Crystal Structure and Characterization of Novel Human Histone H3

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Crystal structure and characterization of novel human histone H3 variants, H3.6, H3.7, and H3.8 Hiroyuki Taguchi, Yan Xie, Naoki Horikoshi, Kazumitsu Maehara, Akihito Harada, Jumpei Nogami, Koichi Sato, Yasuhiro Arimura, Akihisa Osakabe, Tomoya Kujirai, Takeshi Iwasaki, Yuichiro Semba, Taro Tachibana, Hiroshi Kimura, Yasuyuki Ohkawa, and Hitoshi Kurumizaka Biochemistry, Just Accepted Manuscript • Publication Date (Web): 04 Apr 2017 Downloaded from http://pubs.acs.org on April 5, 2017

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Crystal structure and characterization of novel human histone H3 variants, H3.6, H3.7, and H3.8 Hiroyuki Taguchi1, Yan Xie1, Naoki Horikoshi1, Kazumitsu Maehara2, Akihito Harada2, Jumpei Nogami2, Koichi Sato1, Yasuhiro Arimura1, Akihisa Osakabe1, Tomoya, Kujirai1, Takeshi Iwasaki2, Yuichiro Semba2, Taro Tachibana3, Hiroshi Kimura4, Yasuyuki Ohkawa2*, and Hitoshi Kurumizaka1* 1

Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering,

Research Institute for Science and Engineering, and Institute for Medical-oriented Structural Biology, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan 2

Division of Transcriptomics, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan 3

Department of Bioengineering, Osaka City University, Graduate School of Engineering, Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan

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Cell Biology Unit, Institute of Innovative Research, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan

KEYWORDS Histone H3, Histone variant, Nucleosome, Chromatin, Crystal structure, Epigenetics

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ABSTRACT

Non-allelic histone variants are considered as epigenetic factors that regulate genomic DNA functions in eukaryotic chromosomes. In the present study, we identified three new human histone H3 variants (named H3.6, H3.7, and H3.8), which were previously annotated as pseudogenes. H3.6 and H3.8 conserve the H3.3-specific amino acid residues, but H3.7 shares the specific amino acid residues with H3.1. We successfully reconstituted the nucleosome containing H3.6 in vitro, and determined its crystal structure. In the H3.6 nucleosome, the H3.6-specific Val62 residue hydrophobically contacts the cognate H4 molecule, but its contact area is smaller than that of the corresponding H3.3 Ile62 residue. The thermal stability assay revealed that the H3.6 nucleosome is substantially unstable, as compared to the H3.3 nucleosome. Interestingly, the mutational analysis demonstrated that the H3.6 Val62 residue is fully responsible for the H3.6 nucleosome instability, probably by the reduced hydrophobic interaction with H4. We also reconstituted the nucleosome containing H3.8, but its thermal stability was quite low. In contrast, purified H3.7 failed to form nucleosomes in vitro. The identification and characterization of these novel human histone H3 variants provide important new insights toward understanding the epigenetic regulation of the human genome.

INTRODUCTION


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In eukaryotic chromatin, genomic DNA segments (145-147 base pairs) are accommodated within the nucleosome. The nucleosome is a nuclear nucleoprotein complex, in which the histone octamer, composed of two each of the four core histones H2A, H2B, H3, and H4, left-handedly winds the DNA1-3. Short linker DNA segments connect adjacent nucleosomes, and the resulting poly-nucleosomes are folded into the higher-order chromatin conformation4. The chromatin structure enables the accommodation of the large genomic DNA within the nucleus; however, it is generally inhibitory for genomic DNA functions, such as replication, repair, recombination, and transcription4,5. To promote these nuclear events within chromatin, nucleosomes may be disrupted, remodeled, and/or repositioned at the functional loci in chromosomes6-10. To do so, the nucleosome structure must be more dynamic in active chromosomal loci, but may be more stable in inactive loci. Therefore, the nucleosome stability could be a critical factor for the genomic DNA regulation in cells. Histones are post-translationally modified, and these histone modifications are considered to change the nucleosome stability and the nucleosome-nucleosome interactions in chromatin11-18. In addition, many species-specific histone variants exist19-30. These histone variants result in the adaptability of the structures and dynamics of nucleosomes31-46, and may function in genomic DNA regulation as epigenetic factors. In humans, eight histone H3 variants, H3.1, H3.2, H3.3, H3T (H3.4), H3.5, H3.X, H3.Y, and CENP-A (CenH3), have been identified so far47-53. Our previous in silico hybridization screening identified fourteen uncharacterized H3 variant genes in mouse, an uncharacterized H3 variant gene in rat, and three new histone H3 variant genes in humans, which were previously annotated as pseudo-genes21, 54.


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In the present study, we found that these new human histone H3 variants actually exist in certain human tissues. These new human H3 variants were named H3.6, H3.7, and H3.8. Histones H3.6, H3.7, and H3.8 are encoded by the H3F3AP6, HIST2H3PS2, and H3F3AP5 genes, respectively, which were previously annotated as pseudo-genes54. A sequence comparison suggested that H3.6 and H3.8 are H3.3 derivatives. In contrast, H3.7 shares the sequence with H3.1. Consistently, the genomic distribution profile of H3.6 is quite similar to that of H3.3. The nucleosome containing H3.6 was efficiently reconstituted in vitro. H3.8 was incorporated into nucleosomes in vitro, but H3.7 was defective in in vitro nucleosome formation. We then determined the crystal structure of the H3.6 nucleosome. Interestingly, the H3.6 nucleosome is significantly unstable, as compared to the H3.3 nucleosome. In contrast, a structure-based mutational analysis revealed that the H3.6-specific Val62 residue is fully responsible for the instability of the H3.6 nucleosome.

EXPERIMENTAL PROCEDURES Antibodies. Anti-H3.6, -H3.7, and -H3.8 monoclonal antibodies were produced by the method described previously21. Briefly, the monoclonal mouse antibodies targeting the anti-human H3 variants were generated in accordance with the method established by Sado et al.55. The H3.6, H3.7 and H3.8 antigens were synthesized according to their specific sequences, H3.6: CVTIMPKDIQLAHSIRGERA (AA 117-135), H3.7: CVTIMPKDIQLVSRIRGERA (AA 117135) and H3.8: CARTRWTARKSTGGIAPRKQL (AA 1-20) (Sigma-Aldrich). Eight-week-old female C57BL/6 mice (SLC, Shizuoka, Japan) were immunized with an emulsion containing the synthesized oligopeptide conjugated with KLH and Freund's complete adjuvant (Invitrogen).


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After three weeks, cells from the lymph nodes of mice immunized with the antigen were fused with mouse myeloma Sp2/0-Ag14 cells. At seven days postfusion, the hybridoma supernatants were screened by an ELISA against the oligopeptide-conjugated BSA. The specificity of each positive clone was confirmed by verifying the cross-reactivity for the other H3 variant peptides. Positive clones were subcloned and rescreened by an ELISA. Concentrated antibody preparations were prepared from the clones 7C4 (for H3.6), 8E9 (for H3.7), and 1A6 (for H3.8). Large-scale in vitro production of these antibodies was performed by culturing clones in Hybridoma-SFM medium (Invitrogen) containing interleukin-6, and the antibodies were subsequently purified by Hitrap SP ion exchange chromatography (GE Healthcare).

Identification of Human H3 Variants by in silico Hybridization. Human histone H3 variant genes were explored by the in silico hybridization method, as described previously21.

Histone H3 Variant Specific mRNA Detection. mRNA-Seq data of human tissues were obtained from NCBI’s GEO: GSE30611. All single-end reads data were mapped onto the human genome (hg19) using the Bowtie software (version 0.12.8) with the parameters “–v 0 –m 1”, which allow no base mismatches and no multi-hit reads. We counted the mapped reads on the exons of all H3 genes, and normalized the counts as RPM (Reads Per Million mapped reads).

Fluorescence Recovery after Photobleaching (FRAP). HeLa cells in which dox-inducible GFP-fused H3.6 gene was integrated were cultured in the presence of doxycycline for 2 days,


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and then cultured in the absence of doxycycline for 5 days. FRAP analysis using the cells producing GFP-fused H3.6 was performed according to the previous method56. Cells grown on a glass-bottom dish (Mat-tek) was set on to a heated stage (Tokai Hit) under 5% CO2 (Tokken) on a confocal microscope (FV1000; Olympus) with a 60× PlanApoN Oil SC (NA = 1.4) objective lens and a 488-nm Ar ion laser (BA505 emission filter). Images were collected (800 × 800 pixels, zoom factor 2, pixel dwell time 2 µs, pinhole 800 µm, and 0.4% laser transmission) before bleaching. After bleaching one-half of each nucleus (100% laser transmission), 13 images were acquired using the original settings every 5 min for 60 min. The relative fluorescence recovery was measured using the Image J 1.51h software (Rasband, http://rsb.info.nih.gov/ij/), as described previously35,45.

Immunoprecipitation of GFP-Fused H3.6. The chromatin fraction containing GFP-fused H3.6 was prepared as described previously, with some modifications45. Briefly, HeLa cells expressing GFP-fused H3.6 were resuspended in 4 ml of RSB, containing 1% Triton X-100 and 1x protease inhibitor (Nacalai). The cells were homogenized with a Dounce homogenizer (loose pestle; 10 times). Nuclei were isolated and washed twice with 1 ml of buffer A (15 mM HEPESNaOH (pH 7.4), 60 mM KCl, 15 mM NaCl, 0.15 mM spermidine, 0.5 mM spermine, 0.34 M sucrose, 1x protease inhibitor, and 1 mM DTT), and were resuspended in buffer A (10x volume of pellet; 5 x 107 nuclei/ml). CaCl2 (final 1 mM) and micrococcal nuclease (4,000 gel units, NEB) were then added to 500 µl of the nuclear suspension. The sample was incubated at 30°C for 1 h. After adding 5 µl of 0.5 M EDTA (pH 8.0), the sample was centrifuged (10,621x g; 10 min; 4°C), and the pellet was suspended in 450 µl of 10 mM EDTA (pH 8.0). Subsequently, 5 M


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NaCl (50 µl) was added, and the sample was centrifuged (20,000x g; 10 min; 4°C). The supernatant was then collected as the soluble chromatin fraction. For immunoprecipitation, Dynabeads Protein G (50 µl, Life Technologies) were mixed with anti-GFP pAb (2 µg, MBL), in 200 µl of PBS at 25ºC for 1 h. After washing the beads with 1 ml of buffer B (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 5 mM EDTA, and 0.2% Tween 20), 220 µl of the chromatin fraction was added, and the mixture was incubated overnight at 4°C with rotation. The unbound fraction (IP sup) was then collected. CaCl2 (final 1 mM) and micrococcal nuclease (250 gel units, NEB) were then added to the beads. The sample was incubated at 30°C for 30 min. The beads were washed 3 times with 1 ml of buffer B, and the proteins were eluted by adding 22 µl of 2x SDS buffer (100 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.2% bromophenol blue, and 100 mM DTT). Afterward, the sample was incubated at 60°C overnight. For the DNA analysis, DNA was purified using a QIAquick PCR Purification Kit (Qiagen), and was analyzed by electrophoresis on a 2.5% agarose gel in 1x TAE buffer with ethidium bromide staining. For the protein analysis, the samples were separated by 15% SDS-PAGE, and the proteins were detected by Coomassie Brilliant Blue staining and western blotting with a peroxidase-conjugated anti-GFP pAb (1:1,000; SANTA CRUZ; sc-8334). The gel image was acquired with an LAS-4000 image analyzer (GE Healthcare).

Immunohistochemistry. Human tissue sections (multiple organ tissue array) were purchased from US Biomax. Non-specific binding was blocked by rabbit serum, and tissue sections were incubated with the anti-H3.6, -H3.7, and -H3.8 monoclonal antibodies (1:100 dilution) for 24 h


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at room temperature. The endogenous H3.6, H3.7, and H3.8 were visualized using the avidin– biotin complex method.

Chromatin Immunoprecipitation. GFP-fused H3.1, H3.3, and H3.6 expressing HeLa cells were cross-linked by a treatment with 0.1% formaldehyde for 5 min at room temperature, and were suspended in ChIP buffer (10 mM Tris-HCl, 200 mM KCl, 1 mM CaCl2, 0.5% NP-40, 2 µg/ml Aprotinin, 2 µg/ml Leupeptin, and 1 µg/ml Pepstatin A). Chromatin immunoprecipitation (ChIP) assays were performed as described previously21,57.

ChIP Sequencing and Data Analysis. ChIP samples were prepared from GFP-fused H3.1, H3.3, and H3.6 expressing cells. Reads were mapped to the human reference genome (GRCh38) using HISAT2 (v2.0.4)58, with non-unique alignments removed. Peaks were selected with BCP (v1.1)59 by running the executable file BCP_HM, with the options -f 200 -w 200 -p 0.001. The peaks were annotated using HOMER (v4.8)60, by running annotatePeaks.pl with the optionannStats.

Preparation of Recombinant Histones. Recombinant histones (human H2A, H2B, H3.3, H3.6, H3.7, H3.8, and H4) were expressed in E. coli cells61,62, and were purified by the previously described method35-45. The expression vectors for the H3.3 mutants were constructed by site-directed mutagenesis.


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Nucleosome Reconstitution. Freeze-dried histones (1.2 mg of H2A, 1.2 mg of H2B, 1.0 mg of H4, and 1.4 mg of H3 (H3.3, H3.6, H3.7, or H3.8) were mixed in buffer A (20 mM Tris–HCl (pH 7.5), 20 mM 2-mercaptoethanol, and 7 M guanidine hydrochloride), and dialyzed against refolding buffer (10 mM Tris–HCl (pH 7.5), 5 mM 2-mercaptoethanol, 1 mM EDTA, and 2 M NaCl). The resulting histone complexes were subjected to HiLoad16/60 Superdex 200 gel filtration chromatography (GE Healthcare). Fractions containing histone complexes were collected, concentrated, and mixed with the α-satellite DNA (146 base-pair) in 2 M KCl solution. The nucleosomes were then reconstituted by the salt-dialysis method, as described previously3545

. The resulting nucleosomes were separated from the free histones and DNA by non-denaturing

PAGE (Prep Cell, Bio-Rad), in 20 mM Tris-HCl (pH 7.5) buffer containing 1 mM DTT.

The H3.3 and H3.6 Nucleosome Reconstitution for Crystallization and Thermal Stability Assay. The 146 base-pair α-satellite DNA1 was mixed with the purified histone octamer containing either H3.3, or H3.6, reconstituted as described previously35,36, in 2 M KCl solution. The nucleosome was reconstituted as described previously35-45. The resulting nucleosomes were separated from the free DNA and histones by non-denaturing PAGE (Prep Cell, Bio-Rad), in a buffer containing 20 mM Tris-HCl (pH 7.5) and 1 mM DTT.

Crystallization and Structure Determination of the H3.3 and H3.6 Nucleosomes. Crystallization and structural determination of the H3.3 and H3.6 nucleosomes were performed


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by methods similar to those reported previously35. The purified nucleosomes were crystallized by the hanging drop vapor diffusion method. The H3.3 nucleosome crystals were soaked in a cryoprotectant solution, containing 20 mM potassium cacodylate (pH 6.0), 32 mM KCl, 60 mM MnCl2, 30% polyethylene glycol 400, and 5% trehalose. The H3.6 nucleosome crystals were soaked in 20 mM potassium cacodylate (pH 6.0) buffer, 2% trehalose, 28% 2-methyl-2,4pentanediol, 40 mM KCl, and 45 mM MnCl2. These crystals were flash-cooled in a cryo-stream of N2 gas (-180ºC). Diffraction data were collected at the beamline BL41XU station of SPring-8, Harima, Japan, and the beamline BL-5A and BL-17A stations of the Photon Factory (KEK), Tsukuba, Japan. Diffraction data were analyzed by the HKL2000 program63, followed by processing CCP4 program suite64. The structures of the H3.3 and H3.6 nucleosomes were determined by the molecular replacement method, with the search models, PDB ID: 2CV565 and PDB ID: 3AFA35, respectively, using the Phaser program66. The structures of the H3.3 and H3.6 nucleosomes were refined with the PHENIX program67, and the model was built with the COOT program68. The PyMOL program (Schrodinger; http://www.pymol.org) was used for all structure figures.

Thermal Stability Assay of Nucleosomes. The reconstituted nucleosomes were purified by non-denaturing PAGE (Prep Cell, Bio-Rad), as previously described35-45, and were subjected to the thermal stability assay in 19 µl of 20 mM Tris–HCl (pH 7.5), 1 mM DTT, and 5x SYPRO Orange (SIGMA-ALDRICH). The thermal stability assay was essentially performed with the same procedure described previously42,69,70.


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Data Access. The atomic coordinates have been deposited with the RCSB ID code [PDB: 5X7X (for H3.3 nucleosome), 5GXQ (for H3.6 nucleosome)] in the RCSB Protein Data Bank. Deep-sequencing data have been deposited with the accession number [DDBJ: DRA005164] in the DDBJ sequence read archive.

RESULTS Novel Human Histone H3 Variants, H3.6, H3.7, and H3.8. Human histones H3.6, H3.7, and H3.8 are encoded by the H3F3AP6, HIST2H3PS2, and H3F3AP5 genes, respectively. H3.6 and H3.8 share the Ser31 residue, the Ala87-Ala88-Ile89-Gly90 residues, and the Ser96 residue with H3.3, suggesting that these H3 variants are derivatives of H3.3 (Figure 1A). In contrast, H3.7 shares the Ala31 residue and the Ser87-Ala88-Val89-Met90 residues with H3.1 (Figure 1A). Therefore, H3.7 may be a derivative of H3.1. We analyzed the public data (GSE30611) available for various human tissues, and found that these histones were indeed expressed in some of the tissues (Figure 1B-D). The H3.6, H3.7, and H3.8 mRNA productions are detected, but their expression levels are extremely low, as compared to the H3.3 mRNA production from the H3F3B gene (Figure 1E). To test whether the products of these genes are present in human cells, we generated monoclonal antibodies that specifically recognize H3.6, H3.7, and H3.8 (Figure 2A). The specificities of these H3.6, H3.7, and H3.8 antibodies were confirmed by ELISA (Supplementary Figure S1). Immunohistochemical analyses revealed that these antibodies stained human tissue sections, such as ovary, colon, breast, and lung (Figure 2B), suggesting that H3.6 and H3.7 are actually produced in these human tissues. However, it is unclear whether H3.8 exists in these tissues (Figure 2B). Consistently, the amount of the H3.8 mRNA is extremely


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low, although it was detected in some tissues (Figure 1D). These results indicate that the H3F3AP6, HIST2H3PS2, and probably H3F3AP5 genes are not pseudogenes, but actually produce transcripts and proteins.

H3.6, but not H3.7 and H3.8, Efficiently Forms a Histone Octamer with H2A, H2B, and H4. We next tested the histone octamer formation abilities of H3.6, H3.7, and H3.8. Purified histones H2A, H2B, H4, and each of the H3 variants were mixed under the 7 M guanidine hydrochloride conditions, and the histone octamers were assembled by dialysis against reconstitution buffer containing 2 M NaCl (Figure 3A). The reconstituted histone complexes were analyzed by gel filtration chromatography (Figure 3A). As shown in Figure 3B and C, H3.6 formed a histone octamer with H2A, H2B, and H4, like H3.3. Surprisingly, no peaks corresponding to the H3.7-H4 complex were detected, indicating that H3.7 did not form a complex with H4 under the conditions used in this study (Figure 3D). Interestingly, H3.8 formed complexes with H4, but the H3.8-H4 complex did not bind to the H2A-H2B dimer (Figure 3E). These results indicate that H3.7 and H3.8 are defective in the histone octamer formation activity in vitro, but H3.6 efficiently forms a histone octamer, like H3.1 and H3.3.

H3.6 and H3.8 Form Nucleosomes with H2A, H2B, and H4. We next tested whether H3.6, H3.7, and H3.8 possess nucleosome formation activity. To do so, we performed the nucleosome assembly assay by the salt-dialysis method, with the histone complexes eluted from the gel filtration column (Figure 3). We found that H3.6 and H3.8, but not H3.7, formed nucleosomes with H2A, H2B, and H4 (Figure 4A, B). It is quite intriguing that only four amino acid


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substitutions in H3.7 cause such a drastic biochemical difference from H3.1. The H3.7-specific Arg96 residue sterically clashes with H4, when it is superimposed on the H3.3 nucleosome structure (Supplementary Fig. S2). This may be the reason why H3.7 is extremely inefficient in the complex formation with H4. Specific factors that facilitate the histone complex formation between H3.7 and H4 may be required for the incorporation of H3.7 into chromatin in cells. We then tested the stability of the H3.6 and H3.8 nucleosomes. To do so, we employed the thermal stability assay42,68,69. In this assay, the nucleosome is incubated with a fluorescent dye, SYPRO Orange, and thermally dissociated histones are detected as the fluorescence signal emitted from the SYPRO Orange bound to denatured histones (Figure 5A). We found that the H3.6 nucleosome was substantially unstable, as compared to the H3.3 nucleosome (Figure 5B). Non-denaturing polyacrylamide gel electrophoresis revealed that, at 75°C, the DNA wrapped in the H3.3 nucleosome still formed complexes with histones, but a large amount of free DNA was detected in the H3.6 nucleosome sample, indicating that the histones were largely dissociated from the H3.6 nucleosome at this temperature (Figure 5C). The H3.8 nucleosome was extremely unstable (Figure 5D). The H3.8 nucleosome migrated more slowly than the H3.3 and H3.6 nucleosomes, and sub-nucleosomal bands, probably corresponding to tetrasomes and hexasomes, were detected on the native polyacrylamide gel (Figure 4A). These characteristics of the H3.8 nucleosome may also reflect its flexible nature induced by its instability.

H3.6 Is Incorporated into Chromatin in Cells. We then tested whether H3.6 is incorporated into chromatin in human cells. To do so, GFP-fused H3.6 was produced in HeLa cells, and the soluble chromatin fractions were prepared by MNase treatment (Figure 6A, lower and middle


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panels). We found that GFP-fused H3.6 was clearly incorporated into the soluble chromatin (Figure 6A, upper panel). Consistently, our fluorescence recovery after photobleaching experiments demonstrated that the mobility of GFP-fused H3.6 was extremely low (Figure 6B and C). Interestingly, we also detected GFP-fused H3.6, which was incorporated into the Mphase chromosomes (Figure 6D). These results strongly indicate that H3.6 is efficiently incorporated into chromatin in cells. We then analyzed the distribution of H3.6 in the human genome, by the chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) method. Consistent with the sequence similarity data, the genomic distribution of H3.6 was similar to that of H3.3 (Figure 6E), suggesting that H3.6 may be incorporated into chromatin by common chaperone pathway shared with H3.371-74. Notably, the FRAP data suggested that GFP-H3.6 is stably incorporated into chromatin in cells, although the H3.6 nucleosome is thermally unstable. This contradiction may be explained by the absence of a histone chaperone and/or nucleosome remodeler specific for the H3.6 nucleosome, in the cells used in the experiments.

Crystal Structure of the Nucleosome Containing H3.6. We then determined the crystal structure of the nucleosome containing H3.6, at 2.85 Å resolution (Figure 7A, Table 1). H3.6 has three specific amino acid residues, Val62, His128, and Ser129, which correspond to the H3.3Ile62, H3.3-Arg128, and H3.3-Arg129 residues, respectively (Figure 1A). In the H3.3 nucleosome, the H3.3-Ile62 residue forms a hydrophobic cluster with the H4-Ile29, H4-Ala33, H4-Leu37, and H4-Leu58 residues (Figure 7C). In the H3.6 nucleosome, this hydrophobic


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interaction between H3 and H4 may be weakened, because the hydrophobic contact surface of the H3.6-specific Val62 residue with the H4-Ile29, H4-Ala33, H4-Leu37, and H4-Leu58 residues is clearly smaller than that of the H3.3 Ile62 residue (Figure 7B and C). This structural difference between the H3.3 and H3.6 nucleosomes may affect their stability.

The H3.6-Val62 Residue Is Responsible for the Unstable Nature of the H3.6 Nucleosome. We suspected that the weakened hydrophobic interaction around the H3.6-Val62 residue may be responsible for the H3.6 nucleosome instability (Figure 7B and C). We then purified two H3.3 mutants, H3.3 I62V and H3.3 R128H R129S. In these H3.3 mutants, the H3.3-Ile62 residue and the H3.3-Arg128 and -Arg129 residues were replaced by the H3.6-specific residues, Val, His, and Ser, respectively. Our thermal stability assay revealed that the thermal denaturation profile of the H3.3 I62V nucleosome was quite similar to that of the H3.6 nucleosome (Figure 8A). This indicated that the H3.6-Val62 residue contributes to the H3.6 nucleosome instability. To support this conclusion, we tested the nucleosome stability with the H3.3 I62L mutant, in which the H3.3-Ile62 residue is replaced by Leu. We then found that the H3.3 I62L mutation, which did not significantly reduce the hydrophobic contact surface around position 62, did not affect the nucleosome stability (Figure 8B). Interestingly, the mutations of H3.3 R128H R129S did not affect the thermal denaturation profile of the H3.3 nucleosome, indicating that these two residues do not contribute to the H3.6 nucleosome instability (Figure 8C). Therefore, we conclude that the H3.6-specific Val62 residue is fully responsible for the instability of the H3.6 nucleosome.

DISCUSSION


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Histone variants are now recognized as essential factors for the epigenetic regulation of genomic DNA19-30. Previously, we established the in silico screening method for histone variants, and identified fourteen novel H3 variants in mice21. This method has also identified three additional human H3 variants, which were previously annotated as pseudogenes54. Interestingly, except for H3t, the new mouse H3 variants are not conserved in human35,49. Therefore, histone variants may perform species-specific functions in regulating genomic DNA metabolism. We first prepared the H3.6-, H3.7-, and H3.8-specific monoclonal antibodies, and confirmed that these H3 variants actually exist in certain human tissues, at the protein level. mRNA analyses revealed that H3.6, H3.7, and H3.8 have specific expression profiles. H3.6 ubiquitously exists in many tissues, as compared to H3.7 and H3.8. This fact suggests that H3.6 may have a more general function, although the mRNA levels of these H3 variants were very low, as compared to that of H3.3. To understand the functions of these novel human H3 variants, in the present study, we tried to determine the structural and biochemical characteristics of the nucleosomes containing these histone H3 variants. To do so, we prepared H3.6, H3.7, and H3.8 as recombinant proteins, and performed the in vitro nucleosome formation assay. As expected, H3.6 efficiently formed the nucleosome. However, surprisingly, H3.7 was quite defective in the nucleosome formation in vitro. H3.8 forms nucleosomes, but they are extremely unstable. Given that H3.7 and H3.8 are incorporated into chromatin in human cells, as components of nucleosomes, specific histone chaperones for H3.7 and H3.8 incorporation must be required in cells. Alternatively, it is possible that H3.7 and H3.8 may have specific partners, such as other histone variants and/or proteins containing histone-fold domains, to form nucleosomes or nucleosome-like structures


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with H3.7 and H3.8. In future experiments, it will be intriguing to study such H3.7- and H3.8interacting factors. In contrast to H3.7 and H3.8, H3.6 efficiently formed the nucleosome, and we determined the crystal structure of the H3.6 nucleosome. We found that the H3.6 nucleosome is thermally unstable, as compared to the H3.3 nucleosome. Our structural and mutational analyses revealed that the H3.6-specific Val62 residue is fully responsible for the H3.6 nucleosome instability. The H3.3-Ile62 residue hydrophobically contacts the surrounding H4 residues. In contrast, this hydrophobic cluster may be weakened by the H3.6-Val62 residue, because the hydrophobic surface area of the valine side chain is smaller than that of the isoleucine side chain, thus reducing the hydrophobic interaction between histones H3 and H4 in the nucleosome. This may be the structural basis for the H3.6 nucleosome instability. A similar characteristic has been reported for the H3.5-specific Leu103 residue, which corresponds to the H3.3-Phe104 residue44. In contrast to the H3.5 and H3.6 nucleosomes, the human H3 variant H3.Y complexed with H4 associates with DNA more stably than the H3.3-H4 complex45. The nucleosome stability differences have also been reported for other histone variants, including H2A.Z38,43, H2A.B (H2A.Bbd)39,70, H3T35,70, and CENP-A37,42. Intriguingly, the acetylation or crotonylation of the H3-Lys122 residue also reduces the stability of the H3-H4-DNA complex (tetrasome)17. The nucleosome stability is also affected by the mono-ubiquitination of the H2B-Lys120 or H4Lys31 residue18. In contrast, multiple methylations of the nucleosomal DNA did not affect the stability of the nucleosome75. These histone variants and/or modifications, but not the DNA methylation itself, provide the adaptability of the nucleosome stability, and may play important roles in the epigenetic regulation of DNA metabolism by controlling the access to DNA in chromatin.


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In addition to the H3.6 Val62 residue, H3.6 also contains two specific amino acid residues, His128 and Ser129. According to the previous structure of the H3-H4 dimer complexed with a histone-chaperone, Asf176, the H3-Arg129 residue directly binds to Asf1. In the crystal structure of the H3.6 nucleosome, we found no significant structural differences with the H3.3 nucleosome around residue 129. Our thermal stability assay showed that the H3.3 R128H R129S mutations did not affect the nucleosome stability. These facts indicate that the H3.6-specific His128 and Ser129 residues are not involved in nucleosome formation and stability. Therefore, the H3.6specific His128 and Ser129 residues may function to regulate Asf1 binding, which could be a prerequisite for nucleosome assembly, and may affect the H3.6 loading efficiency into chromatin. Further studies are awaited.

AUTHOR INFORMATION Corresponding Authors *Tel +81-3-5369-7315; Fax +81-3-5367-2820; e-mail [email protected]. *Tel +81-92-642-6384; Fax +81-92-642-6562; e-mail [email protected]. Author Contributions H.T., Y.X., N.H., Y.A., A.O., and T.K. purified proteins, prepared nucleosomes, and performed structural and biochemical analyses. K.M., A.H., J.N., T.I., Y.S., T.T., and Y.O. performed immunohistochemical and ChIP-seq analyses. N.H., K.S., and H.Kimura performed fluorescence recovery after photobleaching experiments. Y.O. initially conceived this project. H.Kurumizaka


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conceived, designed, and supervised all of the work, and wrote the paper. All of the authors discussed the results and commented on the manuscript. Funding Sources This work was supported in part by JSPS KAKENHI Grant Numbers JP25116002 [to H.Kurumizaka], JP25250023 [to H.Kurumizaka], JP25116005 [to H.Kimura], JP25116010 [to Y.O.], JP16H01219 [to A.H.], and JP16H01577 [to K.M.], and was also partially supported by grants from the Platform Project for Supporting Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science), from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and the Japan Agency for Medical Research and Development (AMED) [to H.Kurumizaka]. This work was also supported by CREST, JST [to Y.O., H.Kurumizaka, and H.Kimura]. H.Kurumizaka and N.H. were also supported by the Waseda Research Institute for Science and Engineering, and the research programs of Waseda University. H.T. was supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We thank M. Noda (Waseda University) for the preparation of the H3.3 nucleosome crystals. We are also grateful to the beamline scientists for their assistance with data collection at the BL41XU beamline of SPring-8 and the BL-5A, and BL-17A beamline of the Photon Factory.


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The synchrotron radiation experiments were performed with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) [proposal no. 2012B1048] and the Photon Factory Program Advisory Committee [proposal no. 2012G569].

ABBREVIATIONS DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; HEPES, 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid; PAGE, poly-acrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; TBE, Tris-borate-EDTA; Tris, tris(hydroxymethyl)aminomethane.

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(60) Heinz, S., Benner, C., Spann, N., Bertolino, E. Lin, Y. C., Laslo, P., Cheng, J. X., Murre, C., Singh, H. and Glass, C. K. (2010) Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell, 38, 576–589. (61) Tanaka, Y., Tawaramoto-Sasanuma, M., Kawaguchi, S., Ohta, T., Yoda, K., Kurumizaka, H. and Yokoyama, S. (2004) Expression and purification of recombinant human histones. Methods 33, 3–11. (62) Tachiwana, H., Osakabe, A., Kimura, H. and Kurumizaka, H. (2008). Nucleosome formation with the testis-specific histone H3 variant, H3t, by human nucleosome assembly proteins in vitro. Nucleic Acids Res. 36, 2208–2218. (63) Otwinowski, Z. and Minor, W. (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326. (64) Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R., Keegan, R. M., Krissinel, E. B., Leslie, A. G. W., McCoy, A., McNicholas, S. J., Murshudov, G. N., Pannu, N. S., Potterton, E. A., Powell, H. R., Read, R. J., Vagin, A., and Wilson, K. S. (2011) Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235-242, 1–8. (65) Tsunaka, Y., Kajimura, N., Tate, S., and Morikawa, K. (2005) Alteration of the nucleosomal DNA path in the crystal structure of a human nucleosome core particle. Nucleic Acids Res. 33, 3424−3434. (66) McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni L. C. and Read, R.J. (2007) Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674. (67) Adams, P. D., Afonine, P. V., Bunkóczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung, L. W., Kapral, G. J., Grosse-Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., Zwart, P. H., (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221. (68) Emsley, P. and Cowtan, K. (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132. (69) Iwasaki, W., Miya, Y., Horikoshi, N., Osakabe, A., Taguchi, H., Tachiwana, H., Shibata, T., Kagawa, W. and Kurumizaka, H. (2013) Contribution of histone N-terminal tails to the structure and stability of nucleosomes. FEBS Open Bio. 3, 363–369. (70) Taguchi, H., Horikoshi, N., Arimura, Y. and Kurumizaka, H. (2014) A method for evaluating nucleosome stability with a protein-binding fluorescent dye. Methods 70, 119-126. (71) Ray-Gallet, D., Quivy, J. P., Scamps, C., Martini, E. M., Lipinski, M. and Almouzni, G. (2002) HIRA is critical for a nucleosome assembly pathway independent of DNA synthesis. Mol. Cell 9, 1091−1100.


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(72) Tagami, H., Ray-Gallet, D., Almouzni, G. and Nakatani, Y. (2004) Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis. Cell 116, 51−61. (73) Lewis, P. W., Elsaesser, S. J., Noh, K. M., Stadler, S. C. and Allis, C. D. (2010) Daxx is an H3.3-specific histone chaperone and cooperates with ATRX in replication-independent chromatin assembly at telomeres. Proc. Natl. Acad. Sci. U.S.A. 107, 14075−14080. (74) Drané, P., Ouararhni, K., Depaux, A., Shuaib, M. and Hamiche, A. (2010) The deathassociated protein DAXX is a novel histone chaperone involved in the replication-independent deposition of H3.3. Genes Dev. 24, 1253−1265. (75) Osakabe, A., Adachi, F., Arimura, Y., Maehara, K., Ohkawa, Y. and Kurumizaka, H. (2015) Influence of DNA methylation on positioning and DNA flexibility of nucleosomes with pericentric satellite DNA. Open Biol. 5, 150128. (76) Natsume, R., Eitoku, M., Akai, Y., Sano, N., Horikoshi, M., Senda, T. (2007) Structure and function of the histone chaperone CIA/ASF1 complexed with histones H3 and H4. Nature 446, 338-341.


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Biochemistry

FIGURES Figure 1. Expression analyses of novel human histone H3 variants, H3.6, H3.7, and H3.8. (A) Sequence alignment of human histone H3 variants. Amino acid residues specifically found in H3.6, H3.7, and/or H3.8 are shown in white characters with a black background. The secondary structure of H3.1 in the nucleosome is shown at the top of the panel. The α-helices are shown as grey cylinders. (B-E) Graphic representations of the tissue-specific gene expression data, based on the human map (GSE30611). (B) H3.6 (H3F3AP6), (C) H3.7 (HIST2H3PS2), (D) H3.8 (H3F3AP5), (E) H3.3 (H3F3B).

Figure 2. Histochemical analyses of novel human histone H3 variants, H3.6, H3.7, and H3.8. (A) The purified human histones were analyzed by 16% SDS-PAGE with Coomassie Brilliant Blue staining, and were detected by western blotting using specific monoclonal antibodies against H3.6, H3.7, and H3.8. (B) Production of the H3.6, H3.7, and H3.8 proteins in human tissues. The human tissues (ovary, colon, breast, and lung) were immunohistochemically stained with the H3.6-, H3.7-, and H3.8-specific monoclonal antibodies. The bar indicates 10 µm.

Figure 3. Histone octamer formation activities of H3.6, H3.7, and H3.8. (A) Schematic diagram of the octamer formation. The histone complexes were reconstituted with purified histones (H2A, H2B, H4, and H3.6, H3.7, or H3.8). (B-E) The reconstituted histone complexes were subjected to HiLoad 16/60 Superdex 200 prep grade gel filtration column


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chromatography. The elution profiles of the histone complexes, containing H3.3 (B), H3.6 (C), H3.7 (D), and H3.8 (E) are presented. Histone compositions of the peak fractions (indicated by a, b, c, and d with arrows) were analyzed by 18% SDS-PAGE with Coomassie Brilliant Blue staining (inset panels).

Figure 4. Nucleosome formation activities of H3.6 and H3.8. (A) The nucleosomes were reconstituted with the histone fractions eluted from the Superdex 200 gel filtration column (shown in Figure 3), and were purified by non-denaturing PAGE (Prep Cell, Bio-Rad). The purified nucleosomes containing H3.3, H3.6, and H3.8 were analyzed by 0.2xTBE 6% non-denaturing PAGE with ethidium bromide staining. (B) The purified nucleosomes containing H3.3, H3.6, and H3.8 were analyzed by 18% SDS-PAGE with Coomassie Brilliant Blue staining.

Figure 5. Thermal stability of the nucleosomes containing H3.6 and H3.8. (A) Schematic diagram of the thermal stability assay of the nucleosomes. (B) The thermal stability curves of the H3.3 and H3.6 nucleosomes are shown. The mean and standard deviation of measurements performed in triplicate are shown. (C) The H3.3 and H3.6 nucleosomes were incubated at 25ºC and 75ºC, and were analyzed by 0.2xTBE 6% non-denaturing PAGE with ethidium bromide staining. (D) The thermal stability curves of the H3.3 and H3.8 nucleosomes are shown. The mean and standard deviation of measurements performed in triplicate are shown.


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Biochemistry

Figure 6. H3.6 is efficiently incorporated into chromatin in cells. (A) Incorporation of GFP-H3.6 into chromatin. Soluble chromatin fractions prepared by MNase treatment were analyzed by western blotting with an anti-GFP antibody (top, for GFP-H3.6), SDS-PAGE (15%) with Coomassie Brilliant Blue staining (middle, for endogenous histones), and 2.5% agarose gel electrophoresis with ethidium bromide staining (bottom, for MNasedigested genomic DNA). (B) FRAP analysis. The mobility of GFP-fused H3.6 was analyzed by bleaching one-half of a nucleus. Representative images of HeLa cells expressing GFP-H3.6 are presented before bleaching (left panel), 0 min after bleaching (middle panel), and 30 min after bleaching (right panel). The bar indicates 10 µm. (C) The FRAP curve of GFP-H3.6 in cells. Relative fluorescence intensities of GFP-H3.6 after photobleaching are plotted, with the standard deviations (N=13 for GFP-H3.6). (D) A representative image of a mitotic HeLa cell expressing GFP-H3.6. The mitotic chromosomes are stained with anti-GFP (left panel). An image obtained with a differential interference contrast microscope (DIC) is presented (right panel). The bar indicates 10 µm. (E) Genomic distribution of the GFP-fused H3 variants produced in HeLa cells. Localizations in promoter (blue), 5’UTR (orange), exon (yellow), intron (yellowish green), 3UTR (light orange), transcription termination site (TTS) (light blue), intergenic region (grey), CpG-Island (dark green), short interspersed elements (SINE) (green), long interspersed elements (LINE) (light green), long terminal repeat (LTR) (sky blue), DNA transposon (wheat), simple repeat (magenta), satellite (red), and others (white) are presented. For H3.6, two independent experiments were performed, and the results are presented (H3.6 #1 and #2).

Figure 7. The crystal structure of the H3.6 nucleosome.


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(A) The overall structure of the H3.6 nucleosome. H3.6 is shown in magenta. Histones H2A, H2B, and H4 are colored cyan, and the DNA strands are light yellow. Two views are shown. The region enclosed by a black rectangle (right panel) is enlarged and presented in panel B. (B) A close-up view of the H3-H4 interface around the H3.6 Val62 residue (magenta). The H4 residues are shown in cyan. Hydrophobic surfaces of amino acid residues are presented. (C) A close-up view of the H3-H4 interface around the H3.3 Ile62 residue (yellow). The H3.3 nucleosome structure was determined at 2.18 Å resolution, and was used for a structural comparison with the H3.6 nucleosome. Hydrophobic contact surface areas of the H3.6 Val62 and H3.3 Ile62 residues were calculated by PyMOL, and are presented at the bottoms of panels B and C.

Figure 8. The H3.6-specific Val62 residue is responsible for the instability of the H3.6 nucleosome. (A) Thermal stability curves of the H3.6 and H3.3 I62V nucleosomes. (B) Thermal stability curves of the H3.3, H3.6, and H3.3 I62L nucleosomes. (C) Thermal stability curves of the H3.3, H3.6, and H3.3 R128H R129S nucleosomes. For all panels, the means and standard deviations of triplicate measurements are shown.


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Biochemistry

Table 1 X-ray Data Collection and Refinement Statistics Data Collection Space Group Cell Dimensions a, b, c (Å) α, β, γ (°) Resolution (Å) Reflections (Unique) Rmerge (％) I/σ(I) Completeness (％) Redundancy Refinement Resolution (Å) Rwork/free (％) B-factors (Å2) Protein DNA Ligand/Ion Water R.M.S. Deviations Bond Length (Å) Bond Angles (°) Ramachandran Plot residues in favorable regions (％) residues in allowed regions (％) PDB code

H3.3 nucleosome

H3.6 nucleosome

P212121

P212121

a = 98.887 b = 107.509 c = 167.415 α = β = γ = 90 50.00 - 2.18 (2.26 - 2.18) 92136 8.1 (48.3) 19.8 (3.5) 99.5 (98.9) 14.2 (8.9)

a = 106.026 b = 109.764 c = 181.400 α = β = γ = 90 50.00 - 2.85 (2.95 - 2.85) 50454 8.4 (46.4) 13.5 (6.8) 99.6 (100) 5.9 (5.9)

48.600 - 2.18 22.65 / 25.56

38.129 - 2.85 21.91 / 26.18

22.37 47.08 45.73 27.56

44.63 96.37 -

0.006 0.818

0.010 1.147

98.91 1.09 5X7X

97.30 2.70 5GXQ


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Graphic for the Table of Contents (ToC Graphic) For Table of Contents Use Only


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Figure 1 184x186mm (300 x 300 DPI)


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Figure2 184x206mm (300 x 300 DPI)


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Figure 3 174x156mm (300 x 300 DPI)


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Figure 4 90x136mm (300 x 300 DPI)


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Figure 5 184x216mm (300 x 300 DPI)


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Figure 6 184x216mm (300 x 300 DPI)


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Figure 7 90x106mm (300 x 300 DPI)


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Figure 8 184x166mm (300 x 300 DPI)


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Crystal Structure and Characterization of Novel Human Histone H3

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