Gene 476 (2011) 10–14

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Computational analysis suggests a highly bendable, fragile structure fornucleosomal DNA
Tadasu Nozaki a,b, Nozomu Yachie c, Ryu Ogawa a,d, Rintaro Saito a,b,⁎, Masaru Tomita a,b,daInstitute for Advanced Biosciences, Keio University, Tsuruoka, 997–0017, Japan
Systems Biology Program, Department of Environment and Information Studies, Keio University, Fujisawa, 252–8520, Japan
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02445, USAd
Systems Biology Program, Graduate School of Media and Governance, Keio University, Fujisawa, 252–8520, Japanbca r t i c l e

i n f oArticle history:
Accepted 14 February 2011
Available online 19 February 2011
Received by T. Gojobori
Keywords:
Bendability
Cleavage intensity
Nucleosome
DNA structure
HistoneEpigenetics

a b s t r a c t
Eukaryotic chromosomal DNA coils around histones to form nucleosomes. Although histone afﬁnity for DNA
depends on DNA sequence patterns, how nucleosome positioning is determined by them remains unknown.
Here, we show relationships between nucleosome positioning and two structural characteristics of DNA
conferred by DNA sequence. Analysis of bendability and hydroxyl radical cleavage intensity of nucleosomal
DNA sequences indicated that nucleosomal DNA is bendable and fragile and that nucleosome positional
stability was correlated with characteristics of DNA. This result explains how histone positioning is partiallydetermined by nucleosomal DNA structure, illuminating the optimization of chromosomal DNA packaging
that controls cellular dynamics.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction
Eukaryotic chromosomal DNA is packaged into nucleosomes andthen into chromatin, which has a repetitive, compact structure. A
nucleosome comprises about 147 base pairs (bps) coiled around a
histone octamer, which consists of two copies of four core histones(H2A, H2B, H3, and H4) (Luger et al., 1997; Davey and Richmond,2002). Neighboring nucleosomes are separated by linker regions ofDNA (approximately 50 bps) without histones (Van Holde, 1989).
Previous studies have shown that nucleosomes have various
functions besides the packaging of chromosomal DNA. For example,
many cellular processes that involve DNA respond dramatically tochanges in the positions of and modiﬁcations of nucleosomes, and in
the nucleus, the dynamics of the positioning of nucleosomes and theirﬂanking regions play a key role in cellular processes (Strahl and Allis,
2000; Yuan et al., 2005; Segal et al., 2006; Barski et al., 2007; Schones
et al., 2008; Lister et al., 2009; Tolstorukov et al., 2009). For instance,
nucleosome positioning regulates eukaryotic transcription by affecting the accessibility of transcription factors to DNA or by affecting

Abbreviations: ChIP-chip, ChIP combined with microarray technology; ChIP-Seq,
ChIP combined with deep sequencing; GC, guanine-cytosine; PRS, positive reference
set; RRS, random reference set.
⁎ Corresponding author at: Systems Biology Program, Department of Environment
and Information Studies, Keio University, Fujisawa, Kanagawa 252–8520, Japan. Tel.:+81 466 47 5099; fax: +81 466 47 5099.
E-mail address: rsaito@sfc.keio.ac.jp (R. Saito).
0378-1119/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.gene.2011.02.004

transcriptional elongation by polymerase (Yuan et al., 2005; Schones
et al., 2008; Cairns, 2009).
The efﬁciency of the coiling of DNA around histones to form
nucleosomes depends strongly on DNA sequence (Yuan et al., 2005;
Ogawa et al., 2010). In vitro studies have shown that the range of
afﬁnities between histones and DNA is at least 1000-fold and that
DNA sequence pattern is an important factor for accurate nucleosome
positioning (Thastrom et al., 1999). Furthermore, the results of several
other studies suggest that the DNA coiled around the histones isbendable, and these studies have shed light on the importance of DNA
structure for nucleosome positioning (Drew and Travers, 1985;
Balasubramanian et al., 1998; Johnson et al., 2006; Miele and Vaillant,
2008; Rohs et al., 2009). However, no detailed, systematic analysis of
the bendability of coiled DNA has been performed, and whether DNA
bendability is chromatin wide is not clear.
Along with the progress in computational methods for analyzing
nucleosome data (Zhang et al., 2008; Tolstorukov et al., 2009), recent
remarkable progress in chromatin immunoprecipitation (ChIP)
techniques, including ChIP–chip (ChIP combined with microarray
technology) and ChIP–Seq (ChIP combined with deep sequencing),
has permitted detection and prediction of nucleosome positioning at
the whole-genome level (Yuan et al., 2005; Barski et al., 2007; Park,
2009). The increase in the number of large-scale nucleosome data sets
has permitted systematic analysis of nucleosome data in conjunction
with other “omic” data (e.g., genome-wide relationships between
nucleosome positioning and transcription regulation (Schones et al.,2008; Lister et al., 2009)).

T. Nozaki et al. / Gene 476 (2011) 10–14

In this study, we used a computational analysis of a large-scale
nucleosomal data set to evaluate two overall characteristics ofchromosomal DNA structure: DNA bendability and DNA cleavage
intensity. DNA bendability is calculated from parameters for a set oftrinucleotide patterns (Brukner et al., 1995) or tetranucleotide
patterns (Packer et al., 2000) in a given sequence. The parameters
for each trinucleotide pattern were obtained by Brukner et al. (1995)
using DNA binding proteins which generally bind to DNA that bends
toward the major groove. The parameters were derived from 709 DNA
binding regions. Bendability is frequently used to analyze DNA
structure. For example, the location of transcription initiation
promoters is associated with areas of bendable DNA (Abeel et al.,2008; Akan and Deloukas, 2008; Choi and Kim, 2009).
Cleavage intensity indicates the likelihood of DNA cleavage byhydroxyl radicals and provides a map of local variation in the shape of
the DNA backbone. Cleavage intensity is calculated from parameters
for a set of tetranucleotide patterns in a given sequence. The
parameters were derived by Greenbaum et al. (2007) from experiments in which DNA sequences were exposed to hydroxyl radicals;
they analyzed how cleavage of DNA sequences depends on theirsequence patterns. Cleavage intensity also reﬂects the solventaccessible surface area of DNA. This index of DNA fragility has also
been used to characterize structural features of DNA (Tullius andGreenbaum, 2005; Greenbaum et al., 2007).
These two structural parameters cover all combinations oftrinucleotide or tetranucleotide patterns and are sufﬁciently reliable
to analyze DNA structure (Brukner et al., 1995; Greenbaum et al.,2007). We conducted genome-wide computational analysis of the
DNA bendability and cleavage intensity of nucleosome and linker DNA
regions and found that nucleosomal DNA was overall highly bendable
and fragile. We also determined whether the positional stability of
nucleosomes under various cellular conditions depended on these
two structural properties of DNA, and we found that stable
nucleosomal DNA was more bendable and cleavable than unstable
nucleosomal DNA. We discuss which types of DNA structure confer
the optimal DNA packaging in chromosomes and which types are
optional for dynamic cellular processes.
2. Materials and methods
2.1. Genomic DNA and nucleosome positioning data
The reference genome sequence of Homo sapiens (hg18) was
obtained from the University of California at Santa Cruz genome
database (http://genome.ucsc.edu/). To determine genomic histone
positions, we used the previously reported ChIP–Seq data set forhistone H3 of human CD4+ T-cells obtained under two cellularconditions, with and without the T-cell receptor signal, which aredesignated as the activated and resting conditions, respectively(Schones et al., 2008). We modiﬁed a previously developed method(Tolstorukov et al., 2009) to deﬁne nucleosomal positions from the
ChIP–Seq of both resting and activated cells (for details of the method,
see Supplementary methods). We assessed the positional stability of
nucleosomes under the two cellular conditions by computing the
distance of the genomic position of a given nucleosome in the restingcell from its nearest nucleosomal position in the activated cell. For all
the nucleosomes identiﬁed under the resting conditions, we deﬁned
stable nucleosomes as those for which the distance between the
nucleosome under resting conditions and the nearest nucleosome
under activated conditions was within 12 bps, and unstable nucleosomes as those for which that distance ranged from 13 to 73 bps (halfthe size of one nucleosomal DNA unit). Nucleosomes that did not ﬁteither deﬁnition were not used for the positional stability analysis. The
boundary between the two types of nucleosomes (i.e., the 12–13-bp
boundary) was deﬁned from the inﬂection point in the distance
distribution (Supplementary methods).

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2.2. DNA bendability calculation
To assess nucleosomal DNA bendability, we used the trinucleotide
bendability parameter proﬁle obtained by Brukner et al. (1995). For
each of the nucleosomes identiﬁed under the resting conditions(including both stable and unstable nucleosomes), DNA bendability
was calculated for every trinucleotide in the region from − 400 to+400 bps from the center position of the nucleosomal DNA region.
For each position relative to the central position of the nucleosome,the average DNA bendability was calculated.
2.3. DNA cleavage intensity calculation
We used the OH radical cleavage intensity database (ORChID;
http://dna.bu.edu/orchid/) program, which is used to estimate thehydroxyl radical cleavage strength and solvent-accessible surface area
using a tetranucleotide DNA cleavage intensity proﬁle (Greenbaum
et al., 2007), to calculate the DNA cleavage intensity. (The sourcecode of ORChID was kindly provided by its authors; ORChID is a
web-based application.) For each of all the nucleosomes identiﬁed
under the resting conditions, we computed tetranucleotide DNA
cleavage intensities for all the DNA positions from − 400 to+400 bps from the center position by querying to ORChID. For
each position relative to the central position of the nucleosome, theaverage DNA cleavage intensity was calculated.
2.4. Guanine–cytosine content and statistical analysis
For a control analysis with respect to guanine–cytosine (GC)content, we randomly sampled 10 000 out of the ~ 5 000 000
nucleosomes identiﬁed under the resting conditions as a positivereference set (PRS) to reduce the calculation time. For eachnucleosome in the PRS, sliding window averages of DNA bendability,
cleavage intensity, and GC content for the region surrounding thenucleosome (from −400 to + 400 bps) were calculated using a 30-bp
window with 1-bp displacement. To prepare a random reference set(RRS), we randomly selected 1 000 000 genomic positions and usedtheir surrounding sequences (from − 400 to +400 bps) to calculate
DNA bendability, cleavage intensity, and GC content using a 30-bp
sliding window average. Additionally, the DNA bendability andcleavage intensity expected from each GC content value were
calculated from the above data, and we set them as RRS with GC
content. Then, we randomly correlated the sliding window for each of
the 10 000 nucleosomes in the PRS with the sliding windows of the
RRS having the same GC content, so that the DNA bendabilities andcleavage intensities of the same relative sliding window positions
could be compared with the DNA bendabilities and cleavage
intensities expected from the GC content and the genomic context.
For each sliding window position relative to the center of thenucleosomal region, differences in DNA bendability and cleavage
intensity between the PRS and the RRS were evaluated by t-test.
3. Results
3.1. Bendability of nucleosomal DNA
To investigate the structural properties of nucleosomal DNA, we
ﬁrst analyzed the bendability of the DNA sequences surrounding thenucleosomal region. Using all the nucleosomal positions of humanCD4+ T-cells identiﬁed under the resting conditions, we calculated
average DNA bendabilities of the genomic positions relative to thecentral position of the nucleosome (from − 400 to + 400 bps)
(Fig. 1A). Bendability scores within the nucleosomal region weresigniﬁcantly higher than those within the linker regions (P b 10−15;t-test). Moreover, the bendabilities in the nucleosomal region
gradually increased toward the central position of the nucleosome,12

T. Nozaki et al. / Gene 476 (2011) 10–14

DNA bendability pattern, the DNA cleavage intensity pattern for the
nucleosomal DNA region was symmetrical. The cleavage intensity in the
nucleosomal region was higher than that of the linker regions on bothsides (P b 10−15; t-test), and a ~10-bp periodic ﬂuctuation of DNA
cleavage intensity was also observed, especially in the nucleosomal DNA
region (Fig. 2A). The cleavage intensity of the nucleosomal region was
statistically higher than the intensities of the RRS expected from the GC
content, especially at the central position of the nucleosome (Fig. 2B).
Moreover, interestingly, the cleavage intensities in the regions
immediately inside the nucleosome–linker junctions were markedly
higher than average, whereas the intensities in the regions immediately
outside the junctions were markedly lower than average (that is, thedistribution dropped sharply from the inside to the outside of thenucleosome region). However, the difference from the RRS was
statistically signiﬁcant only for the downstream junction (P b 10−5).

3.3. Structural features of nucleosomal DNA and positional stability of
the nucleosome

Fig. 1. Bendability of nucleosomal DNA and the surrounding regions. (A) Trinucleotide
DNA bendability versus genomic position relative to the central position of thenucleosome. The black oval denotes the nucleosome region (from −73 to +73 bps),
and the white diamonds denote linker regions (from −123 to −74 bps and from +74 to+123 bps). The polygonal line and the error bars represent average trinucleotide
bendabilities and standard deviations (s.d.) (Note that s.d. values are shown only for every
5th bp, to improve viewability.) (B) Statistical signiﬁcance (P-values) of differences in DNA
bendability between PRS and RRS as determined by t-test (see Materials and methods).
The polygonal line represents P-values for each genomic position.

which is consistent with the symmetric structure of the histone protein(Luger et al., 1997). The bendability distribution showed a ~10-bp
periodic ﬂuctuation within the nucleosomal DNA region. Even morestriking, DNA bendabilities at the boundaries between the nucleosomal
regions and linker regions on both sides of the nucleosome were quite
high, which implies that the unbending boundary regions also played an
important structural role in nucleosome positioning.
Previous work has suggested that DNA bendability is affected by GC
content (Peckham et al., 2007), and several machine learning
approaches have shown that nucleosome positioning is associatedwith DNA sequence pattern (Peckham et al., 2007; Ogawa et al., 2010).
Therefore, we performed a control analysis of GC content to determine
whether the high bendability of the nucleosomal DNA was aconsequence of high GC content that is possibly correlated with thenucleosomal DNA sequence context. For each position relative to thecentral position of the nucleosome, DNA bendabilities were comparedwith those expected from the RRS (see Materials and methods). P-value
peaks (P b 10−20; t-test) were observed at three positions: the central
position of the nucleosome and at both boundaries between the
nucleosome and the linker regions (Fig. 1B). In contrast, the otherregions did not show statistical signiﬁcance for DNA bendability even
within nucleosomal region, meaning that the DNA bendabilities of theseregions could be expected from GC content alone. The central position of
nucleosomal DNA and the nucleosome–linker junction were suggested
to have high bendabilities that could not be explained by GC content
alone.
3.2. Fragility of nucleosomal DNA
We next computed DNA cleavage intensity, which is an estimate ofhydroxyl radical cleavage strength and the solvent-accessible area, for
the DNA sequence surrounding the nucleosome using ORChID. Like theIf nucleosomal DNA structure determines nucleosome positioning, the positional stability of the nucleosome should correlate withstructural characteristics of nucleosomal DNA. To determine
whether this correlation was in operation, we compared nucleosome positions obtained from the resting state of CD4+ T-cells withthe positions obtained from the activated state, and we then deﬁned
stable and unstable nucleosomes (see Materials and methods). For
each group of stable and unstable nucleosomes, we analyzed DNA
bendability and cleavage intensities of the region surrounding thenucleosome (from −400 to +400 bps) and found that the overall
nucleosomal DNA bendability and the overall cleavage intensity ofstable nucleosomes were markedly higher than those of unstable
nucleosomes (Fig. 3).

Fig. 2. Cleavage intensity of nucleosomal DNA and the surrounding regions. (A) DNA
cleavage intensity. The black oval denotes the nucleosome region (from − 73 to+ 73 bps), and the white diamonds denote linker regions (from − 123 to − 74 bps andfrom + 74 to + 123 bps). The polygonal line and the error bars represent average DNA
cleavage intensity and s.d. (Note that s.d. values are shown only for every 5th bp, toimprove viewability.) (B) Statistical signiﬁcance (P-values) of differences in cleavage
intensity between PRS and RRS as determined by t-test (see Materials and methods).
The polygonal line represents P-values for each genomic position.

T. Nozaki et al. / Gene 476 (2011) 10–14

Fig. 3. Structural characteristics of DNA around stable and unstable nucleosomes. (A)DNA bendability and (B) DNA cleavage intensity. The black oval denotes thenucleosome region (from − 73 to + 73 bps), and the white diamonds denote linker
regions (from − 123 to − 74 bps and from + 74 to + 123 bps).

4. Discussion
In this study, we computationally analyzed two structural featuresof the DNA sequences around nucleosomes and found several
signiﬁcant differences between nucleosomal and linker DNA regions.
The sequence around the nucleosome overall had a higher DNA
bendability score and a higher DNA cleavage intensity score than did
the linker regions. These observations were true overall, but not every
local region of DNA showed exactly the same tendencies. However,
we found many local regions that did show such tendencies (anexample is presented in Supplementary Figure 2). We note that our
preliminary results indicated that these structural features of DNA
sequences associated with nucleosomal and linker regions were also
found in Arabidopsis thaliana (Supplementary Figure 3), which implies
that these structural features may be conserved in other species.
Previous studies have reported the existence of speciﬁc DNA
sequence patterns in the nucleosomal DNA region (Segal et al., 2006;
Peckham et al., 2007; Ogawa et al., 2010). For instance, a previous
support vector machine-based learning approach was used to deriveshort DNA fragments that frequently appear in the nucleosomal
region (Ogawa et al., 2010). However, the correlation between theseshort DNA fragments and nucleosome formation and DNA bendability
scores is far from perfect. Although the trinucleotides CCA/TGG and
CAG/CTG have been reported to have the highest nucleosomeformation potentials (Peckham et al., 2007; Ogawa et al., 2010), thebendability scores of CCA/TGG and CAG/CTG, − 0.246 and 0.175,
respectively, are lower and higher, respectively, than the averagebendability score of −0.019. ATA/TAT, TAA/TTA, and AAA/TTT havebeen suggested as having the potential to inhibit nucleosome
formation (neither of the papers offered any reasons for this low
frequency) (Peckham et al., 2007; Ogawa et al., 2010), but theirbendability scores (0.182, 0.068, and − 0.274, respectively) arehigher, higher, and lower, respectively, than the average. Furthermore, although a previous study suggested that DNA bendability isstrongly correlated with GC content (Peckham et al., 2007), we

13

showed that bendable DNA structures around the center positions of
nucleosomes and in the regions around the nucleosome–linker
junctions cannot be explained by GC content alone. Accordingly, thehigh bendability score of the nucleosomal DNA region is not merely aconsequence of coincidence between DNA bendability parametersand previously known sequence features of nucleosomal DNA, andthis fact supports the plausibility that nucleosomal DNA is bendable.
We also found that high DNA cleavage intensity scores, especiallyat the central positions of the nucleosomes, were independent of GC
content. We also calculated average DNA cleavage intensities oftetranucleotides containing CCA/TGG and CAG/CTG trinucleotides.
Although CCA/TGG and CAG/CTG were the two sequence fragmentswith the highest potentials to form nucleosomes (Ogawa et al., 2010)(as mentioned previously), the average DNA cleavage intensities oftetranucleotides containing CCA/TGG and CAG/CTG were 0.37 and
0.55, respectively, that is, the same as and higher than the averagetetranucleotide cleavage intensity (0.37). This result implies that, likeDNA bendability, the high DNA cleavage intensity score of thenucleosomal DNA region is not due completely to the known
sequence features enriched within the nucleosomal DNA region.
A recent study has suggested a relationship between the bendabilities and cleavage intensities of DNA sequences: these featurescommonly exist within positions of the minor groove of the DNA
structure (Heddi et al., 2009). Our results indicate a similarity
between the distribution patterns of DNA bendability and cleavage
intensity with respect to genomic position relative to the central
position of the nucleosome, and both distributions have ~ 10-bp
periodic ﬂuctuation, which is consistent with length of one pitch of
the double-stranded DNA structure (i.e., the periodicity of the minor
groove positions that face the histone). To determine whether thesimilar distribution patterns were independent from or tightly
associated with each other, we compared trinucleotide bendability
scores and DNA cleavage intensity scores of tetranucleotides. For eachof the trinucleotides having DNA bendability scores, we calculated theaverage DNA cleavage intensity of all tetranucleotides containing thecorresponding trinucleotide in the nucleosomal DNA region. We
found that the Pearson's correlation coefﬁcient between the trinucleotide bendability scores and their corresponding average tetranucleotide cleavage intensity scores was 0.26, which suggests no direct
relationship between bendability and cleavage intensity.
Taken together, our results suggest that the high bendability scoresof the nucleosomal DNA region computed in this study and thesymmetric bendability pattern around the nucleosomes reﬂect the fact
that, regularly repeated, bendable DNA regions have evolved so thatchromosomal DNA can sinuously coil around histones and form thecompact chromatin structure. Moreover, the high cleavage intensity of
the nucleosomal region suggests that histones confer durability tonucleosomal DNA against hydroxyl radicals and that this DNA region
has not needed to gain the durability evolutionarily. Abrupt changes in
DNA bendability and cleavage intensity were found at the nucleosome–linker junctions. In particular, the DNA bendability of thejunction region signiﬁcantly exceeded that expected from the GC
content, which suggests that the DNA at nucleosome–linker junctions
plays an important structural role in the stability of nucleosome
positioning. The overall trends in DNA bendability and cleavage
intensity for the stable nucleosomes were more remarkable than thosefor the unstable nucleosomes; this result supports our speculations
about the biological functions of bendability and cleavage intensity.
Gene expression is thought to be controlled by nucleosomal
positioning and stability (Cairns, 2009). The relationship between
the positional stability of nucleosomes and DNA structural preferences
also suggests that nucleosomal DNA sequences are encoded to control
gene expression. Although known DNA sequence features that aresuggested to facilitate high-efﬁciency formation of nucleosomes andDNA sequences conferring DNA bendability and cleavage intensity
must not be independent from each other, we believe that our results

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T. Nozaki et al. / Gene 476 (2011) 10–14

shed light on how nucleosome positioning's control of cellular
dynamics (e.g., gene expression pattern (Schones et al., 2008; Cairns,2009)) is regulated by DNA structural preferences.
Acknowledgments
This research was supported by research funds from the Yamagata
Prefectural Government and the City of Tsuruoka, Japan, and by agrant-in-aid from the Japan Society for the Promotion of Science(JSPS) to N.Y. (grant no. 1924). During this research, N.Y. was
supported by a JSPS Postdoctoral Fellowship (PD) and a JSPS
Postdoctoral Fellowship for Research Abroad. The source code of
ORChID was kindly provided by Dr. Steve Parker.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
doi:10.1016/j.gene.2011.02.004.
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