Structural Basis of Instability of the Nucleosome Containing a Testis-Specific Histone Variant, Human H3T

Structural basis of instability of the nucleosome containing a testis-specific histone variant, human H3T

Hiroaki Tachiwanaa, Wataru Kagawaa, Akihisa Osakabea, Koichiro Kawaguchia, Tatsuya Shigaa, Yoko Hayashi-Takanakab, Hiroshi Kimurab, and Hitoshi Kurumizakaa,1

aLaboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan; and bGraduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan

Edited by Timothy J. Richmond, Swiss Federal Institute of Technology, Zurich, Switzerland, and approved May 3, 2010 (received for review March 9, 2010)

A histone H3 variant, H3T, is highly expressed in the testis, suggest- physical and structural characteristics of the H3T nucleosome ing that it may play an important role in the chromatin reorganiza- were attributed to the Val111 and Met71 residues that are specific tion required for meiosis and/or spermatogenesis. In the present to H3T. study, we found that the nucleosome containing human H3T is significantly unstable both in vitro and in vivo, as compared to Results the conventional nucleosome containing H3.1. The crystal structure H3T Nucleosome Is Less Stable than the Conventional Nucleosome. of the H3T nucleosome revealed structural differences in the H3T The nucleosome containing human H3T was reconstituted by a regions on both ends of the central α2 helix, as compared to those salt-dialysis method, using human histones H3T, H2A, H2B, of H3.1. The H3T-specific residues (Met71 and Val111) are the and H4, and a 146 base-pair DNA. To prepare a structurally source of the structural differences observed between H3T and homogeneous nucleosome, the reconstituted H3T nucleosome H3.1. A mutational analysis revealed that these residues are was incubated for 2 h at 55 ˚C to disrupt the inappropriate responsible for the reduced stability of the H3T-containing nucleo- histone–DNA interactions (Fig. 1A) and was purified from the some. These physical and structural properties of the H3T-contain- free DNA by gel electrophoresis (Fig. 1B). Conventional H3.1 ing nucleosome may provide the basis of chromatin reorganization nucleosomes were also prepared with the same procedure during spermatogenesis. (Fig. 1B). Histone compositions of the H3T and H3.1 nucleosomes prepared in this procedure were confirmed by SDS-PAGE uring spermatogenesis, dramatic chromatin reorganization (Fig. 1C). We next compared the stabilities of the H3T and H3.1 Doccurs, and most histones are eventually replaced by prota- nucleosomes by examining the gel migration distances of the mines (1). Several histone variants are highly expressed in the nucleosomes exposed to different NaCl concentrations. Exposure testis and are considered to be incorporated into the chromatin to 0.4 M NaCl had no apparent effect on the migration distances in the early stage of spermatogenesis. of both the H3.1 and H3T nucleosomes (Fig. 1D, lanes 1 and 5, In humans, about 4% of the haploid genome in the sperm is respectively). This observation indicates that the nucleosomes reportedly retained in nucleosomes, some containing the testis- were intact at this salt concentration. The H3.1 nucleosome ap- specific histone H2B, hTSH2B/TH2B (2). Interestingly, the nu- peared stable even when exposed to 0.8 M NaCl (Fig. 1D, lane 4), cleosomes retained in the sperm are significantly enriched in loci and only a small fraction of the nucleosome migrated slower. that contain developmentally important genes. In addition, These nucleosomes are probably multimers formed by enforced histone modifications, such as acetylation and methylation, are hydrophobic interactions from higher salt concentrations. By con- likely to occur after the incorporation of the histone variants trast, the band corresponding to the intact nucleosome was nearly during spermatogenesis (1). These observations suggest that absent for the H3T nucleosome that was exposed to 0.6 M NaCl nucleosomes containing testis-specific histone variants, with or (Fig. 1D, lane 6). Instead, multiple, nonnucleosomal bands, con- without chemical modifications, may function as epigenetic taining only H2A/H2B (Fig. S1), were detected (Fig. 1D, lanes markers in the sperm chromatin. 6–8). These results indicate that the H3T nucleosome is less H3T is a variant of histone H3 that is robustly expressed in the stable than the H3.1 nucleosome under high salt concentrations. human testis (3–5). We previously reported that H3T, like the conventional H3.1, can be assembled into nucleosomes with Weaker Association of H3T/H4 Tetramer to H2A/H2B Dimer. To inves- H2A, H2B, and H4 (H3T nucleosome) (6). A histone chaperone, tigate the stability of the H3T nucleosome at physiological ionic Nap2, with 3-fold higher expression in the testis than in other strengths, we examined the Nap1-mediated H2A/H2B disassem- somatic tissues (7), was found to be a more efficient chaperone bly from the H3T nucleosome. It is known that excess amount of for H3T nucleosome assembly than the ubiquitously expressed the histone chaperone, human Nap1 (hNap1), promotes H2A/ histone chaperone, Nap1 (6). Therefore, H3T may be assembled H2B disassembly from the nucleosome (10). The nucleosomal into the chromatin by a specific chaperone-mediated pathway in DNA used in this assay contains two PstI sites that are palindro- the testis. Comprehensive proteome analyses of nuclear extracts mically located in regions 13–18 bases away from both ends of the from HeLa cells suggested that H3T also exists in somatic cells 146 base-pair DNA (Fig. 2A). These PstI sites are close to the (8, 9). However, the nucleosomes containing H3T probably com- prise only a small proportion of the bulk chromatin in somatic Author contributions: H.T. and H. Kurumizaka designed research; H.T., W.K., A.O., K.K., cells, because the amount of H3T in HeLa cells is extremely T.S., Y.H.-T., and H. Kimura performed research; H.T., W.K., H. Kimura, and H. Kurumizaka low. Therefore, H3T may have a limited function in somatic cells analyzed data; W.K., H. Kimura, and H. Kurumizaka wrote the paper. that is currently unknown. The authors declare no conflict of interest. In the present study, we found that the H3T nucleosome is This article is a PNAS Direct Submission. significantly unstable, as compared to the conventional H3.1 nu- Data deposition: The crystallography, atomic coordinates, and structure factors have cleosome, both in vitro and in vivo. The crystal structure of the been deposited in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein H3T nucleosome was determined at 2.7 Å resolution, revealing Data Bank, www.pdb.org (RCSB ID codes 3A6N and 3AFA). that, although the overall structure was similar to that of the con- 1To whom correspondence should be addressed. E-mail: [email protected]. ventional H3.1 nucleosome, structural differences were observed This article contains supporting information online at www.pnas.org/lookup/suppl/ at both ends of the central α2 helix of H3Tand H3.1. The unique doi:10.1073/pnas.1003064107/-/DCSupplemental.

10454–10459 ∣ PNAS ∣ June 8, 2010 ∣ vol. 107 ∣ no. 23 www.pnas.org/cgi/doi/10.1073/pnas.1003064107 Downloaded by guest on October 1, 2021 Fig. 2. H2A/H2B associates weakly with H3T/H4. (A) The crystal structure of the H3.1 nucleosome determined in this study. Locations the PstI and EcoRI sites are indicated. The H2A/H2B and H3.1/H4 molecules are colored in purple and in dark blue, respectively. (B and C) H2A/H2B disassembly assay with hNap1. The nucleosomes were treated with PstIorEcoRI in the presence or absence of excess amount of hNap1 (6.5 μM). The resulting DNA fragments were extracted by Phenol/chloroform, and were analyzed by 10% PAGE with Fig. 1. Instability of the H3T nucleosome. (A) H3T nucleosomes, reconsti- ethidium bromide staining. Arrows indicate the DNA fragment produced by tuted using 1.2 mg∕mL total histones and 0.7 mg∕mL DNA, were analyzed complete PstI digestion. These results were confirmed to be reproduced in by nondenaturing 6% PAGE. Lane 1 indicates naked DNA. Lanes 2 and 3 in- three independent experiments. (B) The H3T nucleosome. (C) The H3.1 nu-

dicate the H3T nucleosomes before and after a 55 °C incubation, respectively. cleosome. (D and E) Interaction between H2A/H2B and H3T/H4 or H3.1/H4. BIOCHEMISTRY DNA was visualized by ethidium bromide staining. Asterisks indicate bands H2A, H2B, H4, and H3T (D) or H3.1 (E) were incubated without DNA in corresponding to nonnucleosomal DNA–histone complexes. (B) The H3T and the presence of 2 M NaCl. The samples were then subjected to HiLoad H3.1 nucleosomes were purified using a Prepcell apparatus, and were 26∕60 Superdex 200 prep grade gel filtration column chromatography. His- analyzed by nondenaturing 6% PAGE with ethidium bromide staining. tone compositions of the peak fractions were analyzed by 18% SDS-PAGE (C) Histone compositions of the purified H3T and H3.1 nucleosomes were with Coomassie brilliant blue staining. The peak fractions denoted as a, b, analyzed by 18% SDS-PAGE with Coomassie brilliant blue staining. (D)Salt and c correspond to H2A/H2B/H3/H4 octamer, H3/H4 tetramer, and H2A/H2B titration. The nucleosomes were incubated in the presence of 0.4 M (lanes dimer, respectively. 1 and 5), 0.6 M (lanes 2 and 6), 0.7 M (lanes 3 and 7), and 0.8 M NaCl (lanes 4 and 8) at 42 °C for 2 h. The samples were analyzed by nondenaturing 6% stably incorporated, as compared with those in the conventional PAGE with ethidium bromide staining. Lanes 1–4 and 5–8 indicate experiments with H3.1 and H3T nucleosomes, respectively. Bands corresponding H3.1 nucleosome. The instability of the H3T nucleosome may be to nucleosome monomers and nucleosome-nucleosome aggregates are primarily caused by a weaker association of the H2A/H2B dimer indicated. Asterisks represent bands corresponding to nonnucleosomal to the H3T/H4 tetramer. It should be noted that the DNA in the DNA-histone complexes. H3.1 nucleosome was more susceptible than that in the H3T nucleosome in the absence of hNap1, and that the PstI digestion binding sites of H2A/H2B within the nucleosome (Fig. 2A). The patterns of the H3.1 and H3T nucleosomes were slightly different DNA also contains a single EcoRI site at the nucleosomal dyad, (Fig. 2 B and C). Differences in the DNA flexibility of the H3.1 which is a binding site for H3/H4 (Fig. 2A). Therefore, if hNap1 and H3T nucleosomes were observed (Fig. S2B), which may be the disassembles H2A/H2B, the DNA is predicted to become more reason for the distinct digestion patterns. susceptible to digestion by PstI. We then tested the interaction between H2A/H2B and H3T/ In the absence of hNap1, PstI partially digested the nucleoso- H4 or H3.1/H4 in the absence of DNA. Gel filtration analysis in mal DNA of both H3T and H3.1 nucleosomes (Fig. 2 B and C, the presence of 2 M NaCl revealed that the H2A/H2B dimers did lane 6), whereas EcoRI only slightly digested the nucleosomal not tightly associate with the H3T/H4 tetramer (Fig. 2D). This DNA (Fig. 2 B and C, lane 4). Therefore, the DNA segment result sharply contrasts with the fact that two H2A/H2B dimers located near the exit of the nucleosome is more susceptible to re- stably associates with the conventional H3.1/H4 tetramer and striction nucleases than that located near the dyad. Interestingly, forms a stable H2A/H2B/H3.1/H4 octamer (Fig. 2E) (11). There- hNap1 substantially increased the PstI susceptibility of the DNA fore, the weaker association between H2A/H2B and H3T/H4 may in the H3T nucleosome (Fig. 2B, compare lanes 6 and 7), probably be responsible for the instability of the H3T nucleosome. A by its H2A/H2B disassembly function. In contrast, this hNap1- possible explanation for this difference could be the fact that the dependent enhancement of the PstI susceptibility was not self-association of the H3T/H4 tetramer prevents the interaction observed in the H3.1 nucleosome under the experimental condi- between the H3T/H4 tetramer and the H2A/H2B dimer. A dy- tions used in this study (Fig. 2C, compare lanes 6 and 7). These namic light scattering analysis revealed that the Stokes radius results suggested that H2A/H2B in the H3T nucleosome is less of the H3T/H4 tetramer in the presence of 2 M NaCl was approxi-

Tachiwana et al. PNAS ∣ June 8, 2010 ∣ vol. 107 ∣ no. 23 ∣ 10455 Downloaded by guest on October 1, 2021 mately twice the size of that of the H3.1/H4 tetramer, which was judged to be monodisperse (Table S1), suggesting that the H3T/ H4 tetramer aggregation competes with the H3T/H4–H2A/H2B interaction.

Rapid Exchange of H3T in Nucleosomes of Living Cells. We next compared the mobility of H3T with H3.1, as GFP-fusion proteins in living cells, by fluorescence recovery after photobleaching (FRAP) (12). Because GFP-H3.1 was stably incorporated into nucleosomes in living cells, only a subtle recovery was observed, even at 20 min after photobleaching (Fig. 3 A and B). By contrast, the fluorescence of GFP-H3T recovered within several minutes (Fig. 3 A and B), suggesting that H3T in the nucleosome is more rapidly exchanged compared to the conventional H3.1 in living cells. GFP-H3Twas detected in the mono- and oligo-nucleosomal fractions prepared by micrococcal nuclease digestion followed by sucrose gradient centrifugation (Fig. 3C), indicating that GFP-H3Twas actually incorporated into chromatin. These in vivo results are consistent with the instability of the H3T nucleosome.

Crystal Structure of the H3T Nucleosome. To understand the structural basis of the instability observed in the H3T nucleosome, the crystal structure of the nucleosome core particle containing H3T was solved at 2.7 Å resolution (Table S2). The overall structure was essentially similar to that of the H3.1 nucleosome (13) (Fig. 4A). There are four amino acid differences between H3T (Val24, Met71, Ser98, and Val111) and the conventional H3.1 (Ala24, Val71, Ala98, and Ala111), and three of them (Met71, Ser98, and Val111) are located in the visible, histone-fold domain (Fig. 4B). The Val24 residue, which is located in the N-terminal tail outside of the histone-fold domain, was not visible in the present H3T nucleosome structure (Fig. 4 A and B). H3.1 and its Fig. 4. Crystal structure of the H3T nucleosome. (A) Two views of the H3T-nucleosome structure are represented. The H3T molecules are shown in red. Locations of the Met71 and Val111 residues are indicated. (B) Struc- tural differences between H3T and H3.1 in the nucleosomes. The H3T and H3.1 structures are superimposed, and the rmsd values for each residue pair is calculated and plotted. The secondary structure of H3T in the nucleosome is shown in the top of the panel. Arrows indicate the locations of the H3T- specific amino acid residues, Met71, Ser98, and Val111. (C and D) Comparison of the H3T structure (red) with the H3.1 structure (green). The side chains of the H3T-M71, H3.1-V71, H3T-V89, H3.1-V89, H3T-V111, H3.1-A111, H3T-R116, H3.1-R116, H3T-D123, and H3.1-D123 residues are represented by space- filling models. The H3T and H3.1 regions containing the amino acid residues 71 (C) and 111 (D) are shown. Arrows in C and D indicate the locations of H3T and H3.1 that are structurally different from each other.

variants contain a central helix (α2) with two shorter α-helices (α1 and α3) flanking both ends of α2. The Met71 (α1) and Val111 (α2) residues of H3Tare located near the L1 and L2 loops, respectively, which connect the flanking helices (α1 and α3) to α2 (Fig. 4B). The Ser98 residue is located near the middle of the α2 helix (Fig. 4B). To identify the finer structural differences between the H3T and H3.1 nucleosomes, we next compared the structures of the H3T and H3.1 histones. Although several H3 structures are available for comparison, differences in crystallization condi- tions and crystal packing may obscure the fine structural differ- Fig. 3. FRAP. HeLa cells expressing GFP-H3.1 or GFP-H3T were subjected to the FRAP analysis. (A) The mobility of GFP-H3.1 or GFP-H3T in living cells was ences. To minimize these possibilities, the H3.1 nucleosome was analyzed by bleaching one-half of the nucleus. (B) The averages of the rela- prepared and crystallized under identical procedures as those of tive fluorescence intensity of bleached area were plotted with the standard the H3T nucleosome, and its structure was determined at a simi- deviations (n ¼ 5). (C) GFP-H3T was incorporated into the HeLa cell chroma- lar resolution (2.5 Å) (Fig. S2 and Table S2). All residues in the tin. (Upper ) DNA fragments of mono-, di-, and trinucleosomes fractionated crystal structures, including the H3T-specific residues, were well by sucrose gradient centrifugation were analyzed by agarose gel electro- structured, as judged from their B factors (Table S3). The H3T phoresis with ethidium bromide staining. (Right and Left) The nucleosome and H3.1 histones were then superimposed, and the rmsd for samples from the HeLa cells with and without GFP-H3T expression, respectively. The sucrose gradient fraction numbers are indicated at the top of each each residue pair was calculated and plotted against each other. panel. Middle panel. Histone compositions of the purified nucleosomes were The largest deviations were found near both ends of the central analyzed by 16% SDS-PAGE with Coomassie brilliant blue staining staining. α2 helix (Fig. 4B). At the N terminus of the H3T α2 helix, the van (Lower) GFP-H3T was detected with anti-GFP monoclonal antibody. der Waals radii of the Met71 residue side chain come in close

10456 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1003064107 Tachiwana et al. Downloaded by guest on October 1, 2021 contact with that of the Val89 residue (Fig. 4C, Left). This close contact is absent in the corresponding location of the H3.1 structure (Fig. 4C, Right). At the other end of the α2 helix, the side chain of the Val111 residue contacts the side chain of the Asp123 residue, which forms a salt bridge with Arg116 (Fig. 4D, Left). This van der Waals interaction between 111 and 123 residues is not observed in the H3.1 structure (Fig. 4D, Right). This difference between H3T and H3.1 appears important for the structural differences observed near the C terminus of the α2 helix (Fig. 4 B and D). All other locations of H3T, including the region around the H3T-specific Ser98 residue, were essentially identical in the main chain structure to those of H3.1 (Fig. 4B). Therefore, the Met71 and Val111 residues in H3T appear to be important for the formation of the specific structure of the H3T nucleosome and may contribute to the reduced stability of the H3T nucleosome.

Contribution of the V71M and A111V Substitutions in the H3T Nucleo- some Stability. In the crystal structure of the H3T nucleosome, we found that Met71 and Val111 are the key residues affecting the H3T-specific conformation (Fig. 4). This structural property of the H3T nucleosome may be responsible for its reduced stability. Therefore, we then sought to determine whether the nucleosome stability is affected by substituting H3T-specific amino acids for the H3.1 types, by creating the H3T-M71V, -S98A, and -V111A mutants. All of the mutants were competent in nucleosome formation (Fig. 5A), and the histone compositions of these nucleosomes were confirmed (Fig. 5B). The nucleosome stability, as judged by the resistance to high NaCl concentrations, was enhanced drastically by the H3T-V111A mutation (Fig. 5C, lanes 13–16) and moderately by the H3T-M71V mutation (Fig. 5C, lanes 5–8). By contrast, the nucleosome stability was not affected by the S98A mutation (Fig. 5C, lanes 9–12). An H3T-M71V/ V111A double mutant exhibited essentially similar stability to the H3.1 nucleosome (Fig. 5C, lanes 17–20). Consistent results were obtained by reciprocal experiments using H3.1 mutants, in BIOCHEMISTRY which Val71, Ala98, and Ala111 were replaced by the corresponding H3T amino acids. The nucleosome stability was decreased drastically and moderately by H3.1-A111Vand H3.1-V71M mutations, respectively (Fig. 5D, lanes 13–16 and lanes 5–8). The H3.1- V71M/A111V double mutant, like the H3T nucleosome, was very – unstable (Fig. 5D, lanes 17 20). As expected, the H3.1-A98S Fig. 5. Mutational analysis of H3T and H3.1 nucleosomes. (A) Nucleosomes mutation did not affect the stability of the H3.1 nucleosome containing H3T and H3.1 mutants were purified using a Prepcell apparatus (Fig. 5D, lanes 9–12). Consistently, the H2A/H2B disassembly and were analyzed by nondenaturing 6% PAGE with ethidium bromide assay revealed that the H3T-V111A and H3.1-A111V mutations staining. (B) Histone compositions of the purified nucleosomes containing suppressed and enhanced, respectively, the H2A/H2B disassembly H3T and H3.1 mutants were analyzed by 18% SDS-PAGE with Coomassie by hNap1 (Fig. S3). These results indicate that the reduced brilliant blue staining. (C and D) Salt titration. Nucleosomes were incubated stability of the H3T nucleosome is attributable mainly to the in the presence of 0.4 M (lanes 1, 5, 9, 13, and 17), 0.6 M (lanes 2, 6, 10, 14, and V111A substitution and partly to the M71V substitution. 18), 0.7 M (lanes 3, 7, 11, 15, and 19), and 0.8 M NaCl (lanes 4, 8, 12, 16, and 20) at 42 °C for 2 h. The samples were analyzed by nondenaturing 6% PAGE with To confirm whether the Val111 residue is responsible for the ethidium bromide staining. Bands corresponding to nucleosome monomers weaker H2A/H2B association to H3T/H4, we performed gel and nucleosome–nucleosome aggregates are indicated. Asterisks represent filtration analyses with these H3 mutants. As shown in Fig. 6C, bands corresponding to nonnucleosomal DNA-histone complexes. (C) The H3.1/H4 containing the H3.1-A111V mutant was significantly stability of H3T mutants. Lanes: 1–4, H3T; 5–8, H3T-M71V; 9–12, H3T-S98A; defective in the H2A/H2B binding in the absence of DNA, like 13–16, H3T-V111A; and 17–20, H3T-M71V/V111A nucleosomes. (D) The H3T/H4 (Fig. 2D). The H3.1-V71M and H3.1-A98S mutations stability of H3.1 mutants. Lanes: 1–4, H3.1; 5–8, H3.1-V71M; 9–12, H3.1-A98S; – – did not significantly affect the H2A/H2B binding of H3.1/H4 13 16, H3.1-A111V; and 17 20, H3.1-V71M/A111V. (Fig. 6 A and B). Reciprocal results were obtained with H3/H4 containing H3T-M71V, H3T-S98A, and H3T-V111A (Fig. 6 D–F). are responsible for the structural differences observed between These biochemical results are consistent with the differences H3T and H3.1. Consistently, mutational analyses revealed that observed in the crystal structure, and therefore, the V111A substi- the reduced stability of the H3T nucleosome is caused mainly tution in H3T may be mainly responsible for the specific structure by Val111 and partly by Met71. Therefore, we conclude that and function of the H3T nucleosome. the Met71 and Val111 residues of H3T are essential for the H3T-specific structure and function. Discussion Our structural and biochemical analyses revealed that the sin- In the present study, we found that a prominent property of the gle amino acid substitution of H3, at position 111, significantly H3T nucleosome is its instability. The crystal structure of the H3T affected the nucleosome stability, probably by weakening the nucleosome revealed that the H3T-specific amino acid residues, association of the H3T/H4 tetramer with H2A/H2B dimers. Met71 and Val111, corresponding to Val71 and Ala111 in H3.1, Whereas the Val111 of H3T does not directly interact with

Tachiwana et al. PNAS ∣ June 8, 2010 ∣ vol. 107 ∣ no. 23 ∣ 10457 Downloaded by guest on October 1, 2021 was distinct from the conventional nucleosome. The H2AL2 nucleosome was quite susceptible to nucleases, suggesting that these nucleosomes have different structural properties, as compared to those of the conventional nucleosome (21). A possible human counterpart of H2AL2, H2A.Bbd, which is highly expressed in the testis, was also suggested to form a specific nucleosome structure with reduced stability (22–24). Furthermore, nucleosomes containing a testis-specific H2B variant, hTSH2B/TH2B, were reportedly unstable, as compared to the conventional nucleosome (25). Therefore, the instability of these nucleosomes formed by the testis-specific histone variants may be a common property required for chromatin reorganization during spermatogenesis. H3T may also be a constituent of sperm chromatin, in which about 4% of the human sperm genome retains nucleosomes (2). These sperm nucleosomes are suggested to have a specific epigenetic function, because they are significantly enriched around developmentally important genes, such as the imprinted gene clusters, microRNA clusters, and HOX gene clusters. Testis-specific H2B variants, hTSH2B/TH2B and H2BFWT, were found in the sperm nuclei (2, 26). H2BFWT was stably incorporated into nucleosomes in vitro and in vivo (27) and was suggested to have telomere-associated function (26). Whereas it remains to be revealed whether sperm chromatin indeed retains H3T nucleosomes, the robust expression of H3T in the testis suggests its presence in the sperm. The specific biochemical and structural properties of the H3T nucleosome, like those containing the Fig. 6. Mutational analyses of interactions of H2A/H2B with H3T/H4 or H3.1/ H2B variants, could also play an important epigenetic role in H4. Gel filtration analyses were performed as described in Fig. 2 D and E, the regulation of genes containing nucleosomes in the sperm. except that a HiLoad 16∕60 Superdex 200 prep grade column was used. Fractions indicated with dots are analyzed by 18% SDS-PAGE with Coomassie Materials and Methods brilliant blue staining. The peak fractions denoted as a, b, and c correspond to Purification of Human Histones. Human H2A, H2B, H3.1, H3T, and H4 were H2A/H2B/H3/H4 octamer, H3/H4 tetramer, and H2A/H2B dimer, respectively. overexpressed in Escherichia coli cells, and were purified by a method (A) H3.1-V71M. (B) H3.1-A98S. (C) H3.1-A111V. (D) H3T-M71V. (E) H3T-S98A. according to previous papers (6, 28) with modifications. Details are described (F) H3T-V111A. in SI Materials and Methods.

H2A/H2B and DNA, a mutation at Arg116 (to His) close to Preparation of the H3T and H3.1 Nucleosomes. Details are described in Val111 reportedly destabilized the nucleosome (14, 15). This SI Materials and Methods. The 146 base-pair DNA (13, 17) was prepared H3-R116H mutation in Saccharomyces cerevisiae has been as described previously (11). Purified H2A/H2B (1.7 mg), H3T/H4 (1.7 mg) identified as a Sin mutation that alleviates the requirement for or H3.1/H4 (1.7 mg), and the DNA (2 mg) were mixed in a solution containing 2 M KCl. The H3T and H3.1 nucleosomes were reconstituted by the salt- the nucleosome-remodeling factor, Swi/Snf, which activates tran- dialysis method and were purified from the free DNA and histones by non- scription (14, 16). A structural study revealed that the H3-Arg116 denaturing polyacrylamide gel electrophoresis, using a Prepcell apparatus residue may not directly interact with the DNA backbone and (Bio-Rad). H2A/H2B, but instead forms a salt bridge with H3-Asp123 (17). This salt bridge may be very important to arrange the residues Salt Resistance Assay. The nucleosomes (240 ng∕μL) were incubated in the appropriately around position 116 for hydrogen bond formation presence of 0.4, 0.6, 0.7, and 0.8 M NaCl at 42 °C for 2 h. After incubation, with the DNA backbone and H2A/H2B (18). Therefore, this NaCl concentration of the samples were adjusted to 0.4 M, and the samples Sin mutation at Arg116 of H3 may destabilize the nucleosome were analyzed by nondenaturing 6% PAGE. by allosterically reducing the histone-DNA and/or histone-histone Crystallization and Structure Determination. Details are described in interactions. In the present study, we found that the Val111residue SI Materials and Methods. Briefly, crystals of the purified H3T and H3.1 nu- of H3Tsterically affected the side chain orientation of the Arg116 cleosomes were obtained by the hanging drop method. The H3T-nucleosome and Asp123 residues within the H3T monomer. The H3T-Val111 crystals belonged to the orthorhombic space group P212121, with unit cell residue may destabilize nucleosomes by a similar mechanism to constants of a ¼ 105.5 Å, b ¼ 109.5 Å, and c ¼ 181.1 Å, and contained one that proposed for the H3-R116H mutation. Consistent with this nucleosome per asymmetric unit. The H3.1-nucleosome crystals also belonged idea, a histone H3-A111G mutant exhibited the Swi/Snf-indepen- to the same space group, with unit cell constants of a ¼ 105.8 Å, b ¼ 109.5 Å, dent phenotype in S. cerevisiae (19). and c ¼ 180.9 Å, and contained one nucleosome per asymmetric unit. High- In the present study, we showed that the H3T nucleosome resolution diffraction data were obtained using the synchrotron radiation source at the beamline BL41XU station of SPring-8, Harima, Japan. structurally and biochemically differs from the conventional The structures of the H3T and H3.1 nucleosomes were initially solved to 2.7 H3.1 nucleosome. The histone H3T variant is highly expressed and 2.5 Å resolutions, respectively, by the molecular replacement method, in the testis (5). Therefore, H3T is anticipated to have a specific using the MOLREP program (29) and the human nucleosome structure function in chromatin reorganization during meiosis and/or the (Protein Data Bank ID code 2CV5) as a guide (13). For H3T nucleosome, postmeiotic maturation of male germ cells. In germ cells, drastic the Ramachandran plot of the final structure showed 93.3% of the residues chromatin reorganization occurs by histone replacement with in the most favorable regions and no residues in the disallowed region. For histone variants, and the histones are eventually replaced by pro- H3.1 nucleosome, the Ramachandran plot of the final structure showed tamines during sperminogenesis (1, 20). The reduced stability of 94.9% of the residues in the most favorable regions and no residues in the disallowed region. Summary of the data collection and refinement the H3T nucleosome may be favorable to promote this global statistics is provided in Tables S2 and S3. All structure figures were created transition of the chromosome architecture during meiotic and/ using the PyMOL program (30). The atomic coordinates of the H3T nucleo- or postmeiotic events. Intriguingly, a mouse testis-specific H2A some and the H3.1 nucleosome have been deposited, with the RCSB ID codes, variant, H2AL2, reportedly formed a specific nucleosome that 3A6N and 3AFA, respectively.

10458 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1003064107 Tachiwana et al. Downloaded by guest on October 1, 2021 hNap1-Mediated H2A/H2B Disassembly Assay. The nucleosomes (0.8 μM) were and 1.5 mM MgCl2 containing 1% Triton X100 and protease inhibitor cocktail incubated with hNap1 (6.5 μM), and were treated with PstI (30 unit) or EcoRI (Nacalai Tesque). After disrupting the cells using Dounce homogenizer (tight (36 unit) in 10 μL of 50 mM Tris · HCl buffer (pH 7.5), containing 10 mM MgCl2 pestle; five times), nuclei were collected and treated with micrococcal nucle- and 100 mM NaCl. After a 120 min incubation at 37 °C, the DNA was ex- ase (2 × 106 Gel units∕mL; New England Biolabs) at 30 °C for 1 h (mixing by tracted with phenol/chloroform. The DNA fragments were then analyzed inverting every 15 min). After adding 10 mM EDTA (pH 8.0) and centrifuga- by 10% PAGE in 0.5 × TBE buffer (45 mM Tris base, 45 mM Boric acid, and tion (10;000 × g; 10 min; 4 °C), the pellet was suspended in 540 μL10mM 1 mM EDTA) (21 V∕cm for 1 h) and ethidium bromide staining. EDTA (pH 8.0). After the addition of 5 M NaCl solution (final 0.3 M), the sample was centrifuged (20;000 × g; 10 min; 4 °C), and the supernatant Superdex 200 Gel Filtration Chromatography. Freeze-dried H3T (2.1 mg), H3.1 was collected. The supernatant was then incubated at 55 °C for 2 h to dena- (2.1 mg), or H3 mutants (2.1 mg) was mixed with H2A (1.9 mg), H2B (1.9 mg), ture nonnucleosomal proteins. After the centrifugation (10;000 × g;5min;4 and H4 (1.5 mg) in 5 mL of 20 mM Tris · HCl buffer (pH 7.5), containing 7 M °C) to remove denatured proteins, the supernatant (nucleosome sample) was guanidine hydrochloride, and 20 mM 2-mercaptoethanol. The samples were collected. The nucleosome sample (0.6 mL) was fractionated in a 12 mL su- dialyzed against 10 mM Tris · HCl buffer (pH 7.5), containing 2 M NaCl, and crose gradient (10–30%) by centrifugation (209;541 × g; 21 h; 4 °C) using a 2 mM 2-mercaptoethanol, and were analyzed by Superdex 200 gel filtration Beckman SW41Ti rotor. Fractions (0.6 mL each) were collected from the top. chromatography (GE Healthcare). The peak fractions were analyzed by 18% For DNA analysis, fractions were mixed with SDS (0.2%) and analyzed by SDS-PAGE. 2% agarose gel electrophoresis in 1 × TAE (40 mM Tris base, 20 mM acetic acid, and 1 mM EDTA) (13.3 V∕cm for 1 h) followed by ethidium bromide Fluorescence Recovery After Photobleaching. HeLa cells stably expressing staining. For protein analysis, fractions containing mono-, di-, and trinucleo- GFP-H3.1 or GFP-H3T were grown on a glass-bottom dish (Mat-tek). FRAP somes were separated by 16% SDS-PAGE, and either stained with Coomassie was performed using a confocal microscope (FV-1000; Olympus) with a brilliant blue or transferred to Hybond-P PVDF membrane (GE Healthcare) 60× UPlanSApo N:A: ¼ 1.35 lens, as described previously (12). Three confocal using a semidry blotting system (BIO CRAFT). The GFP-H3T signals were images of a field containing 4–10 nuclei were collected (800 × 800 pixels, detected with anti-GFP antibody (Nacalai Tesque), peroxidase-conjugated zoom 3, scan speed 2 μs∕pixel, pinhole 800 μm, Kalman filtration for four anti-mouse Ig, and ECL Western Blotting Detection Reagents (GE Healthcare) scans, LP505 emission filter, and 0.1% transmission of 488-nm Ar laser). using a LAS-4000 (Fujifilm). One-half of each nucleus was bleached using 75% transmission of 488 nm and 100% of 514 nm (two iterations), and images were collected using ACKNOWLEDGMENTS. We thank Dr. K. Luger (Colorado State University) for the original setting every 1 min. The fluorescence intensity of the bleached providing the plasmid to prepare the 146 base-pair DNA used in this study. area was measured using Image J 1.39u. After subtracting the background, We also thank Drs. N. Fujikawa and S. Yokoyama (RIKEN) for technical the intensity was normalized to the initial intensity before bleaching. assistance on nucleosome preparation, Dr. T. Fukagawa (National Institute of Genetics) for general discussion, and Dr. S.-Y. Park (Yokohama City Univer- sity) for assistance on dynamic light scattering experiments. We are also Analysis of the GFP-H3T Incorporation into Chromatin in HeLa Cells. Details are grateful to the beamline scientists, Drs. Y. Kawano and N. Shimizu for their described in SI Materials and Methods. Nucleosomes were prepared essen- assistance in data collection at the BL41XU beamline of SPring-8. This work tially according to previous paper (31) with slight modifications. Briefly, HeLa was supported in part by Grants-in-Aid from the Ministry of Education, cells, in which GFP-H3T was exogenously expressed, were collected and Culture, Sports, Science, and Technology, Japan. H. Kurumizaka is a research resuspended in 1 mL ice-cold 10 mM Hepes-NaOH (pH 7.4), 15 mM NaCl, fellow in the Waseda Research Institute for Science and Engineering.

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Tachiwana et al. PNAS ∣ June 8, 2010 ∣ vol. 107 ∣ no. 23 ∣ 10459 Downloaded by guest on October 1, 2021