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 nucleo- somes 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 experi- ments 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.