Late-replicating heterochromatin is characterized by
decreased cytosine methylation in the human genome
Masako Suzuki,1,5Mayumi Oda,1,5,6Marı ´a-Paz Ramos,1Marie ´n Pascual,1Kevin Lau,1
Edyta Stasiek,1Frederick Agyiri,1Reid F. Thompson,1Jacob L. Glass,1Qiang Jing,1
Richard Sandstrom,2Melissa J. Fazzari,1,3R. Scott Hansen,4John A. Stamatoyannopoulos,2,4
Andrew S. McLellan,1and John M. Greally1,7
1Department of Genetics (Computational Genetics), Albert Einstein College of Medicine, Bronx, New York 10461, USA;2Department
University of Washington, Seattle, Washington 98195, USA
Heterochromatin is believed to be associated with increased levels of cytosine methylation. With the recent availability of
genome-wide, high-resolution molecular data reflecting chromatin organization and methylation, such relationships can
be explored systematically. As well-defined surrogates for heterochromatin, we tested the relationship between DNA
replication timing and DNase hypersensitivity with cytosine methylation in two human cell types, unexpectedly finding the
later-replicating, more heterochromatic regions to be less methylated than early replicating regions. When we integrated
gene-expression data into the study, we found that regions of increased gene expression were earlier replicating, as pre-
with early replication. A self-organizing map (SOM) approach was able to identify genomic regions with early replication
and increased methylation, but lacking annotated transcripts, loci missed in simple two variable analyses, possibly encoding
unrecognized intergenic transcripts. We conclude that the relationship of cytosine methylation with heterochromatin is not
simple and depends on whether the genomic context is tandemly repetitive sequences often found near centromeres, which
are known to be heterochromatic and methylated, or the remaining majority of the genome, where cytosine methylation is
targeted preferentially to the transcriptionally active, euchromatic compartment of the genome.
[Supplemental material is available for this article.]
The original definition of heterochromatin was wholly derived
from cytological studies, identifying it as unusually compacted
nuclear material as opposed to the less-condensed euchromatin.
Heterochromatin in the eukaryotic genome is subclassified as fac-
ultative and constitutive. Whereas sites of constitutive heterochro-
matin are present in all cell types (e.g., centromeres, G bands)
(Holmquist 1989), facultative heterochromatin can be present at
different loci in different cell types (e.g., X inactivation in female
mammalian cells). Both types of heterochromatin are usually asso-
ciatedwithtranscriptional silencing, althoughthereisaminorityof
genes that transcribes preferentially in a heterochromatic context
(Vogel et al. 2006). Apart from transcriptional repression, hetero-
chromatin has other functional properties, including associations
with centromeres and telomeres and a role in sister chromatid co-
hesion (Gartenberg 2009). Decades of cytological studies have also
characterized heterochromatin by its late-replication timing within
the cellcycle(Gilbert 2002). From a molecular point of view, certain
variants (e.g., H3K9me3) (Krauss 2008) characterize heterochro-
matin. Methyl-binding domain proteins have also been found to
accumulate in pericentromeric satellite DNA sequences in mouse
of DNA methyltransferase 3B (DNMT3B) mutations cause loss of
cytosine methylation and local decondensation of the heterochro-
matin (Hansen et al. 1999).
It is believed that the DNA within heterochromatin is highly
methylated, the cytosine methylation acting synergistically with
chromatin modifications characteristic of heterochromatin, and a
repository for transposons maintained in a silent state (Henikoff
2000). However, there is reason to question the association of cy-
tosine methylation with facultative heterochromatin formation,
as it has been recognized for some time that the inactive X chro-
et al. 2002), possibly related to the decreased methylation in bodies
of genes (Hellman and Chess 2007) silenced as part of the X in-
Now that we have genome-wide and high-resolution maps of
DNA-replication timing and chromatin constituents characteristic
of heterochromatin, we can study the relationships of heterochro-
matin with other genomic properties more quantitatively. To study
the relationship of cytosine methylation with heterochromatin, we
chose to use DNA replication timing (Gilbert 2002) and DNase hy-
persensitivity as well-characterized indicators of heterochromatin.
Our studies of two human cell lines revealed a paradoxical rela-
tionship between early DNA replication or increased DNase hy-
methylation, attributable in part to the targeting of cytosine
5These authors contributed equally to this work.
6Present address: Center for Integrated Medical Research, Keio Uni-
versity School of Medicine, Tokyo, Japan.
Article published online before print. Article, supplemental material, and pub-
lication date are at http://www.genome.org/cgi/doi/10.1101/gr.116509.110.
21:1833–1840 ? 2011 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/11; www.genome.org
highly repetitive juxtacentromeric sequences of the human genome
represent a special situation, and that in the remainder of the human
genome, heterochromatin is less methylated than euchromatin.
Cytosine methylation and replication timing
In Figure 1, we show a graphical representation of cytosine meth-
ylation status and replication timing in both cell types studied. Vi-
sually, the hypomethylated areas appear to be undergoing late rep-
lication, whereas the relatively hypermethylated, more gene-rich
areas are early replicating. This observation is the opposite of what
correlation between cytosine methylation and replication timing.
Figure 2 shows a contour plot illustrating the relationship between
cytosine methylation and replication timing in 100-kb windows.
The data for both fibroblast and GM06990 cells show a correlation
between cytosine hypomethylation and late replication, and vice
versa (R2= ?0.574 and R2= ?0.334, respectively). The weaker cor-
relation observed in the GM06990 lymphoblastoid cell line may be
explained by greater global hypomethylation in these cells com-
pared with the fibroblast cell line.
Cytosine methylation and gene expression
the transcriptional activity of genes (Zhang et al. 2006; Zilberman
et al. 2007; Backdahl et al. 2009; Ball et al. 2009). Of these prior
studies, Ball et al. (2009) used the same lymphoblastoid cell line that
we describe here as well as human fibroblasts. This observation sug-
gests that cytosine hypermethylation associated with early DNA
tested the relationship between cytosine methylation status and
gene expression status using our own datasets to see whether we
could reproduce the Ball et al. (2009) observations. We tested the
cytosine methylation and gene expression values for each RefSeq
gene annotated in the human genome, looking separately at pro-
moters and gene bodies. We found the promoter regions to be gen-
erally hypomethylated regardless of the gene-expression status, but
consistent methylation of gene bodies was found only in actively
transcribed genes (Supplemental Fig. 3). We confirmed these ge-
nome-wide relationships by locus-specific validation studies, with
the results for two representative loci shown in Supplemental Figure
4 and data from further validated loci listed in Supplemental Table 3.
A100-kb sliding window approach also showed the correlation
of gene expression and cytosine methylation, with actively tran-
scribed regions being relatively hypermethylated, and transcrip-
see whether the hypermethylation of regions containing actively
transcribed genes is solely due to the targeting of cytosine methyl-
ation to active gene bodies, or whether there is also increased cyto-
sine methylation at intergenic loci in these highly transcribed
regions. When we excluded the 100-kb genomic windows that do
not include annotated genes (;50% of the windows genome wide)
and tested what happened to methylation in the gene-containing
windows with the removal of gene body data, we found that the
proportion of windows with overall hypomethylation (log2ratio
HpaII/MspI>0) increases from 32.4% to
64.6%. This indicates that the hypermeth-
ylation in regions of early replication is
substantially, but not solely due to target-
ing of bodies of annotated, actively tran-
scribed genes (Fig. 4; Supplemental Fig. 5).
Gene expression and replication timing
The preceding results indicate that we
should expect to see a positive correlation
between gene expression and early repli-
cation. We tested this formally, showing
the results in Supplemental Figure 6.
Again, using the 100-kb sliding window
approach, we show that the actively tran-
scribed regions are replicated earlier, and
that a small subset of early replicating
regions contain relatively inactive loci
(Supplemental Fig. 6). We also show that
late-replicating regions are largely inactive
in this 100-kb context. This result is con-
cordant with previous observations in
organisms from insects (Schubeler et al.
2002) to mammals (Desprat et al. 2009).
and replication timing
Although the replication timing data
were processed differently in this study,
we confirmed our previous correlation
(Hansen et al. 2010) of increased DNase
Fibroblast data; (bottom) GM06690 lymphoblastoid cell line data. Cytosine methylation is shown as the
HpaII/MspI log2intensity ratio from the HELP assay. Positive values indicate relative hypomethylation,
andnegativevaluesindicate hypermethylationofHpaIIsites.DNAreplication timingdataaregenerated
from raw sequence reads by an arctangent transformation of 1-kb counts comparing early (G1 and S1)
and late (S4 and G2) cell samples, as described in the Methods section. Earlier replicated regions have
higher values than later replicated regions.
Cytosine methylation and replication timing correlate in broad genomic regions. (Top)
Suzuki et al.
hypersensitivity with earlier DNA replication (Supplemental
Self-organizing map analysis
While global correlations are indicative of relationships of genomic
processes, these kinds of analyses may fail to reveal the presence of
a subset of genomic regions diverging from overall genome-wide
relationships. We therefore applied a self-organizing map (SOM)
analysis, wherein we were able to define distinct subsets of loci that
posses similar properties. The SOM is a lossless unbiased clustering
method that projects high-dimensional data onto a two-dimen-
sional map, while at the same time preserving the topology of
the data. To perform an unbiased test of the relationship between
replication timing and other vector elements, we excluded the rep-
lication timing data from the training process. Following the con-
struction of the SOM, the replication timing data were tagged on all
vectors and overlaid to visualize whether the variables used to build
the SOM predicted replication timing. For example, as a negative
control experiment, we examined vectors comprising only the
number of RefSeq genes and HAFs in 100-kb windows and were
unable to observe any discernible clustering in the U-matrix or sep-
aration of replication timing data, as expected (Supplemental Fig. 8).
However, adding gene expression, CpG island number, and cytosine
methylation data to the vectors provided sufficient information
to enable the data to separate into two distinct clusters enriched
in vectors tagged to show either early or late replication (data not
which shows two distinct clusters of loci exhibiting alternative rep-
lication timing patterns. These two clusters mainly consist of loci
with early replication/hypermethylation/high gene expression or
late replication/hypomethylation/low expression (Fig. 5B), as would
be predicted by our previous analyses. However, the SOM analysis
was also able to identify a new group of loci, where early replication
levels of gene expression (Fig. 6A). These low-expression loci con-
sisted not only of regions containing genes expressed at low levels,
but also regions lacking any annotatedRefSeqgenes, Gencode genes
(Harrow et al. 2006), or expressed sequence tags (ESTs, #5) (Fig. 6B).
Adding DNase hypersensitivity data (Hansen et al. 2010) revealed
a substantial proportion of these regions to be nuclease accessible,
indicating these to be euchromatically organized, but retaining
the increased methylation pattern of the
remainder of the early replicating regions
To explore the relationship between het-
erochromatin and cytosine methylation,
wide assays and a number of genomic
sequence feature annotations. The Repli-
seq assay maps DNA-replication timing
(Hansen et al. 2010), while cytosine
methylation was measured using the
HELP assay (Khulan et al. 2006; Oda et al.
2009) and gene expression by microarray
studies in two human cell lines. Our re-
sults show a strong correlation between
cytosine methylation and DNA replica-
tion timing genome wide.
However, this correlation is the opposite of what might have
which functionally defines heterochromatin, is less methylated
than the early replicating, euchromatic compartment. Our prior
were drawn with two-dimensional histograms. Cytosine methylation data and replication timing data
are averaged in 100-kb sliding windows. The cumulative numbers of observations are shown as color-
coded levels to generate the contours. Early replicated regions are more methylated in both the fi-
broblast and lymphoblastoid cell types.
DNA hypermethylation correlates with early DNA replication timing. Filled contour plots
actively transcribed gene regions. Extending the analysis of Supplemental
Figure 3 to a 100-kb sliding window representation continues to show the
relationship between increased gene expression and hypermethylation of
DNA. A two-dimensional histogram of the averaged HpaII/MspI log2ratio
in 100-kb windows and averaged signal intensities of the genes are rep-
resented by filled contour plots.
Broad correlation exists between DNA hypermethylation and
DNA hypomethylation in human heterochromatin
studies showing regions of increased DNase hypersensitivity corre-
lating well with early replication (Hansen et al. 2010) supports the
link between euchromatin and early replication. The finding of in-
creased methylation associated with euchromatin is paradoxical for
known repressive mark in the context of gene promoters, and het-
erochromatin is likewise a repressive environment for gene
transcription, while loci such as pericentromeric satellite DNA
(Gopalakrishnan et al. 2009) are classic examples of heterochro-
matically organized DNA at cytological resolution (Plohl et al. 2008)
any assumption that increased cytosine methylation is a universal
feature of heterochromatic DNA may be questionable based on prior
studies. In the case of mammalian X chromosome inactivation, one
S phase,the active Xchromosome replicates early and the inactiveX
chromosome late (Gribnau et al. 2005), consistent with the hetero-
chromatic organization of the inactive X (Chow and Brown 2003).
The inactivated X chromosome has been reported to be globally
hypomethylated (Viegas-Pequignot et al. 1988), and the gene bodies
on inactive X chromosomes have been found to be hypomethylated
(Hellman and Chess 2007) despite the increased cytosine meth-
ylation at promoters causing gene inactivation of the inactive X
hypomethylation on the inactive X chromosome is consistent with
of the inactive X using methylation-sensi-
tive restriction enzymes (Viegas-Pequignot
et al. 1988). Extending this approach to
the whole genome, a comparison of cyto-
genetic patterns obtained from the diges-
tion of human chromosomes in situ with
methylation-insensitive MspI and methyl-
ation-sensitive HpaII revealed that R bands
(gene-rich, euchromatic) are relatively
methylated, whereas heterochromatic
blocks of sequence can be strikingly un-
Our results associating increased methyla-
tion with earlier replication are therefore
not inconsistent with prior observations.
increased cytosine methylation and early
recent report (Aran et al. 2011). Both
studies concur that increased cytosine
methylation in early replicating regions is
substantially but not solely attributable to
transcription-targeted cytosine methyla-
when we remove RefSeq gene bodies from
the analysis (Fig. 4), and in Supplemental
Figure 5C relative correlation coefficient
from the analysis in a manner similar to
Aran et al. (2011). It is apparent from these
analyses that the exclusion of gene bodies
does not remove all loci with increased
methylation in early replicating regions,
raising the question as to why these sup-
posedly euchromatic genomic compartments have methylation also
targeted to intergenic regions. Our use of the SOM approach revealed
that some of the early replicating, highly methylated loci in the ge-
nome are devoidofannotatedgenesandatthelowestquintileofEST
These SOM-defined regions, which would have been difficult to
identify through the preceding two-variable comparisons, are candi-
dates for being transcribed as nonannotated, noncoding RNAs in
these cell types, causing targeting of cytosine methylation and asso-
ciated with early replication of DNA. Based on these SOM findings, it
occurs in gene-containing regions and helps to account for the
remaining cytosine methylation when annotated gene bodies are
removed from early replicating regions (Fig. 4).
A question that arises is whether cytosine methylation has
a possible role in helping to define the choice of replicationorigins
in the genome. DNA replication is initiated from sites in the ge-
nome calledoriginsof replication. In mammalian cells, replication
is organized into discrete zones of similar replication timing,
which consist of multiple replication origins. The zones are het-
erogeneous in size (30–450 kb, with the most frequent sizes in the
range of 75–150 kb) (Berezney et al. 2000). Since later replication
timing is correlated with closed chromatin, a logical conclusion
would be that the repressive cytosine methylation mark should be
enriched in regions of later replication timing. With the identifi-
cation of specific origins of replication in mammals, direct testing
hypermethylation. We tested how gene-body methylation could be contributing to the patterns shown
in Figure 2, reproducing the fibroblast plot to facilitate comparison in A. C shows the results when 100-
kb windows that do not contain genes are removed, with a decrease in the late-replicating/hypo-
methylated population of signals. Excluding gene bodies, to study only intergenic methylation, gen-
erates a shift in signal distribution toward hypomethylated DNA (B), especially when the analysis is
restricted to the gene-containing regions of the genome (D). These results show that a substantial
proportion of the correlation of cytosine methylation with early replication is due to the methylation
targeting transcribed sequences.
The hypermethylation of early-replicating regions is predominantly due to gene-body
Suzuki et al.
of these loci could be performed to test their methylation status. A
consistent observation has been that nascent strands or defined
replication origins are derived from or located at CpG islands
(Tasheva and Roufa 1995; Rein et al. 1997; Delgado et al. 1998;
Sequeira-Mendes et al. 2009) and, intriguingly, that these CpG is-
lands may be characterized by being methylated (Tasheva and
Roufa 1995; Rein et al. 1997), a characteristic that defines only a
small subset of these genomic elements (Glass et al. 2007). Those
methylated CpG islands are listed in Supplemental Tables 4 and 5.
Our appreciation of the relationship between cytosine
methylation and heterochromatin needs to be refined in terms of
genomic context—the positive association between cytosine
methylation at tandemly repetitive sequences (such as those found
in paracentromeric regions) and heterochromatin is well-estab-
lished, and as such repetitive sequences are not tested by assays
using the microarrays or massively parallel sequencing of the cur-
rent project, our data do nothing to challenge this established re-
lationship. The heterochromatically organized DNA in the re-
maining majority of the genome represents a distinct genomic
context where the relationship with cytosine methylation is the
opposite to that of the tandemly repetitive sequences. This has
implications for the mechanism of drugs such as DNMT inhibitors,
which may promote demethylation and chromosomal instability
primarily in tandemly repetitive DNA, but may have different ef-
other intriguing implication is a context-dependent association of
cytosine methylation with regulators of post-translational modifi-
cations of histones. As the ENCODE project tested the GM06990
representation of the multivariate data set, the top panel shows a U matrix representation of the map derived from genome-wide DNA methylation log2
ratios, RefSeq gene expression, RefSeq gene number, CpG island number, and HpaII-amplifiable fragment number in each 100-kb window. Each node is
shaded using a linear grayscale that represents the mean Euclidean distance of that node vector relative to its immediate neighbors on the map ([white]
most similar; [black] least similar). Overlaying loci with information about late (green) and early (red) replication shows that the parameters tested are
predictive of replication timing, as evidenced by the clear separation of the red and green regions (A). We break out some of the variables used in
generating the SOM (cytosine methylation, gene expression) to illustrate their overall correlations with DNA replication (B).
A self-organizing map analysis correlates DNA replication with methylation and transcription patterns. In this self-organizing map (SOM)
loci where gene expression appeared to be behaving discordantly from the overall relationship with DNA replication and methylation, we represented
early replicating and hypermethylated loci in red and low-expressing loci in green to illustrate these loci in the merged plot as orange (outlined in A). In B
quintile of EST densities annotated for the UCSC Genome Browser. These loci are not only lacking any measurable gene expression, they do not even have
any evidence for any transcriptional potential, regions usually referred to as gene deserts but with DNA-replication characteristics and DNase hyper-
sensitivity (bottom right, green asterisk) that may indicate noncoding, nonprocessed transcription.
Identification of a genomic compartment where early replication and cytosine hypermethylation occur at nongenic regions. To highlight the
DNA hypomethylation in human heterochromatin
cell line in its pilot phase, we were able to correlate a number of
histone modifications with replication timing for the 1% of the
genome surveyed in the pilot phase of the ENCODE project (The
ENCODE Project Consortium 2007). We show these results in
Supplemental Figure 9. We were able to observe one histone mod-
ification in particular to be strongly correlated with late replication,
histone H3 lysine 9 trimethylation (H3K9me3).Thisresultindicates
that histone modifications rather than cytosine methylation are
likely to be responsible for the heterochromatic organization asso-
ciated with late replication, but it also indicates that enriched cyto-
sine methylation and H3K9me3 are not necessarily colocalized in
the genome, despite biochemical (Rottach et al. 2009), genetic, and
regulatory processes in eukaryotes. The function of histone meth-
thus be subject to the DNA sequence composition of specific geno-
mic contexts ratherthan acting in the same mannerthroughoutthe
genome. Overall, we conclude that heterochromatin is inherently
heterogeneous, and that rules that determine relationships within
this compartment may not be universal, but have genomic context
We usedthe GM06990celllineand a human foreskinfibroblast cell
line for these studies. GM06990 is a karyotypically normal lym-
phoblastoid cell line available from the Coriell repository (http://
www.coriell.org/) that has been used by the ENCODE consortium
for a number of studies (The ENCODE Project Consortium 2007),
and was used for our recent genome-wide analysis of DNA replica-
tion timing (Hansen et al. 2010). The GM06690 cells were cultured
as recommended by the Coriell repository. The human fibroblast
cells were grown in Dulbecco’s modified Eagle medium (DMEM)
supplemented with 10% Fetal Bovine Serum, 2 mM Glutamine and
Penicillin–Streptomycin (Invitrogen) in a 37°C incubator with 5%
CO2. The cells were harvested at 80%–90% confluence in 150-cm2
flasks by trypsin-EDTA dissociation. DNA and RNA were extracted
from the cells using standard protocols.
We used our previously published HELP microarray design repre-
senting >1.32 million loci genome wide, representing each HpaII-
amplifiable fragment from 50 to 2000 bp with one to two oligo-
nucleotides encoding unique sequence at each locus (Oda et al.
2009). The human gene expression microarray was a standard
Roche-NimbleGen design (2006-08-03_HG18_60mer_expr).
Microarray sample preparation and hybridization
We performed the HELP assay as previously described (Oda et al.
2009). For expression studies, we converted mRNA to dsDNA using
the SuperScriptDouble-StrandcDNASynthesiskit(Invitrogen) with
and hybridization to the microarrays were performed using a pub-
lished technique (Selzer et al. 2005).
Single-locus quantitative validation assays
Bisulphite conversion and MassArray (Sequenom) were performed
using the same sample of DNA used for the high-throughput assays
above. Bisulphite conversion was performed with the EZ DNA
(http://www.urogene.org/methprimer/) with the following param-
eters: product length (250–450 bp), primer length (23–29 bp) and
primer Tm (56–62°C). PCR was performed in the following condi-
tions with FastStart High Fidelity Taq polymerase (Roche): 95°C for
10 min and 42 cycles of 95°C for 30 sec, primer-specific Tm for 30
sec and 72°C for 1 min, followed by 72°C for 10 min for the final
extension. Primer-specific Tms and primer sequences are provided
in Supplemental Table 1. Bisulphite MassArray assays were per-
formed by the Einstein’s Genomics Core Facility.
Complementary DNA (cDNA) was generated from 2 mg of to-
tal RNA with Superscript III reverse transcriptase (Invitrogen) us-
ing oligo(dT)20. RT–PCR primers were designed with Primer3 soft-
ware (http://frodo.wi.mit.edu/primer3/input.htm). The primer
sequences that we used in this study are provided in Supplemental
Table 2. The quantitative PCR was performed using SYBR Green
(Power SYBR Green PCR Master mix [Applied Biosystems]).
Analysis of cytosine methylation (HELP) assays
HELP assay data analysis was performed using our published
pipeline(Thompson etal. 2008), an open source resource available
through BioConductor (HELP package).
Analysis of gene expression microarray data
Gene expression microarrays performed on the GM06990 and fi-
broblast cells were analyzed using NimbleScan2.3 (NimbleGen)
and R version 2.9.2 (http://www.R-project.org). We selected loci
for validation that appeared to be expressed in one or both cell
types and performed real-time RT–PCR with the ABI7500. The
expression status was normalized using the human GAPDH ex-
pression level. We show the correlation between the microarray
expression intensity and the real-time PCR validation data in
Supplemental Figure 1. The expression microarrays and RT–PCR
results had high correlation values (R = 0.97). Using the RT–PCR
validation we were able to define a threshold for highly expressed
genes as a log intensity of $6.
Timing of replication analysis
The original massively parallel sequencing-based data measuring
timing of replication and DNase hypersensitivity used in this study
have been published previously (Hansen et al. 2010). For the DNA
replication timing, in cell cycle phases G1, S1, S2, S3, S4, and G2,
newly replicated DNA positions were analyzed by massively parallel
sequencing (Hansen et al. 2010). The newly replicated sequences
were counted in windows of 1-kb size. In their correlation of repli-
cation timing with DNase hypersensitivity, they calculated the sum
of read numbers for (G1 + S1) and divided this by the sum of read
numbers for (S4 + G2) to get a single value for each 1-kb window.
Rather than dividing values, we used an approach we recently de-
scribed for our HELP-tagging assay to study cytosine methylation
(Suzuki et al. 2010), transforming the read depth as shown in Sup-
plemental Figure 2, comparing the (G1 + S1) with the (S4 + G2) se-
quence read counts by measuring the inverse tangent (arctangent)
for each data point.
the datawe generatedforcytosine methylationand gene expression,
Suzuki et al.
and added our previously published DNase hypersensitivity and
DNA replication timing data (Hansen et al. 2010) in 100-kb sliding
sites and the mean arctangent DNA-replication timing values per
window were calculated. Two-dimensional histograms were gener-
atedascontour plotsusing R version2.9.2and the areasbetweenthe
contours were filled.
Self-organizing map analysis
To examine the relationships between the variables being tested, we
map (SOM) (Kohonen 2001). Using 100-kb sliding windows, with
a step size of 50 kb, we calculated mean log ratio values from the
HELP assay (indicative of DNA methylation levels), mean RefSeq
gene expression levels, and the cumulative number of HpaII am-
plifiable fragments (HAFs), CpG islands, and genes per window.
These data were used to generate vectors for analysis. A total of 26
experiments were performed to use each of the five vector elements
in all possible two-, three-, four-, and five-element combinations.
on the range of the data, and vectors were then normalized to unit
length. All vectors were tagged as being either late (replication
timing angle 0–30), intermediate (31–60), or early (61–90) replicat-
ing. These tags were provided with the sole purpose of revealing the
whereabouts of vectors of these classes on the maps post-training,
and did not provide any assistanceto the training process itself. The
data were formatted to be compatible with the GACT SOMengine
accompanying Java-based SOM visualization software (AS McLellan,
AA Golden, in prep.), which were used to perform the analysis. This
software produces a SOM analysis using an implementation of the
batch map SOM algorithm, featuring accelerated best-matching unit
(BMU) finding (Kohonen 2001), and is parallelized with openMP
the entire data set of 58,621 vectors. Each trained SOM map was
generated using a 112 3 54 hexagonally arranged grid with random
initialization of the codebook vectors (vectors of the same dimen-
sions as the data vectors that represent each node of the grid). A total
of 10,000 cycles of training was performed, each time presenting the
entire data set to the grid and allowing grid nodes to compete for the
vectors in the data set to which they were most similar (using an
Euclidean distance metric). The SOM software was run on our local
ROCKS/Sun Grid Engine (SGE)-based cluster using all eight pro-
cessors on a single node for each experiment. After each training
cycle, grid vectors were modified to resemble the data vectors ‘‘won’’
by that node with some influence from neighboring nodes. This was
achieved using a Gaussian neighborhood function for updating
codebook vectors at the end of each cycle and Gaussian neighbor-
hood-radius decay with time. An initial neighborhood-radius of 69
was used. After training, all data was reintroduced to the grid a final
in order to examine the distribution of features. For example, repli-
cation-timing status could be examined by coloring the nodes with
a shade and intensity proportional to the number of vectors associ-
ated with each label from green (all late replicating) to red (all early
replicating). Overall clustering patterns in the data were also exam-
ined using a U-matrix representation of the grid, which represents
a similarity graph where a linear grayscale is used to indicate how
similar a node vector is to its immediate neighbors in vector space.
Genome-wide molecular data from HELP microarray experiments
have been submitted to the NCBI Gene Expression Omnibus
(GEO) (http://www.ncbi.nlm.nih.gov/geo), under accession num-
bers GSM679751 (fibroblast HELP), GSM679750 (lymphoblast
HELP), GSM679748 (fibroblast gene expression), and GSM679749
(lymphoblast gene expression).
J.A.S.). WethankShahinaMaqbool PhD,Raul Olea, andGael Westby
of Einstein’s Epigenomics Shared Facility for their contributions, and
Einstein’s Center for Epigenomics.
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Received October 20, 2010; accepted in revised form August 10, 2011.
Suzuki et al.
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