), which is distinct from the PCR products from an unmethylated CpG (CpA or TpG) after bisulfite conversion.
6828 Nucleic Acids Research, 2005, Vol. 33, No. 21

Page 7

to determine the methylation detection sensitivity threshold
for difficult samples.
Development and evaluation of repetitive
element MethyLight reactions
We next developed MethyLight reactions to target methylated
Alu and LINE-1 elements, as well as the centromeric Sata and
juxtacentromeric Sat2 repeats (Figure 4). We also designed
MethyLight reactions specific for the unmethylated versions
of Alu, LINE-1 and Sata sequences (Figure 4); however, we
were unable to successfully develop a MethyLight reaction
specific for unmethylated Sat2 repeats.
Our primers for the unmethylated reactions were designed
by replacing CpG with TpG in the primers and probe. This
design approach does not distinguish between unmethylated
and evolutionarily deaminated CpGs in these repetitive ele-
ments. However, we assume that the fraction and genomic
location of deaminated CpG dinucleotides are fairly constant
in the human population, given the relatively recent evolution-
ary divergence of the human population, compared with the
origin of Alu repeats, which predate the divergence of prim-
ates. Nevertheless, we acknowledge that a low level of inac-
curacy in the measurements of unmethylated reactions may
stem from this inability to discriminate between evolutionary
and bisulfite-induced deamination. This problem does not
arise for the methylation-specific reactions described below.
We designed four reactions toward methylated Alu
sequences: the M1 reaction is designed toward an Alu repeat
within the S100A2 gene; the M2 and M3 reactions are directed
toward the consensus sequence (the locations of the ALU-M2
and ALU-M3 PCR amplicons are indicated in Figure 1); and
the M5 reaction is specific toward an Alu repeat located
upstream of the CASP8 gene. The S100A2 Alu sequence is
similar to AluSx and AluSq subfamilies, and the CASP8 Alu
sequence is most similar to the AluSp subfamily. We also
designed one reaction toward the unmethylated Alu consensus
sequence (ALU-U3). The methylated and unmethylated
Figure 3. EvaluationoftheperformanceoftheAlu-basedcontrolreactioncomparedwithasingle-copycontrolreaction.Serial1:25dilutionsofbisulfite-converted,
M.SssI-treatedDNAwereusedtocomparetheAluandCOL2A1controlreactionsbyreal-timePCR.ThefluorescenceisplottedversusthePCRcyclenumberforboth
reactions and each sample dilution is indicated.
Nucleic Acids Research, 2005, Vol. 33, No. 216829

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6830Nucleic Acids Research, 2005, Vol. 33, No. 21

Page 9

reactions specific for the LINE-1 sequences were based on a
LINE-1 consensus sequence (GenBank accession number
X52235). The Sata and Sat2 reactions were designed toward
sequences specifically on chromosome 1 (GenBank accession
numbers M38468 and X72623, respectively); however,
satellite-specific sequences on other chromosomes may also
be detected. Therefore, we classified these reactions generic-
ally as Sata and Sat2. Similar to the Alu control reaction, the
probes for the Sata and Sat2 reactions also contain a 30MGB-
NFQ moiety to satisfy the TaqMan probe melting temperature
requirements.
We tested the methylation specificities of the methylated
and unmethylated Alu, LINE-1, Sata and Sat2 reactions
on a panel of bisulfite-converted DNA samples (Figure 4).
PBL DNA treated in vitro with the CpG methylase M.SssI
(M.SssI-DNA) served as a methylated DNA template, and
untreated PBL and sperm DNAs were also included. To pre-
pare unmethylated DNA as a negative control for methylation,
we used a strategy that takes advantage of the WGA reaction
used to amplify minute amounts of DNA. An approach similar
to this has recently been described (43). WGA is based on
extending random hexamers annealed to genomic DNA with
the highly processive phi 29 DNA polymerase that contains
both 50–30and 30–50exonuclease (44) as well as strand dis-
placement activities (45,46). As genomic DNA is amplified
by this polymerase, the DNA methylation will be lost. We
amplified sperm and PBL DNAs by WGA, followed by bisul-
fite conversion.
We also included DNA from human cell lines that have been
previously characterized with regards to repetitive element
methylation. ICF lymphoblastoid cell lines were included
because they have extensive hypomethylation of chromosome
1 and 16 Sat2 sequences (47–49). In addition, we analyzed
DNA from HCT116 human colon cancer cells, HCT116
cellscontaining
DNMT1?/?
(D3bKO), DNMT1?/?and DNMT3B?/?cells (DKO) (50,51)
and HCT116 cells after treatment with the DNA methylation
inhibitor 5-aza-20-deoxycytidine (5-Aza-CdR). Global DNA
methylation is largely retained in the DNMT1?/?
DNMT3B?/?HCT116 cells, while DNA from the DKO
HCT116 cells is almost completely hypomethylated (50,51).
Alu sequences are detectably hypomethylated only in the
DKO cells, while Sat2 sequences were hypomethylated in
the single and the DKO cells (51). DNA from HCT116 cells
after 5-Aza-CdR-mediated DNA methylation inhibition should
be hypomethylated relative to the untreated HCT116 cells.
We evaluated the methylation specificity of each repetitive
element MethyLight reaction on the panel of 11 DNA samples
(Figure 4). The methylation values for the reactions directed
toward methylated repetitive elements were expressed as
PMR in which M.SssI-DNA was used as a methylated refer-
ence (39,40). For the unmethylated reactions, the amount of
unmethylated DNA is expressed as a PUR in which a WGA-
sperm sample was used as an unmethylated reference.
(D1KO),
DNMT3B?/?
and
Our results indicated that Alu, LINE-1, Sata and Sat2
repeats were highly methylated in M.SssI-PBL DNA as well
as PBL-DNA (Figure 4). Sperm DNA also showed substantial
methylationofAluand LINE-1sequences. However,Sataand
Sat2 sequences were hypomethylated in sperm relative to
PBL-DNA (Figure 4), consistent with previous reports
(26,33). These results are also in agreement with previous
reports of hypomethylation of Chr1 Sata and Chr1 Sat2
sequences in sperm (47–49). Substantial Alu and LINE-1
methylation was also detected in ICF cells, while Sata and
Sat2 methylation was not detected. While Alu methylation
and Sat2 hypomethylation in ICF cells is consistent with
previously reported studies (22,47,48), we could not detect
unmethylated Sata DNA in the ICF sample. This may reflect
the presence of either partially unmethylated or methylated
Sata DNA in ICF cells, as the unmethylated MethyLight reac-
tions were designed to recognize fully unmethylated target
DNA sequences.
All four unmethylated reactions showed hypomethylation
of the repetitive elements in both WGA-DNA samples, indic-
ating that these samples are appropriate unmethylated DNA
controls (Figure 4). However, centromeric and telomeric
regions of the genome are underrepresented in the WGA assay
(46). Although we detected a decrease in Sata and Sat2 input
levels compared with the Alu and LINE-1 sequences in WGA-
treated DNAs, there was still ample signal to accurately meas-
ure PMR and PUR values (data not shown).
We detected high levels of Alu and LINE-1 methylation in
HCT116 cells, while bothSata andSat2 sequences were hypo-
methylated relative to M.SssI-DNA. The hypomethylation of
satellite repeats in this cancer cell line is consistent with the
very frequent hypomethylation of these sequences in human
cancers (6). Alu and LINE-1 methylation was similar or
slightly reduced in the D1KO and D3bKO cells. Alu and
LINE-1 repeats, as well as both satellite repeats were strongly
hypomethylated in the HCT116 DKO cells, similar to previous
findings by Southern blot analysis (50,51). The HCT116 cells
also showed the expected increase in Alu, LINE-1 Sata and
Sat2 hypomethylation after treatment with 5-Aza-CdR.
Our methylated-specific and unmethylated-specific reac-
tions are designed to recognize fully methylated and fully
unmethylated repetive elements, respectively. However, due
to polymorphisms among the repetitive elements, and due to
variable levels of methylation of these repeats in human DNA
samples, it is difficult to determine exactly how many repeat
units in the genome are recognized by each of our reactions.
Nevertheless, we can obtain a good comparative measure of
the number of copies recognized by each reaction by compar-
ing the C(t) values of each reaction on its cognate optimal
target DNA (M.SssI-treated DNAfor methylated reactions and
WGA DNA for unmethylated reactions). For example, the
ALU-M2 and SAT2-M1 reactions both had C(t) values
more than 10 cycles lower than the LINE-1-M1 and SATa-
M1 reactions. Assuming an up to 2-fold amplification with
Figure 4. Evaluation of MethyLight reactions toward the methylated and unmethylated versions of Alu, LINE-1, Sata and Sat2 sequences on a panel of DNA
samples.Levelsofmethylation(inblack)areexpressedasPMRusingDNAtreatedwithM.SssIasamethylatedreference.LevelsofunmethylatedDNA(inwhite)are
expressedasPURinwhichaWGA-DNAsamplewasusedasanunmethylatedreference.Eachvaluerepresentsthemeanof3–6methylationmeasurements,except
for the ALU-M3 reaction, which is the average of two measurements. Error bars indicate the standard error of the mean and have been omitted for the ALU-M3
reaction,asindicatedbyanasterisk,sincewehaveonlytwoPMRmeasurementsforthisreaction.WedetectedPMRorPURvaluesof<0.01duetocross-reactivityof
the methylated or unmethylated primers to either unmethylated or methylated template DNA, respectively, except for the ALU-M5 reaction, which gave slightly
higher PMR values on the WGA-PBL DNA sample.
Nucleic Acids Research, 2005, Vol. 33, No. 21 6831

Page 10

each PCR cycle, this suggests that the ALU-M2 and SAT2-M1
reactions recognize up to 1000-fold more repeat elements
than either the LINE-1-M1 or SATa-M1 reactions. We anti-
cipate therefore that the ALU-M2 and SAT2-M1 reactions
would be superior surrogate measures of global genomic 5-
methylcytosine content than either the LINE-1-M1 or SATa-
M1 reactions.
Correlation between repetitive element methylation and
global DNA methylation levels
Using the MethyLight assay, each repetitive element reaction
was tested on a panel of DNAs from normal tissues (liver, lung,
kidney, spleen, thyroid and cerebellum) as well as ovarian
carcinoma (OvCa) and Wilms tumor (WT) samples, all of
which had been tested for global DNA methylation levels
by HPLC (Table 2). Global DNA methylation levels in humans
have been shown to be tissue-specific, with a range of
3.43–4.26% of cytosine residues methylated in normal tissues
(32,37). DNA samples were classified as hypomethylated if the
global methylation was between 3.20 and 3.40% and substan-
tially hypomethylated if the amount of global methylation was
<3.20%. However, these levels of hypomethylation were only
used to better describe DNA hypomethylation in human cells
and had no influence on the data analysis in this study.
We compared the PMR values (from reactions aimed at
methylated sequences) or PUR values (from reactions toward
unmethylated sequences) with HPLC-based global DNA
methylation measurements for each sample. While the PMR
values for all four methylated Alu reactions were significantly
associated with global genomic 5-methylcytosine content
(Figure 5A–D), linear regression analysis showed that the
Figure 5. Correlation of MethyLight-based measurements of each repetitive element with HPLC-based global DNA methylation measurements for the samples
describedinTable2.PMRvaluesforthemethylatedAlusequences(A–D),LINE-1(E),Sata(F),Sat2(G),arecorrelatedwithHPLCmeasurements.Thecorrelation
betweenthecompositemeanPMRvaluesofALU-M2andSAT2-M1reactionsandglobalmethylationmeasurementsisshownin(H).ThecorrelationofPURvalues
fortheunmethylatedAlu,LINE-1andSatasequenceswithHPLC-based5-methylcytosinemeasurementsareshownin(I–K).TheHPLCdatarepresentthemeanof
2–3 measurements and the MethyLight data represent the mean of three measurements. The MethyLight and HPLC-based methylation data were correlated using
linear regression analysis for each repetitive element.
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Page 11

ALU-M2 methylation reaction was most closely associated
with global DNA methylation as determined by HPLC
(correlation coefficient, r ¼ 0.70, P < 0.0001, Figure 5B).
ALU-M3, another Alu reaction based on the consensus
DNA sequence, was also correlative with global methylation
measurements (r ¼ 0.51, P ¼ 0.0083, Figure 5C). However,
the ALU-M2 fluorescence was greater than the ALU-M3
reaction (data not shown), suggesting that the ALU-M2 reac-
tion is superior not only in correlating Alu methylation to
global measurements, but also in PCR quality. Methylation
levels of LINE-1 and Sat2 sequences were also significantly
associated with global 5-methylcytosine content (Figure 5E
and G) but there was not a significant association of Sata
methylation with global DNA methylation (Figure 5F).
The ALU-M2 and SAT2-M1 reactions gave the best
correlation with global 5-methylcytosine content, as we had
anticipated from their relatively low C(t) values. Since these
two reactions both recognize a relatively large number of
repeat units, but represent quite distinct types of repetitive
elements, we considered that their combined measurement
could potentially provide a superior assessment of global
DNA methylation across various human genomic DNA sam-
ples. Both measurements are expressed as PMR values, so we
compiled a composite measurement by calculating the mean
PMR for the two reactions for each sample. This composite
measure yielded a remarkable improvement in the correlation
with HPLC measurements of 5-methylcytosine content
(r ¼ 0.85, P < 0.0001) (Figure 5H). We therefore recommend
that this composite measure be used for MethyLight-based
estimates of genomic 5-methylcytosine content.
The reactions directed toward unmethylated versions of Alu
and LINE-1 sequences were of borderline significance when
compared with global DNA methylation measurements
(Figure 5I and J). Unmethylated Sata repeats were also not
significantly correlated with HPLC-based global DNA
methylation measurements (Figure 5K). However, as expec-
ted, there was a clear trend of increased PUR measurements
with decreasing global DNA methylation levels for all
unmethylated repetitive element reactions.
Correlation between Sat2 MethyLight measurements
and Sat2 Southern blot-based hypomethylation
scores
We showed here a statisticallysignificant relationshipbetween
Sat2 methylation and global DNA methylation levels
(Figure 5G). Sat2 methylation had been previously determined
for these samples using Southern blot analysis in which
Sat2 hypomethylation was graded on a scale of 0 (methylated)
to 3 (extensively hypomethylated) using quantitation of
Phosphorimager data and evaluation of band patterns from
autoradiograms (33). We compared the MethyLight PMR
measurements with the corresponding DNA hypomethylation
scores from Southern blots for the same normal tissues, as well
as ovarian carcinoma and Wilms tumor samples (Figure 6).
Two ICF cell line DNAs were also included, as these samples
were previously shown to harbor Sat2 hypomethylation
(47–49). We found a statistically significant correlation
between the Sat2 MethyLight PMR measurements and the
Southern blot-based hypomethylation score (P < 0.0001),
suggesting that the MethyLight-based assay for Sat2
methylation measurements are highly consistent with the
Southern blot-based assay to determine Sat2 hypomethylation.
DISCUSSION
Repetitive elements comprise ?45% of the human genome,
withthe1millionAlusequencesaloneoccupying?10%ofthe
genome, and LINE-1 elements also representing a substantial
portion of the genome. Since these interspersed repetitive
elements as well as tandem repeated centromeric and juxta-
centromeric repeats contain numerous CpG dinucleotides, the
methylation status of these sequences is relevant to under-
standing global DNA methylation. It is generally thought
that repetitive elements are heavily methylated in normal
somatic tissues, but are methylated to a lesser extent in malig-
nant tissues, driving the global genomic hypomethylation
commonly found in human cancers.
In order to evaluate the repetitive element methylation in
normal and malignant tissues, we designed MethyLight reac-
tions specific for methylated Alu, LINE-1, and the Sata and
Sat2 repeats, as well as unmethylated versions of Alu, LINE-1
and Sata repeats. These reactions were first evaluated on a
panel of DNAs to test their methylation specificities. These
samples included HCT116 colon cancer cells as well as
HCT116 cells harboring gene-targeted disruptions of DNMT1,
DNMT3B or both DNMT genes (50,51). In support of previous
findings (50,51), DNMT1?/?and DNMT3B?/?cells retained
Alu methylation, but were hypomethylated in the HCT116
DKO cells. Sat2 hypomethylation had been described for
both the single and double knockout HCT116 cells when com-
pared with wild-type HCT116 cells (51). Our MethyLight data
directed toward this repeat showed similar findings.
More evidence of the specificity of the MethyLight-based
Sat2 repetitive element reaction was shown with the analysis
of cells from ICF patients. ICF cells exhibit hypomethylation
of Sat2 repeats on chromosomes 1 and 16; however, global
methylation is generally retained and Sata hypomethylation is
found in only some patients (47–49). Here, we also show that
Sat2 sequences were hypomethylated in ICF cells, while
Figure 6. MethyLight data (PMR) versus Southern blot-based Chr1 Sat2
hypomethylation densitometry scores. A score of 0, no hypomethylation;
1, small amounts of hypomethylation; 2, moderate hypomethylation; 3, strong
hypomethylation on 7 normal tissues, two ICF cell lines, one control cell line
and20cancertissuesamples(Wilmstumorsandovariancarcinomas).Thedata
points are indicated by the squares (hypomethylation score ¼ 0, upward
triangles (hypomethylation score ¼ 1), downward triangles (hypomethylation
score ¼ 2)anddiamonds(hypomethylationscore ¼ 3).MeanPMRvaluesare
indicated by the horizontal bars. The significance of the association of both
types of data after ANOVA analysis is shown.
Nucleic Acids Research, 2005, Vol. 33, No. 216833

Page 12

substantial levels of Alu and LINE-1 methylation remained.
While DNMT3B is required for normal Sat2 methylation
in vivo, both DNMT1 and DNMT3B physically interact
(52) and cooperatively maintain genomic methylation (51).
The MethyLight measurements for each repetitive element
were also compared with global 5-methylcytosine measure-
ments by HPLC for a panel of normal and tumor tissue sam-
ples. Of all the MethyLight reactions tested, the methylated
ALU-M2 reaction was most closely correlated with genomic
5-methylcytosine content, although all four reactions targeting
methylated Alu sequences exhibited a significant association
with global DNA methylation. MethyLight-based Sat2 and
LINE-1 methylation levels were also significantly correlated
to global DNA methylation. Therefore, methylation of diverse
repetitive elements may serve as surrogate markers for
genomic 5-methylcytosine content. Indeed, a composite meas-
ure combining ALU-M2 and SAT2-M1 PMR values yielded
much better correlation with genomic 5-methylcytosine con-
tent (r ¼ 0.85) than either measure alone. We recommend that
this composite measure be used for MethyLight-based estim-
ates of genomic 5-methylcytosine content.
A recent study from Yang et al. (18) describes the use of
COBRA and pyrosequencing assays to determine Alu and
LINE-1 methylation. Their PCR strategy can capture the com-
posite methylation information of ?15000 Alu sequences,
which represents ?0.1% of the genome. The authors suggest
that this measurement could serve as a surrogate for global
DNA methylation measurement, but they do not confirm this
with a validation using 5-methylcytosine determinations in the
same samples. Instead they show that 5-aza-CdR treatment
causes a reduction in their Alu measurement, which would be
expected, regardless of whether their assay is a good surrogate
for global DNA methylation or not. In our study, we have
included this important validation with HPLC measurements
of 5-methylcytosine content. Our MethyLight-based approach
is the first to provide quantitative estimates of the degree of
correlation between these repetitive element measurements
and 5-methylcytosine content.
An important other innovation of our MethyLight-based
methylation-specific assays is that the accuracy of these assays
is not affected by deamination at CpG dinucleotides, whether
by evolution or by bisulfite conversion, since our methylation-
specific reactions only recognize fully methylated, non-
deaminated CpG dinucleotides. The Yang method relies on
an indirect subtraction to distinguish between evolutionary
and bisulfite-mediated deamination.
Both COBRA and pyrosequencing require extensive post-
PCR manipulation, thereby substantially increasing the labor
intensiveness of the assay, and introducing the potential for
contamination of future reactions by PCR products. The
MethyLight assay is finished as soon as the PCR has been
completed, requires no post-PCR processing and can easily be
applied to hundreds or thousands of samples. These repetitive
element reactions represent advances in the MethyLight assay
not only for determining the methylation of individual repet-
itive elements, but also in serving as markers for global
5-methylcytosine content.
Pyrosequencing, COBRA and MethyLight provide dif-
ferent perspectives on DNA methylation determinations:
pyrosequencing and COBRA are used to quantitatively meas-
ure methylation levels of individual CpGs within a given
locus, while MethyLight assays measure the percentage of
molecules in which all of the CpGs of a specific locus (usually
between 4 and 10 CpGs) are either methylated or unmethyl-
ated. COBRA and pyrosequencing (18) platforms showed that
Alu or LINE-1 methylation provides a marker for 5-Aza-CdR-
mediated demethylation in human cancer cell lines. Unlike the
MethyLight data shown here, it is indefinite whether COBRA
and pyrosequencing assays could serve as surrogate markers
for determining global methylation.
The measurements of repetitive element hypomethylation
targeted by unmethylated MethyLight reactions were not sig-
nificantly correlated with 5-methylcytosine content, although
there was a trend of increasing levels of unmethylated repeat
sequenceswith decreasing 5-methylcytosinecontent.Thepoor
correlation may be due to relatively low numbers of com-
pletely unmethylated repetitive elements in vivo. In addition,
these reactions generated low output fluorescence signals, and
this may be due to difficulties in design and performance of
MethyLight reactions specific for unmethylated sequences.
These sequences have a higher A:T content than methylated
sequences due to the fact that all unmethylated cytosines are
converted to thymines after bisulfite conversion. This may
reduce primer-template specificity and PCR efficiency.
The MethyLight-based Sat2 methylation data were signi-
ficantly associated with HPLC-based global DNA methylation
levels and also with Southern blot-based Sat2 hypomethyla-
tion scores. Therefore, Sat2 methylation analysis by MethyL-
ight is a strong indicator of global DNA methylation meas-
urements. This is in agreement with previous findings that the
Southern blot-based Sat2 hypomethylation index is also stat-
istically significantly associated with global DNA hypo-
methylation levels in numerous human cancer tissues
(27,29,37,53)
We did not find a statistically significant correlation
between Sata methylation by MethyLight and genomic 5-
methylcytosine content. Previously, a significant association
between Southern blot-determined Sata hypomethylation and
global DNA methylation was shown in ovarian cancers,
Wilms tumors and breast cancers (27,29,53). The discrepancy
between these sets of data may be due to inherent assay dif-
ferences. The MethyLight reaction design is based on a short
DNA sequence and may not be specific toward the Chromo-
some 1 Sata sequence when compared with the hybridization
probe used for the Southern blot assay. Moreover, the relat-
ively high C(t) value for the Sata MethyLight reaction indic-
ates that a relatively small number of Sata repeats contribute
to the MethyLight measurement.
We also show here that Sata and Sat2 repeats are unmethyl-
ated in sperm. These data are consistent with previous findings
of satellite hypomethylation in sperm based on Southern blot
assays (26,33). However, the findings of Hassan et al. (49)
demonstrated that there was sporadic methylation of Sat2
sequences in sperm, using bisulfite genomic sequencing. Par-
tial Sat2 methylation may not be identified by this MethyLight
assay, since the PCR specifically targets sequences containing
multiple fully methylated CpGs.
The findings presented here also provide further insight into
DNA hypomethylation in human cancers. Hypomethylation of
diverse classes of repetitive elements, such as interspersed and
tandemly repeated sequences, occurs together with regional
CpG island hypermethylation in cancer cells (33,37). DNA
6834Nucleic Acids Research, 2005, Vol. 33, No. 21

Page 13

hypomethylation is not restricted to satellite sequences and
other tandem DNA repeats (6,54), but also includes the inter-
spersed Alu and LINE elements. Repetitive element methyla-
tion, however, may be influenced by the local chromatin
structure, especially for Alu and LINE-1 sequences, which
are interspersed throughout the genome and can be embedded
within genes. A study from Kondo and Issa (55) showed evid-
ence of histone H3 lysine 9 methylation, a marker for inactive
heterochromatin, at numerous Alu repeat sequences.
While Alu methylation was most closely associated with
global methylation levels in our study, not all Alus are
hypomethylated in human cancers. An Alu sequence located
upstream of the extensively methylated CDKN2A promoter
was found to be hypermethylated in cancer cell lines (22),
and an Alu sequence located in intron 6 of TP53 showed
extensive methylation (22,23). In support of this, a recent
study showed that NBL2, a tandem repeat found in acrocentric
chromosomes, can become either hypermethylated or hypo-
methylated in human cancers (54).
We also describe here the development of a novel strategy to
designanAlu-basedcontrolreactionfor measuringthe levelsof
bisulfite-converted input DNA for MethyLight assays by which
bisulfite and evolutionary deaminated sequences could be dis-
tinguished. The Alu-based control reaction shows fluorescence
above background levels at ?15 cycles earlier than the single-
copy COL2A1 control reaction, suggesting an increased detec-
tion of four orders of magnitude (?10000 copies). This rep-
resents ?1% of total Alu content. Using human colon cancer
cells,Yangetal.(18)havededucedthatalargenumberofCpGs
in Alu sequences are evolutionarily deaminated, supporting our
results in which a large number of deaminated Alu repeats were
detected using this control reaction.
Although the Alu-based control reaction does not increase
the sensitivity of the MethyLight methylation reactions and
hence the sensitivity of methylation detection, it is useful for
determining relative DNA amounts in specimens where the
quantity and/or quality of DNA may be limited, such as
formalin-fixed, paraffin-embedded tissues or free tumor DNA
in plasma/serum. Therefore, a methylation detection sensitiv-
ity threshold for such samples can be determined. More
importantly, the Alu control reaction is expected to be a more
stable and reliable measure of input bisulfite-converted DNA
levels than reaction toward a single-copy sequence when ana-
lyzing tumor samples with local amplifications or deletions,
since single-copy genes may not always be present at diploid
copy number levels in human cancers, where chromosomal
alterations are frequent events. The high copy number of
interspersed Alu repeats in the human genome makes it
unlikely that cancer-related sequence abnormalities would
substantially influence their PCR yield. The use of the Alu-
based control reaction should therefore result in a more
stable determination of PMR/PUR values. Moreover, Alu
and other high copy repetitive element sequences may also
be useful in measuring changes in gene dosage, such as gene
amplifications.
In conclusion, the design and application of MethyLight
assays to measure repetitive element methylation represent
novel technical advancements. Their use as surrogate markers
for global DNA methylation makes them attractive in analyz-
ing the effects of DNA methylation on human disease in
population-based studies.
ACKNOWLEDGEMENTS
The authors thank Dr Bert Vogelstein for providing the
HCT116, HCT116 DNMT1?/?, HCT116 DNMT3B?/?, and
HCT116 DNMT1?/?, DNMT3B?/?double knockout cells.
This work was supported by NIH grants CA 81506 (to
M.E.) and ES 011672 and CA 96958 (to P.W.L.).
Conflict of interest statement. None declared.
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