Expanded methyl-sensitive cut counting reveals
hypomethylation as an epigenetic state that
highlights functional sequences of the genome
Alejandro Colaneria,1, Nickolas Staffaa, David C. Fargob, Yuan Gaoc, Tianyuan Wanga, Shyamal D. Peddadad,
and Lutz Birnbaumera,1
aLaboratory of Neurobiology,bLibrary and Information Services, anddBiostatistics Branch, National Institute of Environmental Health Sciences, National
Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC 27709; andcDivision of Genomics, Epigenomics, and
Bioinformatics, The Lieber Institute for Brain Development, and Neuroregeneration and Stem Cell Program, Institute for Cell Engineering, The Johns Hopkins
University, Baltimore, MD 21205
Contributed by Lutz Birnbaumer, April 13, 2011 (sent for review March 3, 2011)
Methyl-sensitive cut counting (MSCC) with the HpaII methylation-
sensitive restriction enzyme is a cost-effective method to pinpoint
unmethylated CpGs at single base-pair resolution. However, it has
the drawback of addressing only CpGs in the context of the CCGG
site, leaving out the remainder of the possible 16 XCGX tetranu-
cleotides in which CpGs are found. We expanded MSCC to include
three additional enzymes to address a total of 5 of the 16 XCGX
combinations. This allowed us to survey methylation at about one-
third of all a mammalian genome’s CpGs. Applied to mouse liver
DNA, we correctly confirmed data reported with other methods
showing hypomethylation to be concentrated at promoters and in
CpG islands (CGIs), with gene bodies and intergenic regions being
mostly methylated. Grouping unmethylated CpGs, characterized
by high MSCC scores (7% false discovery rate), we found a large
number of unmethylated regions not qualifying as CGIs located in
intergenic and intronic regions, which are highly enriched in func-
tional DNA sequences (open regulatory annotation database) as
well as in noncoding yet highly conserved mammalian sequences
thought to be important but with as yet unknown function. About
50% of MSCC-defined unmethylated regions do not overlap algo-
rithm-defined CGIs and offer a novel search space in which new
functionalities of DNA may be found in health and disease.
methylome|single CpG|genome-wide|functional annotation|repetitive
a widespread modification in the vertebrate genomes (1).
A family of DNA methyltransferases whose primary targets are
the cytosines located at CpG dinucleotides catalyzes this chem-
ical modification (2). Many CpGs are not distributed at random,
because a significant proportion of them have coalesced into
what has been called CpG islands (CGIs), where they are mostly
hypomethylated, whereas CpGs outside of CGIs are mostly
methylated (1, 3–5). CGIs are identified with computer algo-
rithms that search for shared distinctive properties; traditionally
CpG and (G + C) richness (5–8). A different approach selects
CGIs between clusters of CpGs whose maximum inter-CpG
distances are below a threshold (e.g., median genomic inter-CpG
distance) (9). The filtering criteria used by all these programs
seek to optimize the possibility that the selected CGIs are not the
product of chance but the result of evolutionary processes. The
most widely accepted explanation for the origin of CGIs is based
in the tendency of 5-methylcytosines to undergo spontaneous
deamination to uracil producing C-to-T mutations. This process
drove a nonselective purge of CpGs from the broadly methylated
genomic sequences with no evolutionary constraints (10, 11) (SI
Appendix, Tables S1, S2, and S3). However, this purge did not
occur in regions rich in regulatory elements that have been
protected from being methylated. According to this simplified
and generally accepted hypothesis, the functionality of a CGI is
measured by the probability of finding it unmethylated. For ex-
ample, the program called CpGCluster uses a statistical criterion
ethylation of DNA at position five of the cytosine ring is
(P value) to select for clusters with low probability of having been
formed by chance (9). This means that these loci have retained
their CpG density during evolution, presumably because of their
prevalence in an unmethylated state. CGIs, including CpGCluster
CGIs with the lowest P values, are more frequently found over-
lapping promoters (12), which supports connections between
evolutionary origin, unmethylated state, and functionality. The
idea that active promoters protect their CpGs from being meth-
ylated is supported by site-specific mutagenesis experiments. For
example, mutations that prevent the transcription factor Sp1 from
recognizing and binding its target sequences in a particular CGI
remove the protection of that CGI from DNA methylation (13).
of CGIs aim to identify functionally relevant epigenetic loci in de-
velopment and disease; as a consequence, CGIs still constitute the
framework on which the majority of researchers base their high-
throughput methylation analyses. However, the filtering criteria
that can be disrupted or activated by changes in methylation.
Inspection of the mammalian genome shows it to be divided
into two classes of subsequences. In one class (85% of the ge-
nome), CpGs are sparse (one every 250 bp). The other class
(15% of the genome) concentrates half of the total genomic
CpGs at an average inter-CpG distance of 40 bp. At this density,
methylation has been shown to have a deleterious effect on the
functionality of the DNA elements (14). These relative CpG-rich
subsequences accommodate the totality of the CGIs, regardless
of the algorithms used to define them, and overlap with 95% of
the RefSeq-defined transcription start site (TSS) regions (−3 Kb
to +2 Kb from the TSS).
Our goal was to design a method that allows us to identify
targets of methylation-mediated epigenetic processes throughout
the genome without having to select a priori candidate sub-
sequences. We developed a high-throughput sequencing-based
DNA methylation analysis, which consists of an expanded (more
comprehensive) version of the methyl-sensitive cut counting as-
say (MSCC) (15, 16). Applied to mouse, our method identifies
the methylation status of 6 million CpGs (one-third of all existing
CpGs) and covers 58% of the CpG-rich subsequences.
We found that a surprising proportion (50%) of our unme-
thylated regions (UMRs) do not meet the traditional CGI cri-
Author contributions: A.C. designed research; A.C. performed research; N.S., D.C.F., Y.G.,
and T.W. contributed new reagents/analytic tools; A.C., N.S., D.C.F., T.W., S.D.P., and L.B.
analyzed data; and A.C. and L.B. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or birnbau1@
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1105713108/-/DCSupplemental. Tab-delimited file for Table S6 available upon
| June 7, 2011
| vol. 108
| no. 23
teria. Interestingly, most of these non-CGI UMRs are located in
noncoding DNA outside of promoters and are more enriched in
experimentally determined regulatory sequences than CGI-like
UMRs (UMRs that overlap predicted CGIs). At least 10% of the
UMRs identified through MSCC contain literature-published
liver-specific and liver-related regulatory sequences.
Results and Discussion
Coverage. CpG tag libraries prepared from DNA digested with
four methylation-sensitive restriction enzymes (4Enz; SI Appen-
dix, SI Materials and Methods) were sequenced by Illumina Ge-
nome Analyzers reporting the sequences returned by individual
sequencing channels of their flow cells. The data analyzed here
were obtained from sequencing three CpG tag libraries, yielding
three datasets that we called Slax1, Slxa2, and Slxa3 (SI Appen-
dix, Table S4). The majority of the data analyzed below are from
the Slxa3 dataset returned to us from five sequencing channels,
which, when pooled, rendered 29 million reads that mapped to
unique nonrandom CpGs (SI Appendix, Tables S4 and S5). The
number of reads per identified CpG (MSCC score, see below)
was found to vary from site to site because of disparities in the
level of methylation, but a minimum coverage is required to be
able to perceive these differences. We used the reads recovered
from lambda phage (lambda) CpGs to gauge the coverage and
followed the behavior of the 1,202 unmethylated lambda CpGs
addressable by 4Enz during the preparation of our CpG tag li-
braries. A read frequency map of the 22,728 reads that mapped
to the lambda genome is shown in Fig. 1A. We identified 92%
(1,107 hits) of the 4Enz CpGs with at least 1 read and an average
reads-per-hit ratio of 20.5 ± 16.1 (mean ± SD). Thus, although
the measures come from equally unmethylated sites, not all were
identified with similar frequency. The read values were scattered
between a minimum of 0 (8%) and a maximum of 99. For the
mouse genome, whose methylation status is unknown, it is nec-
essary to discriminate between highly methylated CpGs and
CpGs that were poorly covered. Because we aligned the reads
returned by each of the five channels separately, we classified
the CpGs according to the number of channels in which they
were identified, revealing a relation between the experimental
coverage and the number of channels in which each CpG was
identified. The hits were grouped into three classes: one- to
three-channel hits, four-channel hits, and five-channel hits (Fig. 1
B and C). We compared the number of CpGs identified in the
mouse and lambda genomes according to these criteria. At a
sequencing depth of about 30 million mapped reads from five
channels, for lambda (100% unmethylated), the majority of the
CpGs (61%) were identified by reads that came from all five
channels and only a small portion (16%) from one to three
channels. The proportion of lambda one- to three-channel hits
plus the proportion of not identified CpGs estimate the failure
rate of the method. In contrast to lambda, we found only 24% of
mouse hits in five channels. The majority of which (63%) were in
one to three channels at an average of 2.5 reads per hit, which
reflects the widely methylated status of the genome and the
sensitivity of the method to detect CpGs even when they are
heavily methylated (Fig. 2). The box-and-whisker plots in Fig. 1
B and C show that CpGs located in the same class were identified
with a similar level of reads. Interestingly, the MSCC scores
(reads per hit) recorded for mouse or lambda five-channel hits
show a similar distribution of values (median MSCC score of
24.5 for lambda and 23.2 for mouse). The distributions of MSCC
scores obtained from the mouse and lambda genomes are com-
pared in Fig. 1D, which reflect the difference between a com-
pletely unmethylated genome and one with different levels of
hypomethylation. For the mouse genome, we obtained higher
frequencies of CpGs identified with lower numbers of reads.
However, when we compared lambda CpGs against those lo-
mouse. (A) Plots of reads per hit (identified CpGs) along the lambda genome.
(Upper) Reads-per-hit plot of identified forward and reverse tags. (Lower)
Combined forward and reverse reads per hit. (B) Box-and-whisker plots
(median, quartiles, and fifth and 95th percentiles) representing the reads-
per-hit distribution as a function of the number of channels in which the
CpG-identifying reads were found in the lambda genome. (C) Same as in B
but for CpGs detected in the mouse genome. (D and E) Frequency histo-
grams of the reads per hit recovered for CpGs identified with 4Enz in the five
sequencing channels. ch, channel. (D) Distribution of read recovery from the
lambda genome is compared with that of reads recovered from the whole-
mouse genome. (E) Distribution of read recovery from the lambda genome
is compared with that of the reads per hit found in CpGs that were located in
UMRs of the mouse genome validated by bisulfite sequencing analysis.
Analysis of reads that mapped to lambda DNA: comparison with
a genome-wide scale. The MSCC data from the genomic region
spanning the Gnas complex locus are shown. The promoters for
Nesp, Nespas, Gnasxl, and Exon1A lie within differentially
methylated regions (DMRs) that have been identified in the lo-
DMR containing exon 1A. Tick bars at the bottom of the figure
indicate the positions of CpGs and 4Enz CpGs. The majority of
the sites are largelyresistantto4Enz, except for those located in
three regions colocalizing with the described promoters. Low
avgMSCC scores for two regions outside the three major UMRs
Bisulfite sequencing analysis confirms this conclusion.
Single CpG resolution profile of hypomethylation on
| www.pnas.org/cgi/doi/10.1073/pnas.1105713108Colaneri et al.
cated in the newly discovered mouse UMRs, the MSCC score
distributions showed no difference (Fig. 1E). By comparing dif-
ferent CpG tag libraries in which the same amount of lambda
DNA was introduced as an internal standard, we found that
certain sites systematically perform better or worse than others
(SI Appendix, Fig. S2). Although this systematic bias increases the
variability above that expected from the Poisson distribution, it is
shown below that the level of methylation of a CpG under study
is the primary parameter determining the final number of reads.
Assigning Methylation Status at Single-CpG Resolution. The 4Enz
set of methylation-sensitive restriction enzymes accurately tar-
gets 6 million CpGs located in the context of five different pat-
terns (CCGG, ACGT, GCGC, CCGC, and GCGG) and collects
information about their methylation state. The number of CpGs
and the genomic regions that can be studied (addressable CpGs)
depend not only on the number of restriction enzymes used but
on the number of CpGs with unique tags (SI Appendix, Tables
S1, S2, S3, S4, and S5). Although the abundance of any in-
dividual tag in the CpG tag library is expected to be inversely
proportional to the methylation state of the addressed CpG (15,
16), the demonstrated local bias impairs the usefulness of the
method to perceive moderate variations in levels of methylation
within a genome (SI Appendix, Fig. S2). Despite this limitation,
the method is sensitive to detect the small fraction of hypo-
methylated CpGs in the genome (Fig. 2). We used receiver op-
erating characteristic (ROC) analysis to visualize and compare
the performance of the method to classify CpGs in different
categories of methylation according to their MSCC scores. ROC
curves (Fig. 3) were built following the strategy described in SI
Appendix, SI Materials and Methods using the MSCC scores and
methylation status of a panel of 358 CpGs validated by bisulfite
Fig. 3 shows the area under the curve (AUC) plots for two
situations: detection of sites <75% methylated and detection of
sites <25% methylated. The AUC in Fig. 3A is 0.89 ± 0.02 and
represents the probability that a randomly selected hypomethy-
lated CpG (<75% methylation) will score a higher number of
reads than a randomly selected heavily methylated CpG (>75%
methylation). We found a MSCC score of 11 as the optimal
cutoff for this classification (SI Appendix, Fig. S3A). A similar
analysis to detect mostly unmethylated sites (<25% methylation)
(Fig. 3B and SI Appendix, Fig. S3B) showed that using an MSCC
score of 17 as a cutoff allows us to classify 67% of CpGs correctly
as mostly unmethylated with a false discovery rate (FDR) of
7.2% (Fig. 3C). Single-CpG MSCC scores are affected by local
systematic bias (SI Appendix, Fig. S2); however, CpGs with a
tendency to be overestimated or underestimated are randomly
distributed in the genome. When the level of methylation of
a discrete region of the genome is measured by averaging the
counts from the individual CpGs, the bias tends to cancel. For
this reason, the larger the number of CpGs that can be covered
in the MSCC analysis, the higher is the accuracy with which the
method evaluates the level of unmethylation of a given region.
The average MSCC (avgMSCC) score was thus used to
quantify the level of hypomethylation of two regions known to
differ by 50% in their methylation status as a result of imprinting
(17) (SI Appendix, Fig. S4). As recorded in three independent
experiments, the differentially methylated 1A domain of the
Gnas locus produced an avgMSCC score that was one-half of
that scored for the neighboring completely unmethylated exon 1
domain (SI Appendix, Fig. S4). We concluded that avgMSCC
scores reflect the hypomethylation status of a region accurately
Distribution of Hypomethylation in the Genome. We mapped 29
million reads to 3 million unique CpG tags (SI Appendix, Table
S5) and built a read frequency table (all-CpGs-all-hits frequency
table; SI Appendix, Table S6) listing all addressable CpG tags
(forward and reverse) and the number of times that each unique
tag was identified. The MSCC threshold values derived from
ROC analysis were used to assign methylation status to each
CpG listed in this table, giving a distribution of hypomethylation
throughout the entire genome.
The landscape that emerged from this analysis is in complete
agreement with the known genome-wide mosaic pattern showing
heavily methylated sites sharply separated from hypomethylated
sites (Fig. 2 and SI Appendix, Fig. S4). However, the hypomethy-
lated regions corresponded poorly with predicted CGIs. We an-
alyzed the degree of hypomethylation in four different sets of
CGIs: Gardiner-Garden and Frommer (GG&F), Takai and Jones
(T&J), CpGCluster CGIs, and Epi-CGIs, with the last being based
on an algorithm that combines the GG&F criterion with in-
formation gathered from epigenetic marks (6–9) (Fig. 4).
Although our method can interrogate, on average, one of
every two CpGs located in CGI-like loci, not all CGIs are in-
terrogated to the same extent (Fig. 4 A and Inset). We included
in our analysis those CGIs in which we can address at least one-
third of the total CpGs (79% of T&J, 52% of GG&F, 56%
of CpGCluster CGIs, and 60% of Epi-CGIs; SI Appendix,
Table S8). Fig. 4A shows the distribution of the degree of
unmethylation (avgMSCC score) for 16,731 of the mouse
genome’s T&J CGIs. According to the scores, the T&J set par-
titions into 14,993 (∼90%) hypomethylated islands [MSCC
score: 21.5 ± 4.6 (mean ± SD)] and 1,738 (∼10%) heavily
methylated islands. Bisulfite sequencing confirmed this partition
of CGIs (SI Appendix, Fig. S5). The majority of T&J CGIs are
located in promoter regions (SI Appendix, Table S7). This bias is
probably what makes the T&J set very specific in terms of pre-
dicting UMRs; however, sensitivity is a concern for this algo-
rithm. We performed the same analysis for the methylation
status of CGIs originated with the CpGCluster algorithm (Fig.
4B). This set is three times larger than the T&J set, but 58%
(39,022) of them were found to be heavily methylated, whereas
only 32% (21,386) were found to be unmethylated [avgMSCC
score: 20.7 ± 6.4 (mean ± SD)]. Notice that whereas the T&J
algorithm was more effective in predicting UMRs, it missed
data. (A) MSCC scores are used to classify CpGs as hypomethylated (<75%
methylation) or heavily methylated (>75% methylation). (B) Same as in A
but classifying CpGs as mostly unmethylated (<25% methylation) or not. (C)
Summary of the results when an MSCC score of 11 or 17 is selected as the
optimal cutoff for the classification process. The true-positive rate indicates
CpGs with a methylation rate <75% (25%) and an MSCC score >11 (or 17)/
CpGs with a methylation rate <75% (or <25%). The true-negative rate
indicates CpGs with a methylation rate >75% (or 25%) and an MSCC
score <11 (or 17)/total CpGs with a methylation rate >75% (or 25%). The
false-negative rate indicates CpGs with a methylation rate <75% (or 25%)
and an MSCC score <11 (or 17). The false-positive rate indicates CpGs with
a methylation rate >75% (or 25%) and an MSCC score >11 (or 17).
ROC analysis generated from MSCC scores and bisulfite sequencing
Colaneri et al.PNAS
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| vol. 108
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classifying a substantial number of CGIs compared with
CpGCluster (8,926 CGIs). We found that 70% of these 8,926
unmethylated loci are located in introns or intergenic regions. We
extended this analysis to GG&F CGIs and Epi-CGIs (SI Appendix,
Table S8). The methylation profile of the GG&F CGIs resembled
that of CpGCluster CGIs (Fig. 4B); however, the number of
unmethylated loci increased to 23,318 (SI Appendix, Table S8). The
Epi-CGI algorithm appears to be as effective as the T&J algo-
rithm in predicting unmethylated islands (67% of the Epi-CGIs
analyzed); even though it finds the smallest number of unme-
thylated loci (11,191), these are less biased toward TSS regions
compared with the T&J set (SI Appendix, Tables S7 and S8).
Reproducibility. The avgMSCC score of five-channel hits in our
dataset (Slxa3) was 28.3. Thus, five-channel hits identify unme-
thylated CpGs. We found that of 199,618 HpaII five-channel hits
with an avgMSCC score of 29.5, 104,701 hits mapped to 15,098
(unmethylated) T&J CGIs. In an independent experiment se-
quenced to a similar depth (Slxa2), we found that 90,301 HpaII
five-channel hits mapped to 14,303 T&J CGIs. A common set of
14,054 T&J CGIs was identified in the two experiments (Fig.
4C), indicating that the MSCC approach is a highly reproducible
tool to identify UMRs.
Genome-Wide Annotation of Experimentally Determined Hypomethyl-
ated Regions. We made the CpGCluster program (9) specific for
it to cluster all CpGs identified in five channels. This group com-
scored more than 10 reads (Fig. 1C). The modified CpGCluster
distances (SI Appendix, Fig. S6). If all 6 million addressable CpGs
were randomly distributed, thedistancesbetween neighboring sites
should follow the geometrical distribution with a mean intersite
separation of 311 bp. We set the distance to 300 bp to search for
hypomethylated CpG clusters (w300 clusters) and found 559,901
hypomethylated CpGs grouped in 64,266 clusters (SI Appendix,
Table S9). Although the five-channel CpGs included in these clus-
an avgMSCC score of 6.9. This finding is an indication that certain
clusters could have a considerable number of CpGs with high rates
ofmethylation(SIAppendix, Fig. S7B and SI Appendix, Table S10).
Isolation and Analysis of UMRs. We showed that single CpGs with
MSCC scores ≥17 can be classified as mostly unmethylated
(<25% methylation), generating a low number of false-positive
results (7% FDR). We reasoned that a hypomethylated region
with an avgMSCC score ≥17 will have the majority of its CpGs in
an unmethylated state. After calculating and applying this cutoff,
we ended with a set of 46,804 UMRs that span 22 million bp of
the mouse genome and include 1.3 million unmethylated CpGs
(SI Appendix, Tables S9 and S11). A number of studies have
shown that CpGs located in close proximity tend to share
a common methylation state (15, 18, 19). The penetrance of this
correlation increases with the proximity of neighboring CpGs.
We found that 97% of nonaddressable CpGs located in UMRs
have at least 1 addressable CpG within a distance of 100 bp. The
correlation between methylation states for CpGs within a dis-
tance of 100 bp has been estimated to be ∼75% (18). Bisulfite
sequencing analysis of randomly selected UMRs confirms the
comethylation phenomenon (SI Appendix, Figs. S7 and S8). We
conclude that the UMRs with avgMSCC scores ≥17 constitute
a set of mostly unmethylated sequences.
We analyzed the colocalization of UMRs with predicted CGIs
and classified them in non-CGI UMRs (do not overlap) and CGI-
like UMRs (overlap). The result showed a poor correspondence.
For example the T&J and CpGCluster CGIs fail to detect 75%
and 60%, respectively, of the experimentally determined UMRs
(SI Appendix, Table S9). Indeed, 52% of the UMRs could not
be detected by any of the CGI-defining algorithms (Fig. 5A). The
high failure rate, even after combining different CGI sets, sug-
gests that the algorithms are failing to include one or more critical
features. We hypothesize that protein-binding DNA elements
must be a ubiquitous feature shared by all the UMRs.
The original quantitative criteria used to define CGIs were
based on a small set of sequences, likely biased by the limited size
of the 1985 GenBank database (4, 7). Later programs readjusted
thresholds and changed how the edges of CGIs are defined
(5, 8). However, most of these new computational methods still
CpGs represent one-third of the total CpGs, were selected for this analysis.
At each CGI, the average reads-per-hit ratio was calculated, and the distri-
bution of these ratios is represented as a frequency histogram (black dots).
The red curve is the result of fitting a Gaussian model to the data. Means
and SDs were calculated from this model. (Inset) Distribution of the frac-
tional addressability of CpGs among T&J CGIs. The box-and-whisker plot
depicts the median, first and third quartiles, and fifth and 95th percentiles.
(B) Same as in A but analyzing CGIs predicted by CpGCluster. (C) Repro-
ducibility in the identification of unmethylated T&J CGIs with HpaII hits in
two experiments (SI Appendix, Table S4). The 5-ch HpaII hits of the Slxa2
experiment collected 4,396,101 reads; those of the Slxa3 experiment col-
lected 5,904,634 reads. 5-ch, five-channel.
Analysis of methylation at CGIs. (A) T&J CGIs, wherein addressable
liver genome. (A) Distribution of UMRs in the major genomic compartments:
TSS regions (−3 Kb to +2 Kb of the TSS) and 3′ region of genes (3 Kb). For this
segmentation, the RefSeq definition of genes was used. If a UMR is not
completely included in one of the five categories, it was labeled as “other.”
UMRs were classified as overlapping or not in the combined set of CGIs (T&J,
GG&F, or CpGCluster). (Inset) Distributions of the average inter-CpG distance
calculated for each UMR, represented as box-and-whisker plots depicting
the median, quartiles, and fifth and 95th percentiles. (B) Size distribution of
UMRs and comparison with CGIs. Box-and-whisker plots (median, quartiles,
and fifth and 95th percentiles) represent the distribution of sizes for UMRs
that were completely included in the indicated genomic regions. (C and D)
Scatter plots represent the (C + G) content vs. Obs/Exp CpG ratio in UMRs
that overlap and do not overlap CGIs. Note the large proportion of UMRs
that do not meet CGI criteria. Obs/Exp, observed/expected.
CGI-like and non-CGI UMRs as detected genome-wide in the mouse
| www.pnas.org/cgi/doi/10.1073/pnas.1105713108Colaneri et al.
rely on the three initially considered DNA features: Obs/Exp
CpG ratio, G + C content, and length. We found these three
parameters to be highly variable among the UMRs (Fig. 5). For
example, many non-CGI UMRs meet the first two criteria
mentioned, but all of them have sizes below the 500 bp required
by the T&J algorithm (Fig. 5C, second quadrant). Also, many
UMRs do not overlap CGIs but meet the size required by dif-
ferent algorithms (SI Appendix, Table S9). This shows that the
size (base pairs) of a region rich in CpGs should not be used as
a filter to select for putatively functionally important unmethy-
lated sequences. The size of the UMRs was found to be primarily
a function of their genomic location, with larger UMRs over-
lapping TSSs and smaller UMRs being located in intergenic
regions (Fig. 5B). On the other hand, there are UMRs that
overlap predicted CGIs but the CpG and G + C richness are
below the CGI thresholds (Fig. 5D, quadrants 1, 3, and 4), sug-
gesting that in addition to colocalizing, they cover substantially
different sequences. The inability to predict the edges of CGIs
seems not to be trivial. It has been reported that tissue- and
cancer-specific differentially methylated regions map to the
shores of CGIs (20). Recently, computational algorithms have
incorporated hidden Markov models (HMMs) to improve the
detection of CGI borders. Many of the HMM CGIs incorporated
the shores of previously defined islands (5). Although this new
strategy predicts a relative large number of CGIs, the majority
(75%) do not overlap our experimentally determined UMRs.
Our description of thousands of UMRs not qualifying as CGIs
outside of promoter regions is in agreement with the previous
discovery of nonpromoter UMRs subject to tissue-specific de
novo methylation and suggests the existence of additional tissue-
specific UMRs not evident in liver (21).
The relation between CpG density and methylation has been
studied in promoter and nonpromoter regions. Whereas pro-
moters with high CpG density (more than one CpG every 20 bp)
are found to be primary unmethylated, the methylation rate at
single-copy DNAs outside promoters was found to increase with
the CpG density until a threshold value of 0.025 (one CpG every
40 bp) was reached; beyond this threshold, the methylation rates
fell sharply (19). Interestingly, we found that CGI-like UMRs
and non-CGI UMRs differ in their CpG density. CGI-like
UMRs have a median inter-CpG distance of 18 bp, which is just
above the expected distance for the CpG dinucleotide in a DNA
sequence with identical base composition (A = T = C = G). In
contrast, the median distance between CpGs in the non-CGI
UMRs is 34 bp (Fig. 5A, Inset). Whether these differences have
a relationship to the functionality of the underlying sequences has
to be determined in future experiments. On the other hand, there
is a clear pattern in how these two kinds of UMR partition among
different noncoding compartments of the genome (Fig. 5A).
Whereas 55% of CGI-like UMRs localize to TSS regions, only
12% of non-CGI UMRs do. Whereas less than 35% of CGI-like
in exons, introns, and intergenic regions was unexpected. Introns
and intergenic sequences account for almost 98% of mammalian
genomes, and most of the CpGs located in these are heavily
methylated (15). Mammalian DNA regulatory sequences are
principally located in noncoding sequences but concentrated
preferentially in the 5′-flanking regions of genes, leaving introns
However, the comparison of complete genomes has revealed
a large number of conserved non–protein-coding DNA sequences
biological function remains unknown (22).
We hypothesized that UMRs are highlighting functional
sequences, which, if proven, emphasizes the usefulness of our
method as a tool to identify loci at which epigenetic mechanisms
could influence complex phenotypes and diseases. To provide
genome-wide evidence in favor of our hypothesis, we evaluated
the functional significance of our UMRs by asking if they overlap
with highly conserved mammalian sequences and/or with ex-
perimentally determined protein-binding sites. To test the po-
tential functionality of our UMRs, we used two tracks of the
University of California, Santa Cruz Genome Browser (23). One
is the mammal most conserved (MMC) track of conserved se-
quences or elements based on whole-genome alignments of dif-
ferent mammalian species. The other is the track for open
regulatory annotation (ORegAnno), which includes literature-
curated regulatory regions and transcription factor-binding sites
(24). We reasoned that the sequences of these tracks could be
used as probes that would allow us to follow the partitioning of
regulatory sequences between the methylated regions and UMRs
of the genome. We found that our UMRs are indeed enriched in
both MMCs and ORegAnno sites (SI Appendix, Table S12). For
ORegAnno sites, 25% populating 10% of the UMRs and non-
CGI UMRs (with the majority in intronic and intergenic regions)
reached enrichments of 100-fold compared with the concentra-
tion at which these regulatory elements are found in the whole
genome (Fig. 6 and SI Appendix, Table S12). The likelihood that
this enrichment occurred by chance is less than 4 in 100,000.
Interestingly, 98% of all ORegAnno elements overlapping
UMRs belong to binding sites for Esr1 and Foxa2. The Foxa 2
gene codes for the forkhead box protein A2, which is a tran-
scriptional activator for liver-specific genes (25), and the Esr1
gene codes for the nuclear estrogen receptor α. This is the major
estrogen receptor expressed in the liver, where it regulates glu-
cose homeostasis as well as lipid metabolism (26). Among the
remaining 2% of regulatory elements, we also found tissue-
related transcription-binding sites (i.e., Hnf4A). The hepatocyte
nuclear factor 4α is a transcription factor found upstream in the
regulation pathways of several hepatic genes (27). Surprisingly,
the non-CGI UMRs produced, on average, a threefold greater
enrichment in regulatory elements than the CGI-like UMRs.
Relying on the way the protein-binding sites were partitioned,
we believe that most of the UMRs represent regions rich in reg-
tures. (A) Distribution of CGI-like and non-CGI UMRs in genomic regions.
UMRs at TSS regions (−3 kb to +2 kb of TSS), in gene bodies (non-TSS exons,
introns, and +3 kb of 3′ not-transcribed regions), and in intergenic DNA do
not add up to 100 because only those UMRs with >90% overlap were con-
sidered. (B) Enrichment of ORegAnno sites and MMC sequences in the UMRs
located in the indicated genomic regions. Enrichments are compared with
abundance in the undiluted genome (numerical values are provided in SI
Appendix, Table S12). **P < 0.005; ***P < 0.0001.
Genomic distribution of UMRs and enrichment of annotated fea-
Colaneri et al.PNAS
| June 7, 2011
| vol. 108
| no. 23
ulatory elements, with many of them being liver-related and pos- Download full-text
sibly liver-specific. We used the “David Bioinformatics Functional
Annotation Tool” Web application to bin UMR-containing genes
according to functional annotation and analyzed the enrichments
in three selected categories: biological process, molecular func-
tion, and tissue specificity (28). We took thetop 5% ofourUMRs,
ranked according to their avgMSCC score. In each functional
category, we sorted the results according to significance (smaller
P values on top). For “tissue specificity,” liver is the most sig-
nificantly enriched tissue (P value of 2 × 10−12.). For “biological
process,” genes related to system development are at the top
(P value of 1 × 10−8). Finally, for “molecular function,” genes
related to steroid hormone receptor activity are the most
enriched class of function (P value of 1 × 10−5).
In conclusion, our screening for hypomethylated CpGs showed
that CGIs are weak predictors of sensitive epigenetic loci and,
in addition, revealed a large unexpected number of non-CGI
UMRs with the highest enrichment in regulatory elements. The
fact that 50% of the UMRs have one-half of the average CpG
content of traditional CGIs prompts a rethinking of the re-
lationship between CpG density, unmethylation, and function-
ality of the genomic subsequences. A mechanistic link between
these variables is beginning to emerge. A recent report shows
that the genomic insertion of a promoterless CpG cluster is able
to recruit the Cfp1 protein, which can bind to unmethylated
CpGs and attract the Setd1 histone H3K4 methyltransferase
complex, which, in turn, creates a new focus of H3K4me3
modification (29). There is evidence that this modification repels
the methyltransferase involved in de novo methylation. However,
only one-half of the cells carry the insertion-acquired methylation,
indicating that CpG density, per se, is insufficient to maintain the
unmethylated state. Our hypomethylation map showing thousands
of UMRs not qualifying as CGIs but with the highest enrichment
in DNA regulatory motifs supports the idea that methylation is
the default state of CpGs, except for those than are protected
from de novo methylation, which may be mediated by the action
of DNA-binding proteins.
The idea that DNA-containing regulatory elements are fur-
nished with a critical CpG density working as a local signal to
recruit proteins able to create epigenetic marks that highlight
functional sequences is simple and attractive. In this scenario,
the unmethylated genomic regions (UMRs) could reflect the
footprint of regulatory DNA-binding proteins that protected
local sequences from the activity of de novo methylases.
4Enz MSCC vs. HpaII MSCC. The MSCC approach was first pub-
lished in 2009 by Ball et al. (15) in a proof-of-principle report.
The average reads-per-hit values (MSCC score) reported for
unmethylated CCGGs was 5.0 ± 15.4, which identified 69% of
the addressable sites with at least one read. In discussing the
usefulness of the approach, the authors used statistical criteria to
conclude that by increasing the sequencing depth, the method
would allow for the identification of the UMRs of a genome.
Having sequenced to a greater depth, which reached average
MSCC scores for HpaII of 10.4 ± 10.0 for all CCGGs identified
at least once, we sought to determine to what extent the HpaII
hits are able to report on the hypomethylation measured using
4Enz five-channel hits and a window of 300 bp. Our all-channel
HpaII hits allow for formation of 109,877 of 300-bp–based
clusters, which overlapped with only 25,373 (54.2%) of 46,804 of
our UMRs and with 7,207 (18%) of 24,399 of our non-CGI
UMRs. We thus conclude that only the expanded 4Enz MSCC
reports reliably on the genome’s hypomethylation.
Materials and Methods
CpG-tag libraries were prepared according to strategies similar to those
outlined in ref. 15 except that four instead of one methylation-sensitive
restriction enzyme was used, and the tags were retrieved with EcoP15I and
not MmeI. CpG tag libraries were sequenced by Illumina Inc. using Genome
Analyzer's Solexa technology. Illumina Inc returned approximately 1 Giga-
base of sequence partitioned into 36-nt long reads per library. After mapping
to the mm9 Mus musculus reference genome, the data were analyzed by
creating frequency histograms showing the number of times reads were
found that identified any given CpG. For further details on methods and data
analysis, including materials, acknowledgements, 12 tables, and 8 figures, see
S1 Appendix, SI Materials and Methods.
ACKNOWLEDGMENTS. This study was supported by the Intramural Research
Program of the National Institutes of Health (2009 National Institutes of
Health Director’s Challenge Award and Project Z01 ES101643 to L.B.).
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| www.pnas.org/cgi/doi/10.1073/pnas.1105713108Colaneri et al.