Crystal structure and stable property of the cancer-associated heterotypic nucleosome containing CENP-A and H3.3

The centromere-specific histone H3 variant, CENP-A, is overexpressed in particular aggressive cancer cells, where it can be mislocalized ectopically in the form of heterotypic nucleosomes containing H3.3. In the present study, we report the crystal structure of the heterotypic CENP-A/H3.3 particle and reveal its “hybrid structure”, in which the physical characteristics of CENP-A and H3.3 are conserved independently within the same particle. The CENP-A/H3.3 nucleosome forms an unexpectedly stable structure as compared to the CENP-A nucleosome, and allows the binding of the essential centromeric protein, CENP-C, which is ectopically mislocalized in the chromosomes of CENP-A overexpressing cells.

I n eukaryotes, chromatin compacts genomic DNA with the nucleosome, as the basic repeating unit. Histones H2A, H2B, H3, and H4 are the protein components of the nucleosome. The histone octamer, containing two each of histones H2A, H2B, H3, and H4, left-handedly wraps about 150 base pairs of DNA around itself in the nucleosome 1,2 . In addition to the canonical types of histones, non-allelic isoforms of histones H2A, H2B, and H3 have been identified as histone variants in many species 3 , and are suggested to have specific functions in the regulation and maintenance of genomic DNA in chromatin [4][5][6] .
In humans, eight types of histone H3 genes, encoding H3.1, H3.2, H3.3, H3T (also named H3.4), H3.5, H3.X, H3.Y, and CENP-A (also named CenH3), have been identified 3,7 . H3.1, H3.2, and H3.3 are constantly produced in somatic cells. H3.1 and H3.2 are the canonical types of histone H3, and are expressed in the S-phase of the cell cycle 8 . On the other hand, H3.3 is continuously produced throughout the cell cycle. During the transcription and DNA repair processes, H3.3 functions as a replacement histone that is incorporated into chromatin regions by the HIRA histone chaperone, after the depletion of the canonical H3.1 [8][9][10] . In addition, H3.3 is specifically localized in the telomeric and pericentromeric regions of chromosomes, by a complex containing the histone chaperone DAXX and the nucleosome remodeler ATRX proteins 11,12 . Therefore, H3.3 may have dual functions as a replacement histone and an architectural component of distinct chromosome domains.
Among the histone H3 variants, CENP-A is the most distant isoform, and is conserved from yeasts to humans. CENP-A specifically localizes to centromeres and epigenetically specifies the sites for kinetochore assembly [13][14][15][16][17][18] . CENP-A expression is tightly controlled in normal cells, and its chromosome localization is strongly restricted within centromeric regions. However, CENP-A is reportedly overexpressed in particular aggressive cancer cells 4,[19][20][21][22] . In Drosophila cells, overexpressed CENP-A (CID) is mislocalized into noncentromeric euchromatic regions and leads mitotic delays, anaphase bridges and chromosome fragmentation 23 . Similarly, in human cells, CENP-A overexpression can lead to its ectopic localization to chromosome regions with active histone turnover, as shown in cancer cell lines 19,24 . At these ectopic loci, CENP-A forms heterotypic nucleosomes, containing one each of the histone H3 variants, CENP-A and histone H3.3, within a single nucleosome 24 . These heterotypic nucleosomes occlude CTCF binding, and their presence may increase DNA damage tolerance in cancer cells 24 . However, the structural features of the heterotypic CENP-A/H3.3 nucleosome are unknown.
In the present study, we report the crystal structure of the human CENP-A/H3.3 nucleosome at 2.67 Å resolution. The CENP-A/H3.3 nucleosome forms a ''hybrid structure'', in which the physical characteristics of CENP-A and H3.3 are independently conserved within the same nucleosome. Unexpectedly, we found that the heterotypic CENP-A/H3.3 nucleosome is more stable than the CENP-A nucleosome, and its stability is very similar to that of the H3.3 nucleosome. In addition, the CENP-A/H3.3 nucleosome retains the ability to bind to the essential centromeric protein, CENP-C, whose ectopic localization may also be harmful for the proper regulation of cell division and chromosome integrity.
The structural characteristics of CENP-A and H3.3 are conserved independently within the heterotypic CENP-A/H3.3 nucleosome. We then determined the crystal structure of the heterotypic CENP-A/H3.3 nucleosome at 2.67 Å resolution ( Fig. 2a and Supplementary  Table 1). In the crystal structure, the CENP-A and H3.3 molecules were clearly distinguishable. For example, the side chain moiety of the CENP-A-specific His104 residue, corresponding to the H3.3 Gly102 residue, was clearly observed in the CENP-A molecule in the heterotypic nucleosome ( Fig. 2b and Supplementary Fig. 2b and c). On the other hand, the H3.3-specific Gln68 residue, corresponding to the CENP-A Ser68 residue, was distinctly visible in the H3.3 molecule ( Fig. 2b and Supplementary Fig. 2b and c). In the homotypic CENP-A nucleosome, the CENP-A aN helices were shorter than the canonical H3.1 aN helices in H3.1 homotypic nucleosomes 27 . This CENP-A-specific aN structure is perfectly conserved in the CENP-A/H3.3 nucleosome, in which the CENP-A aN helix is one helical turn shorter than the H3.3 aN helix ( Fig. 2b and Supplementary Fig. 2a).
The structural asymmetry of the CENP-A and H3.3 molecules induces the asymmetric wrapping of the DNA in the CENP-A/ H3.3 nucleosome (Fig. 2a). The different electron densities observed at the two ends suggested that the DNA end on the CENP-A side is more flexible than that on the H3.3 side in the CENP-A/H3.3 nucleosome. This was confirmed by differential micrococcal nuclease (MNase) digestion at the ends ( Fig. 3a and Supplementary Fig. 3a) and by a treatment with Exonuclease III (ExoIII), which digests one strand of duplex DNA from the 39 end, and degraded only one end of the DNA in the CENP-A/H3.3 nucleosome ( Fig. 3b and Supplementary Fig. 3b). In contrast, with the homotypic CENP-A nucleosome ( Fig. 3a and Supplementary Fig. 3a), MNase attacked about 20 base pairs of DNA ( Fig. 3a and Supplementary Fig. 3a), and ExoIII degraded about 10 bases from both DNA ends ( Fig. 3b and Supplementary Fig. 3b).
Therefore, in the CENP-A/H3.3 nucleosome, the DNA end close to CENP-A could be more flexible than the DNA end close to H3.3. These results are consistent with a previous in vivo MNase analysis, which demonstrated that the average DNA length tightly wrapped within the CENP-A/H3.3 nucleosomes is shorter than that of the canonical H3 nucleosome, but longer than that of the homotypic CENP-A nucleosome 24 .
The heterotypic CENP-A/H3.3 nucleosome is more stable than the homotypic CENP-A nucleosome. To assess the biological significance of the CENP-A/H3.3 nucleosome, we examined its thermal stability. For this, we utilized a fluorescent dye, SYPRO Orange, which binds to denatured proteins by hydrophobic interactions 28 . In the assay, the fluorescence signal from SYPRO Orange was monitored as a function of increasing temperature. An increase in the fluorescence signal indicates that the histones have dislodged from the nucleosome and become denatured 29,30 (Fig. 4a).
For the H3.3 nucleosome, a biphasic histone dissociation curve (Fig. 4b, upper panel) was observed, with first and second Tm values of 70-71uC and 83-84uC, respectively (Fig. 4b, lower panel). The first and second thermal dissociation curves reflect the stepwise dissociation of the H2A-H2B dimer and the H3.3-H4 tetramer, respectively 30 . The CENP-A nucleosome exhibited a monophasic dissociation curve (Fig. 4c, upper panel) with a Tm value of about 71-72uC (Fig. 4c, lower panel). This suggested that the CENP-A-H4 tetramer may be unstably incorporated into the nucleosome, as compared to the H3.3-H4 tetramer, and may simultaneously dissociate with the H2A-H2B dimer.
To test the unstable association of the CENP-A-H4 tetramer with DNA, we reconstituted tetrasomes, containing the CENP-A-H4 tetramer or the H3.3-H4 tetramer with a 146 base-pair DNA fragment (CENP-A tetrasome and H3.3 tetrasome, respectively), in the absence of the H2A-H2B dimer. After the salt dialysis reconstitution, the tetrasomes were fractionated by native PAGE. The bands corresponding to the H3.3 tetrasomes were smeared, probably by the unusual histone-DNA binding and/or the multiple positions of the H3.3-H4 tetramer in the tetrasomes (Fig. 4d, lanes 1-3). The H3.3 tetrasomes ran as a single band, when the sample was treated at 45-65uC for 2 hr (Fig. 4d, lanes 4-6), suggesting the formation of the stably positioned H3.3 tetrasome. In contrast, the CENP-A tetrasomes had a tendency to form positioned tetrasomes at lower temperatures (Fig. 4d, lanes 7-10), but the positioned CENP-A tetrasomes were substantially disrupted by the 55-65uC treatment (Fig. 4d, lanes 11-12). These results confirmed that the association of the CENP-A-H4 tetramer with DNA was less stable than that of www.nature.com/scientificreports the H3.3-H4 tetramer. Therefore, the homotypic CENP-A nucleosome ectopically mislocalized in the chromosome arms could easily be removed in cells, although it should be stably maintained with additional proteins in functional centromeres.
Surprisingly, although the DNA end is asymmetrically detached in the CENP-A/H3.3 nucleosome, we found that the CENP-A/H3.3 nucleosome was very stable, in contrast to the CENP-A nucleosome (Fig. 4e). The CENP-A/H3.3 nucleosome generated a biphasic histone dissociation curve (Fig. 4e, upper panel), and the first and second Tm values (70-72uC and 82-84uC) were very similar to those of the H3.3 nucleosome (Fig. 4e, lower panel). Consistently, a gel retardation assay revealed that the CENP-A nucleosome was disrupted at lower temperatures (72 and 79uC), as compared to the H3.3 and CENP-A/H3.3 nucleosomes (Fig. 4f).

Conclusion
We conclude that the structural characteristics of CENP-A and H3.3 are conserved in the CENP-A/H3.3 nucleosome, which forms a surprisingly stable structure. This stable existence of the CENP-A/H3.3 nucleosome may cause ectopic kinetochore assembly, which could lead to neocentromere formation and chromosome instability in cancer cells [19][20][21][22][23][24] . The unique structural and physical properties of the CENP-A/H3.3 nucleosome provide important insights toward understanding the chromosome instability observed in cancer progression, thus offering a basis for potential drug development.

Methods
Purification of recombinant human histones. The DNA fragment encoding human histone H3.3 was inserted between the NdeI and BamHI sites of the pET21a-NHSP2 vector 35 , in which the His 6 -tag sequence, the Saccharomyces cerevisiae SUMO homolog, Smt3, and a PreScission protease cleavage sequence are located just upstream of the H3.3 coding sequence. Then, the N-terminally His 6 -SUMO tagged H3.3 was expressed in Escherichia coli BL21(DE3) cells in the presence of isopropylb-D-thiogalactopyranoside (final concentration 1 mM). His 6 -SUMO tagged H3.3 was recovered from inclusion bodies with 50 mM Tris-HCl (pH 8.0) buffer, containing 7 M guanidine hydrochloride, 500 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, and 5% glycerol, and was purified by nickelnitrilotriacetic acid (Ni-NTA) agarose chromatography (Qiagen) under denaturing conditions with 6 M urea. His 6 -SUMO tagged H3.3 was then purified by Mono S column chromatography (GE Healthcare) under denaturing conditions with 6 M urea. The purified His 6 -SUMO tagged H3.3 was dialyzed against water containing 2 mM 2-mercaptoethanol, freeze-dried, and stored at 4uC.
Crystallization and structure determination. The purified CENP-A/H3.3 nucleosome was dialyzed against 20 mM potassium cacodylate (pH 6.0) buffer, containing 1 mM EDTA, and 1 ml of the 3 mg/ml CENP-A/H3.3 nucleosome solution (concentration of DNA) was mixed with 1 ml of 20 mM potassium cacodylate (pH 6.0) buffer, containing 50 mM KCl and 90 mM MnCl 2 . The sample was equilibrated against a reservoir solution of 20 mM potassium cacodylate (pH 6.0), 40 mM KCl, and 65 mM MnCl 2 for a month. The resulting CENP-A-H3.3 nucleosome crystals were cryoprotected with a 30% polyethylene glycol 400 solution, containing 20 mM potassium cacodylate (pH 6.0), 40 mM KCl, 50 mM MnCl 2 , and 5% trehalose, and were flash-cooled in a stream of N 2 gas (100 K). The diffraction data were collected at the beamline BL17A (wavelength: 0.97319 Å ) at the Photon Factory (Tsukuba, Japan), and were processed using the HKL2000 and CCP4 programs 37,38 . The CENP-A/H3.3 nucleosome structure was determined by the molecular replacement method, with the human H3.1 nucleosome 39 (PDB ID: 2CV5) as the search model, using the PHASER program 40 . Crystallographic refinement was performed using the PHENIX program 41 . The model rebuilding was performed using the Coot program 42 . All structural graphics were displayed using the PyMOL program (http://pymol.org). The atomic coordinates of the CENP-A/H3.3 nucleosome have been deposited in the Protein Data Bank, with the ID code 3WTP. In the refined model, 97.5% of the residues are in the favored regions of the Ramachandran plot, with 0.1% in the disallowed regions.
MNase and exonuclease III treatment assays. The H3.3, CENP-A, or CENP-A/H3.3 nucleosomes (0.4 mM) were treated with MNase (Takara) or ExoIII (Takara). For the MNase assay, the nucleosome samples were incubated with MNase (0, 0.3, 0.5 and 0.7 units) for 5 minutes at 25uC in 10 ml of 44 mM Tris-HCl (pH 8.0) buffer, containing 15 mM NaCl, 2.5 mM CaCl 2 , and 1.9 mM dithiothreitol. After the MNase treatment, the reactions were stopped by the addition of 55 ml of 0.5 mg/ml proteinase K (Roche) solution, containing 20 mM Tris-HCl (pH 8.0), 20 mM EDTA, and 0.25% SDS. The resultant DNA fragments were extracted by phenol/chloroform/isoamyl alcohol, and were precipitated by ethanol. The purified DNA fragments were subjected to 10% native PAGE in 0.5 3 TBE buffer (11.1 V/cm for 1 hr). For the ExoIII assay, the nucleosome samples were incubated with or without 2.0 units of ExoIII for 2.5, 5, and 7.5 minutes at 37uC in 10 ml of 63 mM Tris-HCl (pH 8.0) buffer, containing 5 mM MgCl 2 , 5 mM KCl, and 2.45 mM dithiothreitol. After the ExoIII treatment, the reactions were stopped by the addition of 55 ml of 0.5 mg/ml proteinase K (Roche) solution, containing 20 mM Tris-HCl (pH 8.0), 20 mM EDTA, and 0.25% SDS. The resultant DNA fragments were extracted by phenol/chloroform/ isoamyl alcohol, and were precipitated by ethanol. The purified DNA fragments were subjected to 14% native PAGE in 0.5 3 TBE buffer containing 7 M urea (11.1 V/cm for 3.5 hr).
Thermal stability assay of nucleosomes. The stabilities of the H3.3, CENP-A, and CENP-A/H3.3 nucleosomes were evaluated by a thermal stability assay in the presence of SYPRO Orange, by the previously described method 29,30 . The thermal stability assay was performed in 10 ml of 20 mM Tris-HCl (pH 7.5) buffer, containing 1 mM dithiothreitol and 100 mM NaCl. The StepOnePlus TM Real-Time PCR unit (Applied Biosystems) was used to detect the fluorescence signals with a temperature gradient from 26uC to 95uC, in steps of 1uC/min. Raw fluorescence data were adjusted to normalized % values as (F(T) 2 F 26uC )/(F 95uC 2 F 26uC ), where F(T), F 26uC , and F 95uC indicate each fluorescence at a particular temperature, the fluorescence at 26uC, and the fluorescence at 95uC, respectively.