OPENSUBJECT AREAS:HISTONE VARIANTS
SAXS
CHROMOSOMES

Received
19 August 2013
Accepted
29 November 2013
Published
16 December 2013

Correspondence andrequests for materials

Structural basis of a nucleosome
containing histone H2A.B/H2A.Bbd thattransiently associates with reorganizedchromatin
Yasuhiro Arimura1, Hiroshi Kimura2,3, Takashi Oda4, Koichi Sato1, Akihisa Osakabe1, Hiroaki Tachiwana1,
Yuko Sato2,3, Yasuha Kinugasa2, Tsuyoshi Ikura5, Masaaki Sugiyama6, Mamoru Sato4,7& Hitoshi Kurumizaka1
1

Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 wakamatsu-cho,Shinjuku-ku, Tokyo 162-8480, Japan, 2Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka
565-0871, Japan, 3JST, CREST, 1-3 Yamadaoka, Suita, Osaka, 565-0871, Japan, 4Graduate School of Medical Life Science,
Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan, 5Department of Mutagenesis, Division of
Chromatin Regulatory Network, Radiation Biology Center, Kyoto University, Yoshidakonoe, Sakyo-ku, Kyoto 606-8501, Japan,
6
Research Reactor Institute, Kyoto University, Kumatori, Osaka, 590-0494, Japan, 7RIKEN SPring-8 Center, 1-1-1 koto, Sayo,
Hyogo 679-5148, Japan.

should be addressed to
H.Kimura (hkimura@fbs.osaka-u.ac.jp) or
H.Kurumizaka(kurumizaka@
waseda.jp)

Human histone H2A.B (formerly H2A.Bbd), a non-allelic H2A variant, exchanges rapidly as compared to
canonical H2A, and preferentially associates with actively transcribed genes. We found that H2A.B
transiently accumulated at DNA replication and repair foci in living cells. To explore the biochemicalfunction of H2A.B, we performed nucleosome reconstitution analyses using various lengths of DNA. Twotypes of H2A.B nucleosomes, octasome and hexasome, were formed with 116, 124, or 130 base pairs (bp) of
DNA, and only the octasome was formed with 136 or 146 bp DNA. In contrast, only hexasome formation
was observed by canonical H2A with 116 or 124 bp DNA. A small-angle X-ray scattering analysis revealed
that the H2A.B octasome is more extended, due to the flexible detachment of the DNA regions at the entry/
exit sites from the histone surface. These results suggested that H2A.B rapidly and transiently forms
nucleosomes with short DNA segments during chromatin reorganization.

Eukaryotic genomic DNA is organized into chromatin, with the nucleosome as the elemental unit. The
protein components of the nucleosome are the core histones H2A, H2B, H3, and H4. In the canonical
nucleosome, two each of the H2A–H2B and H3–H4 dimers associate as the histone octamer, and 145–147
base pairs of DNA are wrapped around it1–3. In general, nucleosomes are biochemically very stable structures invitro, but they are dynamically reorganized during transcription, replication, recombination, and repair in living
cells4–7. During the nucleosome remodeling and assembly/disassembly processes, unusual nucleosomes, such as ahexasome lacking one H2A–H2B dimer from the canonical nucleosome (octasome), are also formed, especiallyduring the transcription initiation and elongation steps8–12. This structural plasticity of the nucleosome appears tobe essential for gene expression and genomic DNA maintenance.
Recent studies have revealed that non-allelic variants of histones exhibit distinct dynamic behaviors. The
structural basis underlying the differential nucleosome stabilities of H3 variants has been extensively studied13–17.
Among nucleosomes containing human H3 variants, the H3T (H3.4) nucleosome is extremely labile both in vitro
and in vivo14, and the centromere-specific H3 variant, CENP-A (cenH3), forms an unusual nucleosome structure,in which the DNA segments located at the entry/exit sites are flexibly detached from the histone surface16.
Therefore, the natural amino acid substitutions in histone variants contribute toward the diversity of nucleosome
structures and dynamics, in combination with post-translational modifications18–20.
In addition to the H3 variants, several H2A variants have been identified in humans, including H2A.X, H2A.Z,
macroH2A, and H2A.B (H2A.Bbd; Barr-body deficient)21,22. H2A.B is a distant H2A variant, and shares about 50%
amino acid identity with the canonical H2A23,24. In living cells, H2A.B tagged with the green fluorescent protein(GFP) is highly mobile, as compared to the canonical H2A tagged with GFP25. Chromatin immunoprecipitation
SCIENTIFIC REPORTS | 3 : 3510 | DOI: 10.1038/srep03510

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coupled with genomic microarray or next generation sequencing
analyses revealed that H2A.B is preferentially localized on the bodies
of actively transcribed genes26,27. Biochemical studies demonstrated
that H2A.B is efficiently incorporated into nucleosomes, and the
DNA segments at the entry/exit sites of the H2A.B nucleosome are
more susceptible to endonuclease28,29. Consistently, electron microscopic and atomic force microscopic analyses indicated that the DNA
entry/exit sites are more flexible in the H2A.B nucleosome, as compared to the canonical H2A nucleosome29,30. The H2A.B nucleosome
has a propensity to form open chromatin in the nucleosome array31.
Based on these studies, H2A.B has been implicated in the nucleosome
dynamics associated with chromatin opening.
In the present study, we further analyzed the properties of H2A.B
in de novo assembly. In living cells, GFP-H2A.B transiently accumulated at DNA replication and repair foci. In a reconstituted system,
we found that H2A.B forms two types of nucleosomes (i.e., octasomeand hexasome) with shorter DNA fragments, such as a 124 bp DNA,
which allows only hexasome formation with the canonical H2A. A
small-angle X-ray scattering analysis revealed that the H2A.B octasome structure contained extra volumes on both sides of the nucleosome, as compared to the canonical H2A octasome, probably
reflecting the flexible DNA segments at the entry/exit sites. These
data suggested that H2A.B may form an intermediate nucleosome
with a shorter DNA segment, when chromatin is newly formed or
reorganized after DNA replication, repair, and transcription.

Results
Behavior of GFP-H2A.B during the cell cycle. We first established
HeLa cells stably expressing GFP-H2A.B. GFP-H2A.B was localized
to the chromosomes in mitotic cells (Fig. 1a, arrow) and distributedin euchromatin in interphase nuclei (Fig. 1a), as previously shown23
and suggested by chromatin immunoprecipitation analyses26,27.
Within a cell population, however, cells exhibiting GFP-H2A.B concentrated in discrete foci were also observed (Fig. 1a, arrowheads).
Since these foci resembled DNA replication foci32, we investigatedthe localization of GFP-H2A.B during the cell cycle. We establishedcells expressing both GFP-H2A.B and PCNA-mCherry, as a marker
of DNA replication complexes formed during S phase32. Time-lapse
microscopy revealed that GFP-H2A.B became more concentrated in
PCNA foci during S phase (Fig. 1b; Supplementary Movie S1). These
results suggested that H2A.B is assembled into chromatin just after
DNA replication, and is soon replaced with the canonical H2A. Thisis consistent with the lower stability of nucleosomal H2A.B in living
cells, as previously detected by fluorescence recovery after photobleaching (FRAP)25.
We also confirmed the rapid recovery of GFP-H2A.B, as compared to the canonical GFP-H2A, after photobleaching with a 488nm laser line (Fig. 2a and b). After bleaching one-half of the nucleus,
the exchange of GFP-H2A.B was almost complete (i.e., the intensitiesof the bleached and unbleached areas reached the same level) within
20 min, while only subtle recovery in the bleached area was observedwith GFP-H2A (Fig. 2a). Although the expression levels of these
GFP-tagged histones varied from cell to cell, their FRAP recovery
kinetics did not depend on the original fluorescence intensity(Supplementary Fig. S1), probably because the GFP-tagged versions
are equilibrated with the endogenous histone molecules that arepresent in great excess4. The FRAP data also revealed the differencein the amounts of chromatin-free, diffusible fractions between GFPH2A.B and GFP-H2A. If free molecules are present, they diffuse in
and out of the bleached area during bleaching, and this causes theintensity of the unbleached area to decrease from the original level, ina phenomenon called fluorescence loss in photobleaching (4; andreferences therein). In addition, under the slow acquisition conditions used, free unbleached molecules can diffuse into the bleachedarea before the acquisition of the first post-bleach image. Under long(,39 s) bleaching conditions (Fig. 2a), the post-bleach intensity of
SCIENTIFIC REPORTS | 3 : 3510 | DOI: 10.1038/srep03510

Figure 1 | Localization of GFP-H2A.B in living cells. (a) GFP-H2A.B
distribution in living HeLa cells imaged by confocal microscopy. GFPH2A.B is localized to interphase nuclei and mitotic chromosomes (arrow).
In some nuclei, GFP-H2A.B is concentrated in foci (arrowheads). (b) GFPH2A.B is concentrated in replication foci during S phase. HeLa cells
expressing both GFP-H2A.B and PCNA-mCherry were grown on a glassbottom dish under a confocal microscope. A cell exhibiting the early
replication pattern of PCNA-mCherry was chosen for time-lapse imaging.
Confocal images of GFP-H2A.B and PCNA-mCherry were obtained every
5 min (see Supplementary Movie S1). The elapsed time (hh:mm) from thestart of recording is shown on each panel. During mid- to late-S phase,
GFP-H2A.B becomes clearly concentrated in PCNA foci, where DNA
replication occurs. Bars, 10 mm.

GFP-H2A.B in the unbleached area dropped to 0.83, while that of
GFP-H2A was close to the original level (0.98). Consistently, thepost-bleach intensity of GFP-H2A.B in the bleached area was higher
than that of GFP-H2A (0.38 vs. 0.27; Fig. 2a). These data suggested
the presence of a large pool of chromatin-free GFP-H2A.B, in contrast to GFP-H2A. The rapid recovery and the larger free pool of
GFP-H2A.B were also observed in short-term FRAP experiments(Fig. 2b). Under these rapid (110 ms) strip-bleach conditions, thepost-bleach intensity in the unbleached region was not affected, but2

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the intensity of GFP-H2A.B in the bleached area was higher than thatof GFP-H2A.
As GFP-H2A.B is concentrated in replication foci during S phase,
the FRAP recovery kinetics could depend on the cell cycle. To clearly
distinguish S-phase cells from non-S phase (G1 or G2) cells, we used
HeLa cells expressing both GFP-H2A.B and PCNA-mCherry, adefinitive indicator of replication foci32. GFP-H2A.B in non-S phasecells exhibited higher recovery than in S phase cells, but this difference was due to the different amounts in the free pools (i.e., the levelat the first post-bleach point), and the curve shapes (exchange rates)
were very similar (Supplementary Fig. S2). The lower level of thechromatin-free fraction in S phase is likely to be a consequence ofthe massive assembly of GFP-H2A.B into replicated chromatin in
addition to the basal level of incorporation into transcribed chromatin26,27. As the FRAP curve shapes were similar between S andnon-S phase cells, the dissociation rates are apparently comparable
even in replication-coupled assembly.
Interestingly, when cells were microirradiated using a 405-nm
laser line that can induce DNA damage, GFP-H2A.B accumulated
in the irradiated area within 2 min (Fig. 2c). In contrast, the canonical GFP-H2A remained relatively immobile under the same conditions (Fig. 2c), similar to the results from photobleaching using a
488-nm laser line (Fig. 2b). Once again, the curve shapes were similar
between S and non-S phase cells (Supplementary Fig. S3). These
results suggested that H2A.B is specifically assembled into chromatinduring DNA damage repair. This may simply be due to the presenceof more chromatin-free H2A.B molecules than H2A; however, specific mechanisms might also be involved. Although the deposition
complex of H2A.B has not been characterized yet, a specific chaperone may facilitate the assembly of H2A.B to the damaged chromatin.
Alternatively, H2A.B incorporation might be coupled with H2AX
evicion, which occurs after DNA damage in a ubiquitylation-dependent manner33.
Taken together with previous data26,27, H2A.B appears to be transiently assembled into chromatin after DNA replication, DNA
repair, and transcription, which all involve chromatin reorganization, including H2A-H2B dimer exchange4,5,8,12. To obtain mechanistic insights into the transient H2A.B assembly, we performed
structural and biochemical analyses using a reconstituted system.

Figure 2 | Kinetics of GFP-H2A.B in living cells. The kinetics of GFPH2A.B and GFP-H2A were analyzed by bleaching with a 488-nm laser (aand b), and by irradiation with a 405-nm laser to induce DNA damage (c).
Fluorescence intensities of bleached or damaged (closed circles) andunbleached or undamaged (open circles) areas are plotted as relative values
to the initial intensity of the same area before bleaching or damaging(averages with standard deviations; N 5 14–24). (a) One-half of thenucleus was bleached using a 488-nm laser for ,38 s, and fluorescence
images were obtained for 50 min. The first post-bleach image was acquired
at ,45 s after the beginning of bleaching. (b) A 2 mm strip was bleachedusing a 488-nm laser for 110 ms, and fluorescence images were obtainedfor 2.5 min. The first post-bleach image was acquired at 1.8 s after the
beginning of bleaching. (c) A 2 mm strip was irradiated using a 405-nm
laser for 3.6 s, and fluorescence images were obtained for 2.5 min. The firstpost-bleach image was acquired at 4.9 s after the beginning of irradiation.
Bars, 10 mm.
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De novo nucleosome formation by H2A.B with 116, 124, and130 bp DNAs. Previous reports found that, in the H2A.B nucleosome, about 118–130 bp of DNA are protected from digestion by
micrococcal nuclease (MNase)28,29. This abbreviated contact of DNA
with histones may explain the instability of H2A.B once it isassembled within a nucleosome; however, it remained uncertain
whether H2A.B could form a nucleosome with a short DNA
segment de novo. To determine whether the H2A.B nucleosome isactually assembled with short DNA fragments, we performed
nucleosome reconstitutions with various lengths of DNA fragments (116, 124, 130, 136, and 146 bp DNAs) and human histones
purified as bacterially expressed recombinant proteins (Fig. 3a). The
nucleosome reconstitution was performed with H2A.B–H2B (or
H2A–H2B) and H3–H4 by the salt-dialysis method (Fig. 3b). As
expected, both H2A and H2A.B formed a nucleosome with 146 bp
DNA (Fig. 3c, lanes 5 and 10). However, we found that H2A.B
formed two types of nucleosomes, which migrated differently onnative PAGE when a 116, 124 or 130 bp DNA fragment was usedas the substrate for nucleosome reconstitution (Fig. 3c, lanes 6–8). In
contrast, H2A only formed one type of nucleosome when a 116 or
124 bp DNA fragment was used (Fig. 3c, lanes 1 and 2). When the
nucleosome reconstitution was performed with H2A in the presenceof a 130 or 136 bp DNA fragment, extra bands, with slower
migration than the major (lower) band, were detected (Fig. 3c,lanes 3 and 4). These extra bands may correspond to the tetrasome3

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Figure 3 | Reconstitution of the H2A.B and H2A nucleosomes withvarious lengths of DNAs. (a) Purified human recombinant histonecomplexes were analyzed by 18% SDS-PAGE with Coomassie Brilliant
Blue staining. (b) Schematic representation of the nucleosome
reconstitution experiments by the salt dialysis method. (c) Nucleosomesreconstituted with 116 bp (lanes 1 and 6), 124 bp (lanes 2 and 7), 130 bp(lanes 3 and 8), 136 bp (lanes 4 and 9), and 146 bp (lanes 5 and 10) DNAs
were analyzed by non-denaturing 6% PAGE, and the gel was stained withethidium bromide after PAGE. Lanes 1–5 indicate the nucleosomesreconstituted with H2A–H2B and H3–H4, and lanes 6–10 indicate thenucleosomes reconstituted with H2A.B–H2B and H3–H4. The 146 bp
DNA was the palindromic satellite DNA derivative.

containing H3–H4 and/or the complex formed by improper histoneDNA binding (Supplementary Fig. S4).
H2A.B forms both an octasome and a hexasome de novo with a
124 bp DNA fragment. To determine the histone compositions of
the H2A and H2A.B nucleosomes reconstituted with a 124 bp DNA
fragment, we electrophoretically purified the upper and lower bandsof the reconstituted H2A.B nucleosome (Fig. 4a). The histonecompositions were analyzed by SDS-PAGE and Coomassie
Brilliant Blue staining (Fig. 4b). Consistent with our previous
study34, the H2A nucleosome reconstituted with a 124 bp DNA
fragment contained about one-half the amount of H2A–H2B, as
compared to that of H3–H4 (Fig. 4b, lane 3, and c), indicating
hexasome formation. Similarly, the lower band of the H2A.B
nucleosome with a 124 bp DNA fragment contained a reduced
amount (about 50%) of H2A.B–H2B (Fig. 4b, lane 5, and c). In
contrast, the upper band contained similar amounts of H2A.B–
H2B (Fig. 4b, lane 6, and c) relative to the control H2A.B octasome(Fig. 4b, lane 4, and c). These results indicated that H2A.B forms both
SCIENTIFIC REPORTS | 3 : 3510 | DOI: 10.1038/srep03510

Figure 4 | H2A.B forms both an octasome and a hexasome with a
124 bp DNA fragment. (a) Nucleosomes reconstituted with a 124 bp
DNA were purified using a Prep Cell apparatus, and were analyzed bynon-denaturing 6% PAGE, and the gel was stained with ethidium
bromide after PAGE. Lanes 1 and 4 indicate the 124 bp DNA used in the
nucleosome reconstitution. Lanes 2 and 3 indicate the H2A nucleosome
samples before (input) and after (lower band) Prep Cell purification,
respectively. Lane 5 indicates the H2A.B nucleosome sample before(input) purification, and lanes 6 (lower band) and 7 (upper band)
indicate the H2A.B nucleosomes after Prep Cell purification. (b) The
histone compositions of the purified nucleosomes were analyzed by 18%
SDS-PAGE with Coomassie Brilliant Blue staining. Lane 1 indicatesmolecular mass markers. Lanes 2 and 4 represent the histones included in
the purified fractions of the H2A and H2A.B octasomes reconstituted
with a 145 bp 601 DNA fragment, respectively. Lane 3 depicts thehistones included in the purified fraction of the H2A nucleosome with a
124 bp DNA fragment, as shown in panel a, lane 3. Lanes 5 and 6 show
the histones included in the purified fractions of the H2A.B nucleosomeswith a 124 bp DNA fragment. Lanes 5 and 6 indicate the lower and upperband fractions, as shown in panel a, lanes 6 and 7, respectively. (c) Lineintensity profiles (a.u.) of the gel shown in (b). Arrowheads indicate
bands with decreased intensity, due to the lack of a single H2A–H2B or
H2A.B–H2B dimer in the hexasome.
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hexasomes and octasomes de novo, but H2A only forms a hexasome
with a shorter DNA fragment, such as 124 bp.
Solution structure of the H2A.B nucleosome. To gain further
insights into the structural differences between the H2A.B nucleosome and the canonical H2A nucleosome, we performed dynamic
light scattering (DLS) measurements. In this analysis, we used the
145 bp 601 DNA fragment, which reportedly forms a stably positioned nucleosome3. Consistent with previous electron and atomic
force microscopic analyses29, the mean particle size of the H2A.B
octasome with the 145 bp 601 DNA fragment was clearly larger
than that of the canonical H2A octasome (Fig. 5a and b). TheseDLS data suggested that the DNA segments may be unwrapped at
the entry/exit sites of the H2A.B octasome. We then performed asmall-angle X-ray scattering (SAXS) analysis of the H2A.B octasome
with the 145 bp 601 DNA fragment (Fig. 5c, d, and e). The radius of
gyration (Rg 5 49.4 6 0.4 Å) and the maximum diameter (Dmax 5
195 Å) of the H2A.B octasome were significantly larger than those of
the canonical H2A octasome (Rg 5 43.4 6 0.3 Å, Dmax 5 125 Å). The
shape of the distance distribution function (P(r)) of the H2A.B

octasome has a long tail, suggesting that the structure of the
H2A.B octasome is not compact. Based on these data, we constructed a dummy atom model of the H2A.B octasome structure(Fig. 5e). In this model, extra volumes were observed on both sidesof the H2A.B octasome, as compared to the canonical H2A octasomemodel. These extra volumes may reflect the flexible DNA segments at
the entry/exit sites of the H2A.B octasome. It should be noted that
nucleic acids scatter more strongly than proteins. Despite thedifferences in X-ray scattering by nucleic acids and proteins, theknown crystal structure of the canonical nucleosome and the SAXS
envelope are consistent with each other (Fig. 5e, right panels)35. This
indicated that the SAXS data could be used to build low-resolution
structural models of nucleosomes.

Discussion
H2A.B (originally named H2A.Bbd) was found as a distant H2A variant that is excluded from the inactive X-chromosome (Barr body)23.
A genome-wide analysis revealed that H2A.B is preferentially associated with actively transcribed genes26,27. It was previously reported
that 118–130 bp DNAs are protected in the H2A.B nucleosome from

Figure 5 | Solution structure of the H2A.B octasome. (a) Schematic representations of the relationship between the hydrodynamic diameter in the DLS
analysis and the nucleosome structure. (b) Particle size distribution profiles in the DLS analysis of the H2A and H2A.B octasomes with a 145 bp 601 DNA
fragment. Closed and open circles represent the particle size distributions of the H2A.B and H2A octasomes, respectively. The nucleosome concentration
was 1.0 mg DNA/ml. The hydrodynamic diameters (Z-average size) of the H2A and H2A.B octasomes were 10.598 nm (error: 1.4658 nm) and11.682 nm (error: 1.6588 nm), respectively. Both octasomes were detected as monodisperse. (c) The SAXS intensity curves of H2A and H2A.B octasomeswith a 145 bp DNA fragment. (d) Distance distribution functions P(r) of the H2A and H2A.B octasomes. (e) Dummy atom models of the H2A.B
octasome (left) and the H2A octasome (right). Side and top views are presented in the upper and lower panels, respectively. The crystal structure of thecanonical nucleosome (PDB ID: 3AFA) is superimposed on the solution structures of the H2A and H2A.B octasomes. Putative flexible DNA segments are
enclosed by dashed circles. Arrows indicate the extra volumes, which may be the mobile areas of the flexible DNA segments.
SCIENTIFIC REPORTS | 3 : 3510 | DOI: 10.1038/srep03510

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MNase digestion28,29. In the present study, we found that H2A.B
intrinsically forms both a hexasome and an octasome with shorterDNA segments, such as 116–130 bp. In contrast, the canonical H2A
did not form an octasome with a 124 bp DNA fragment. Therefore,
the octasome formation with shorter DNA segments may be a specificcharacteristic of the H2A.B nucleosome. This property of H2A.B might
facilitate rapid and transient nucleosome reformation with shorterDNA segments available after transcription, replication, and repair,
before more stable nucleosomes are formed with the canonical H2A.
The transient formation of H2A.B nucleosomes may protect DNA, by
preventing the access of specific or non-specific DNA binding proteinsthat can alter the chromatin structure and/or epigenetic status. The
alteration of the linker DNA orientation, which is caused by DNA
flexibility at the entry/exit regions of the nucleosome, may also affectlinker histone binding. Indeed, the H2A.B nucleosome is reportedly
defective in linker histone H1 binding30. Histone H1 binds to nucleosomal and linker DNAs, and forms a stem-like structure36 to establish ahigher-order chromatin structure. H2A.B nucleosomes may prevent
histone H1 binding and maintain the open chromatin structure after
temporary nucleosome disruption by transcription, replication, andrepair, although H2A.B may play more specific roles27.
H2A.B is reportedly expressed in spermatogenic cells and thenucleosomal chromatin fraction of human sperm37. H2A.B does
not form a stable histone octamer without DNA28, and nucleosomal
H2A.B exchanges rapidly in nuclei25. Consistently, H2A.B confers amore flexible nucleosome structure, as compared to the other mammalian histone H2A variants, H2A.X, H2A.Z, macroH2A, andcanonical H2A38. Intriguingly, these characteristics are common tohuman histone H3T14, which is also highly expressed in testis39–41.
Therefore, the unstable nature of the testis-specific nucleosomes may
play an essential role in the formation of the specific chromatin
structure packaged within the sperm nucleus.

Methods
Cells and microscopy. HeLa cells were routinely grown in Dulbecco’s modified
Eagle’s medium (DMEM; Nacalai Tesque) containing antibiotics (10 U/ml penicillin
and 50 mg/ml streptomycin; Sigma-Aldrich) and 10% fetal calf serum (FCS). Cells
expressing GFP-H2A.B were selected in 1 mg/ml G418 (Nacalai Tesque) after
transfection with the GFP-H2A.B expression vector, which was constructed byinserting a PCR product into the pEGFP-C1 vector (Clontech). The cells expressing
GFP-H2A were previously described42. Cells expressing both PCNA-mCherry and
GFP-H2A.B or GFP-H2A were generated by cell fusion32,43. HeLa cells expressing
GFP-H2A.B or GFP-H2A (G418 resistant) and those expressing PCNA-mCherry(puromycin resistant) were fused using polyethylene glycol (Roche), and single
colonies were selected in the presence of 1 mg/ml G418 and 0.5 mg/ml puromycin.
For live cell analysis, cells were grown on a glass-bottom dish (Mat-Tek) without
synchronization. When necessary, the PCNA-mCherry pattern was used to judge thecell cycle point of each cell. The dish was placed on a confocal microscope (FV-1000;
Olympus), equipped with a culture system (Tokai Hit) at 37uC under a 5% CO2
atmosphere. For time-lapse imaging, confocal images were collected every 5 min for
14 h, using a PlanApoN 603 OSC (NA 5 1.4) oil-immersion lens (512 3 512 pixels;
4 ms/pixel; 4 line Kalman; pinhole 110 mm; line sequential scanning with 488- and
543-nm lasers). Fluorescence recovery after photobleaching (FRAP) and laser microirradiation were performed using a confocal microscope (FV-1000; Olympus) with a
PlanApoN 603 OSC (NA 5 1.4) oil-immersion lens. For Fig. 2a, after 2 images wereobtained every 30 s (0.3% 488-nm laser transmission; 4 ms/pixel; 512 3 512 pixels;
pinhole 800 mm; 1.53 zoom), one-half of each nucleus was bleached (100% 488-nm
laser transmission; 20 ms/pixel; 2 iterations), and images were obtained using theoriginal settings. For Fig. 2b and 2c, after 5 images were obtained (0.1% 488-nm lasertransmission; 4 ms/pixel; 512 3 512 pixels; pinhole 800 mm; 83 zoom), a 2 mm width
strip was bleached (100% 488-nm laser transmission with 4 ms/pixel for FRAP, or100% 405-nm laser transmission with 200 ms/pixel for micro-irradiation), and 95
more images were obtained using the original settings. Fluorescence intensity measurements were performed using Image J version 1.45 s (http://rsb.info.nih.gov/ij/).
The net intensities of the bleached and unbleached areas were obtained by subtracting
the background intensity outside nuclei in each time frame. To obtain relative
intensities to the initial intensity of the same area, the net intensities were normalized
to the average intensity of pre-bleach images.
Purification of recombinant human H2A, H2B, H3.1, and H4. Human histones
H2A, H2B, H3.1 and H4 were expressed as the N-terminally His6-tagged proteins inEscherichia coli cells44, and purified according to the method described previously14–16.
Briefly, the His6-tagged histones were recovered from the insoluble fraction in
50 mM Tris-HCl buffer (pH 8.0), containing 7 M guanidine hydrochloride, 500 mM

SCIENTIFIC REPORTS | 3 : 3510 | DOI: 10.1038/srep03510

NaCl, and 5% glycerol, and purified by Ni-NTA agarose chromatography (Qiagen)under denaturing conditions. The His6-tag was then removed by cleavage withthrombin protease (1 unit/mg of histones; GE Healthcare), and further purified by
Mono S column chromatography (GE Healthcare). The purified histones weredialyzed against water, freeze-dried, and stored at 4uC.
Preparation of the H2A.B–H2B complex. The human H2A.B gene was amplified
from a human testis cDNA pool (Clontech) by polymerase chain reaction (PCR). The
resulting DNA fragment was ligated into the same vector used for the H2A
expression44. The recombinant H2A.B was expressed in Escherichia coli BL21(DE3)codon(1)RIL cells (Stratagene), as the N-terminally His6-tagged protein. The His6tagged H2A.B was recovered from the insoluble fraction in 50 mM Tris-HCl buffer(pH 8.0), containing 7 M guanidine hydrochloride, 500 mM NaCl, and 5% glycerol,and purified by Ni-NTA agarose chromatography (Qiagen) under denaturingconditions. The purified His6-tagged H2A.B was mixed with purified H2B (1 mg
protein/ml) at a 151 stoichiometry, and the sample was dialyzed against 20 mM TrisHCl buffer (pH 7.5), containing 7 M guanidine-HCl and 20 mM 2-mercaptoethanol,for 4 hours, followed by overnight dialysis against 10 mM Tris-HCl buffer (pH 7.5),
containing 2 M NaCl, 1 mM EDTA, and 2 mM 2-mercaptoethanol. The sample wasthen dialyzed against the buffers containing 1 M NaCl for 4 hours, 0.5 M NaCl for 4
hours, and 0.1 M NaCl overnight. The His6-tag was removed from H2A.B by cleavage
with thrombin protease (1 unit/mg of His6-tagged H2A.B). The H2A.B–H2B
complex was further purified by Superdex200 (GE Healthcare Biosciences) gel
filtration chromatography with 10 mM Tris-HCl buffer (pH 7.5), containing 2 M
NaCl, 1 mM EDTA and 2 mM 2-mercaptoethanol.
Preparation of the H2A–H2B and H3.1–H4 complexes. The H2A–H2B and H3.1–
H4 complexes were reconstituted according to the previously published methods14–16.
Purified H2A and H2B were mixed (1 mg protein/ml) at a 151 stoichiometry in
20 mM Tris-HCl buffer (pH 7.5), containing 7 M guanidine-HCl and 20 mM 2mercaptoethanol. Purified H3.1 and H4 were also mixed (1 mg protein/ml) at a 151
stoichiometry in 20 mM Tris-HCl buffer (pH 7.5), containing 7 M guanidine-HCl
and 20 mM 2-mercaptoethanol. These H2A–H2B and H3.1–H4 mixtures weredialyzed against 10 mM Tris-HCl buffer (pH 7.5), containing 2 M NaCl, 1 mM
EDTA, and 2 mM 2-mercaptoethanol. The resulting histone H2A–H2B and H3.1–
H4 complexes were further purified by Superdex200 (GE Healthcare Biosciences) gel
filtration chromatography.
Preparation of 116–146 bp DNA fragments for nucleosome reconstitution. The
116 bp, 124 bp, 130 bp, 136 bp, and 146 bp DNA fragments, containing the asatellite sequence, were prepared by self-ligation of the 56 bp, 60 bp, 63 bp, 66 bp,
and 71 bp DNA segments containing an additional four-base overhang at one 59 end(proximal end), which is located at the center of the DNA fragment after self-ligation.
The 56 bp, 60 bp, 63 bp, and 66 bp DNAs were produced by deleting 15 bp, 11 bp,
8 bp, and 5 bp from the distal end of the 71 bp DNA, respectively. These DNA
fragments were bacterially produced, and were purified by the method described
previously45. Sixteen repeats of each of these DNA segments were inserted into thepGEM-T Easy Vector (Promega). The plasmids were amplified in E. coli cells. The
DNA fragments containing the a-satellite sequence were isolated from the plasmid
vector by digestion with EcoRV. The ends of the EcoRV fragments were
dephosphorylated with calf intestine alkaline phosphatase (CIAP; Nippon Gene)treatment. After the CIAP treatment, the proximal end was digested with EcoRI, andthe four-base overhang was created. Each of these DNA fragments was self-ligated,
and the 116 bp, 124 bp, 130 bp, 136 bp, and 146 bp DNA fragments were prepared.
The 145 bp 601 DNA was bacterially produced, and was purified by the method
described previously45,46. Eight 145 bp 601 DNA fragments were tandemly ligatedinto the pGEM-T Easy vector. The purified plasmid containing eight 601 DNA
repeats was digested with EcoRV, and the resulting 145 bp 601 DNA fragment was
treated with CIAP.
Reconstitution of nucleosomes. The nucleosomes were reconstituted by the salt
dialysis method, as previously described14. H2A–H2B or H2A.B–H2B and H3–H4
were mixed with a DNA fragment (5.2 mM of the molecule for biochemical
experiments or 6.8 mM of the molecule for DLS and SAXS analyses), at a molar ratioof H2A–H2B (or H2A.B–H2B):H3–H4:DNA 5 252.651, in a solution containing
2 M KCl. The KCl concentration was gradually reduced to 250 mM with a peristaltic
pump. The reconstituted nucleosomes were incubated at 55uC for 2 hours, toeliminate the non-specific histone-DNA binding. The nucleosomes were further
purified by non-denaturing polyacrylamide gel electrophoresis (PAGE), using a Prep
Cell apparatus (Bio-Rad). The nucleosomes were eluted with 20 mM Tris-HCl buffer(pH 7.5), containing 1 mM EDTA and 1 mM dithiothreitol.
Dynamic light scattering (DLS) measurements. DLS measurements of thenucleosomes (1.0 mg DNA/ml) containing H2A.B or H2A were performed at 20uC
with a Zetasizer Nano mV system (Malvern Instruments), in 20 mM Tris-HCl buffer(pH 7.5), containing 1 mM EDTA and 1 mM dithiothreitol. For the DLS
experiments, the 145 bp 601 DNA was used.
Small-angle X-ray scattering (SAXS) analysis. The SAXS camera (SAXES) installed
at BL10C of KEK-PF (Ibaraki, Japan) was used to collect SAXS intensity data. The
SAXS intensities of H2A and H2A.B nucleosome solutions were measured with an RAXIS IV11 imaging plate detector at 20uC with an exposure time of 600 s and a6www.nature.com/scientificreports
sample-to-detector distance of 2040.060 mm, which was calibrated by the powder
diffraction from silver docosanoate. The nucleosome samples were reconstituted withthe 145 bp 601 DNA. Circular averaging of the SAXS intensity data was thenperformed to obtain the one-dimensional intensity data I(q) as a function of q (q 5
4psinh/l, where 2h is the scattering angle and the X-ray wavelength l 5 1.488 Å). To
correct the inter-particle interference effect, I(q) data were collected at three
nucleosome concentrations (0.5, 1.0, and 3.0 mg/mL), and were extrapolated to zeroconcentration. A SAXS intensity measurement of the buffer solution (20 mM TrisHCl buffer, pH 7.5, containing 1 mM EDTA and 1 mM dithiothreitol) for
background subtraction was also performed, using the same conditions andprocedure as those used for the nucleosome solutions.
The radius of gyration, Rg, was estimated by fitting the I(q) data using the Guinier
approximation I(q) 5 I(0) exp(2q2Rg2/3), where I(0) is the forward scattering at thezero scattering angle, in the smaller angle region of qRg , 1.3. The error of Rg wasestimated from the least-squares fitting. The distance distribution function P(r) andits error were calculated by the program GNOM47. The maximum dimension, Dmax,
was estimated from the P(r) function as the distance r, where P(r) 5 0, and its error
was estimated from the errors of the P(r) values around P(r) 5 0. All data were
processed and analyzed using the software applications embedded in the ATSAS
package48.
Construction of dummy atom models of nucleosomes containing H2A.B or H2A.
The low-resolution dummy atom models of the nucleosomes containing H2A.B or
H2A were produced by DAMMIN, using the scattering data49. Ten independently
calculated, low-resolution dummy atom models were averaged with DAMAVER foreach nucleosome50.
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Acknowledgments
We thank M.C. Cardoso for the PCNA-GFP expression vector, and T. Kamimoto for
constructing the GFP-H2A.B expression vector. This work was supported in part by
Grants-in-Aid from the Japanese Society for the Promotion of Science (JSPS), and theMinistry of Education, Culture, Sports, Science and Technology (MEXT), Japan. H.
Kurumizaka was also supported by the Waseda Research Institute for Science andEngineering.

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Author contributions

Additional information

Y.A., K.S., A.O. and H.T. purified the histones, and performed biochemical analyses. H.
Kimura, Y.S. and Y.K. performed the cell-based analysis. Y.A., T.O., M. Sugiyama and M.
Sato performed the SAXS analysis. T.I. contributed to the preliminary FRAP analysis. H.
Kimura and H. Kurumizaka conceived the study, supervised all of the work, and wrote themanuscript.

Supplementary information accompanies this paper at http://www.nature.com/
scientificreports
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Arimura, Y. et al. Structural basis of a nucleosome containing
histone H2A.B/H2A.Bbd that transiently associates with reorganized chromatin. Sci. Rep. 3,
3510; DOI:10.1038/srep03510 (2013).
This work is licensed under a Creative Commons Attribution 3.0 Unported license.
To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0

SCIENTIFIC REPORTS | 3 : 3510 | DOI: 10.1038/srep03510

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