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 biochemical function of H2A.B, we performed nucleosome reconstitution analyses using various lengths of DNA. Two types 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 it1,2,3. In general, nucleosomes are biochemically very stable structures in vitro, but they are dynamically reorganized during transcription, replication, recombination and repair in living cells4,5,6,7. During the nucleosome remodeling and assembly/disassembly processes, unusual nucleosomes, such as a hexasome lacking one H2A–H2B dimer from the canonical nucleosome (octasome), are also formed, especially during the transcription initiation and elongation steps8,9,10,11,12. This structural plasticity of the nucleosome appears to be 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 studied13,14,15,16,17. 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 modifications18,19,20.
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 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., octasome and 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.
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 distributed in 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 investigated the localization of GFP-H2A.B during the cell cycle. We established cells 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. This is 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 488-nm 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 intensities of the bleached and unbleached areas reached the same level) within 20 min, while only subtle recovery in the bleached area was observed with 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 are present in great excess4. The FRAP data also revealed the difference in the amounts of chromatin-free, diffusible fractions between GFP-H2A.B and GFP-H2A. If free molecules are present, they diffuse in and out of the bleached area during bleaching and this causes the intensity of the unbleached area to decrease from the original level, in a phenomenon called fluorescence loss in photobleaching (4; and references therein). In addition, under the slow acquisition conditions used, free unbleached molecules can diffuse into the bleached area before the acquisition of the first post-bleach image. Under long (~39 s) bleaching conditions (Fig. 2a), the post-bleach intensity of 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, the post-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, the post-bleach intensity in the unbleached region was not affected, but the intensity of GFP-H2A.B in the bleached area was higher than that of 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, a definitive indicator of replication foci32. GFP-H2A.B in non-S phase cells exhibited higher recovery than in S phase cells, but this difference was due to the different amounts in the free pools (i.e., the level at the first post-bleach point) and the curve shapes (exchange rates) were very similar (Supplementary Fig. S2). The lower level of the chromatin-free fraction in S phase is likely to be a consequence of the 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 and non-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 chromatin during DNA damage repair. This may simply be due to the presence of 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.
De novo nucleosome formation by H2A.B with 116, 124 and 130 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 is assembled 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 is actually 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 on native PAGE when a 116, 124 or 130 bp DNA fragment was used as 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 presence of 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 tetrasome containing H3–H4 and/or the complex formed by improper histone-DNA 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 bands of the reconstituted H2A.B nucleosome (Fig. 4a). The histone compositions 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 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). These DLS data suggested that the DNA segments may be unwrapped at the entry/exit sites of the H2A.B octasome. We then performed a small-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 = 49.4 ± 0.4 Å) and the maximum diameter (Dmax = 195 Å) of the H2A.B octasome were significantly larger than those of the canonical H2A octasome (Rg = 43.4 ± 0.3 Å, Dmax = 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 sides of the H2A.B octasome, as compared to the canonical H2A octasome model. 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 the differences in X-ray scattering by nucleic acids and proteins, the known 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.
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 MNase digestion28,29. In the present study, we found that H2A.B intrinsically forms both a hexasome and an octasome with shorter DNA 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 specific characteristic of the H2A.B nucleosome. This property of H2A.B might facilitate rapid and transient nucleosome reformation with shorter DNA 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 proteins that 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 affect linker 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 a higher-order chromatin structure. H2A.B nucleosomes may prevent histone H1 binding and maintain the open chromatin structure after temporary nucleosome disruption by transcription, replication and repair, although H2A.B may play more specific roles27.
H2A.B is reportedly expressed in spermatogenic cells and the nucleosomal 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 a more flexible nucleosome structure, as compared to the other mammalian histone H2A variants, H2A.X, H2A.Z, macroH2A and canonical H2A38. Intriguingly, these characteristics are common to human histone H3T14, which is also highly expressed in testis39,40,41. 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.
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 μg/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 by inserting 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 μg/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 the cell cycle point of each cell. The dish was placed on a confocal microscope (FV-1000; Olympus), equipped with a culture system (Tokai Hit) at 37°C under a 5% CO2 atmosphere. For time-lapse imaging, confocal images were collected every 5 min for 14 h, using a PlanApoN 60× OSC (NA = 1.4) oil-immersion lens (512 × 512 pixels; 4 μs/pixel; 4 line Kalman; pinhole 110 μm; line sequential scanning with 488- and 543-nm lasers). Fluorescence recovery after photobleaching (FRAP) and laser micro-irradiation were performed using a confocal microscope (FV-1000; Olympus) with a PlanApoN 60× OSC (NA = 1.4) oil-immersion lens. For Fig. 2a, after 2 images were obtained every 30 s (0.3% 488-nm laser transmission; 4 μs/pixel; 512 × 512 pixels; pinhole 800 μm; 1.5× zoom), one-half of each nucleus was bleached (100% 488-nm laser transmission; 20 μs/pixel; 2 iterations) and images were obtained using the original settings. For Fig. 2b and 2c, after 5 images were obtained (0.1% 488-nm laser transmission; 4 μs/pixel; 512 × 512 pixels; pinhole 800 μm; 8× zoom), a 2 μm width strip was bleached (100% 488-nm laser transmission with 4 μs/pixel for FRAP, or 100% 405-nm laser transmission with 200 μs/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 in Escherichia coli cells44 and purified according to the method described previously15,16,14. 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 NaCl and 5% glycerol and purified by Ni-NTA agarose chromatography (Qiagen) under denaturing conditions. The His6-tag was then removed by cleavage with thrombin protease (1 unit/mg of histones; GE Healthcare) and further purified by Mono S column chromatography (GE Healthcare). The purified histones were dialyzed against water, freeze-dried and stored at 4°C.
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(+)RIL cells (Stratagene), as the N-terminally His6-tagged protein. The His6-tagged 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 denaturing conditions. The purified His6-tagged H2A.B was mixed with purified H2B (1 mg protein/ml) at a 1:1 stoichiometry and the sample was dialyzed against 20 mM Tris-HCl 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 was then 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 methods14,15,16. Purified H2A and H2B were mixed (1 mg protein/ml) at a 1:1 stoichiometry in 20 mM Tris-HCl buffer (pH 7.5), containing 7 M guanidine-HCl and 20 mM 2-mercaptoethanol. Purified H3.1 and H4 were also mixed (1 mg protein/ml) at a 1:1 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 were dialyzed 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 α-satellite 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 5′ 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 the pGEM-T Easy Vector (Promega). The plasmids were amplified in E. coli cells. The DNA fragments containing the α-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 and the 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 ligated into 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 μM of the molecule for biochemical experiments or 6.8 μM of the molecule for DLS and SAXS analyses), at a molar ratio of H2A–H2B (or H2A.B–H2B):H3–H4:DNA = 2:2.6:1, 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 55°C for 2 hours, to eliminate 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 the nucleosomes (1.0 mg DNA/ml) containing H2A.B or H2A were performed at 20°C with a Zetasizer Nano μV 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 R-AXIS IV++ imaging plate detector at 20°C with an exposure time of 600 s and a sample-to-detector distance of 2040.060 mm, which was calibrated by the powder diffraction from silver docosanoate. The nucleosome samples were reconstituted with the 145 bp 601 DNA. Circular averaging of the SAXS intensity data was then performed to obtain the one-dimensional intensity data I(q) as a function of q (q = 4πsinθ/λ, where 2θ is the scattering angle and the X-ray wavelength λ = 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 zero concentration. A SAXS intensity measurement of the buffer solution (20 mM Tris-HCl buffer, pH 7.5, containing 1 mM EDTA and 1 mM dithiothreitol) for background subtraction was also performed, using the same conditions and procedure 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) = I(0) exp(−q2Rg2/3), where I(0) is the forward scattering at the zero scattering angle, in the smaller angle region of qRg < 1.3. The error of Rg was estimated from the least-squares fitting. The distance distribution function P(r) and its 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) = 0 and its error was estimated from the errors of the P(r) values around P(r) = 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 for each nucleosome50.
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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 the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. H. Kurumizaka was also supported by the Waseda Research Institute for Science and Engineering.
The authors declare no competing financial interests.
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Arimura, Y., Kimura, H., Oda, T. et al. Structural basis of a nucleosome containing histone H2A.B/H2A.Bbd that transiently associates with reorganized chromatin. Sci Rep 3, 3510 (2013). https://doi.org/10.1038/srep03510
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