Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 162-8480, Japan.
J Biol Chem. 2010 Apr 16;285(16):11913-21. doi: 10.1074/jbc.M109.083238. Epub 2010 Feb 18.
NASP (nuclear autoantigenic sperm protein) is a member of the N1/N2 family, which is widely conserved among eukaryotes. Human NASP reportedly prefers to bind to histones H3.H4 and the linker histone H1, as compared with H2A.H2B, and is anticipated to function as an H3.H4 chaperone for nucleosome assembly. However, the direct nucleosome assembly activity of human NASP has not been reported so far. In humans, two spliced isoforms, somatic and testicular NASPs (sNASP and tNASP, respectively) were identified. In the present study we purified human sNASP and found that sNASP efficiently promoted the assembly of nucleosomes containing the conventional H3.1, H3.2, H3.3, or centromere-specific CENP-A. On the other hand, sNASP inefficiently promoted nucleosome assembly with H3T, a testis-specific H3 variant. Mutational analyses revealed that the Met-71 residue of H3T is responsible for this inefficient nucleosome formation by sNASP. Tetrasomes, composed of the H3.H4 tetramer and DNA without H2A.H2B, were efficiently formed by the sNASP-mediated nucleosome-assembly reaction. A deletion analysis of sNASP revealed that the central region, amino acid residues 26-325, of sNASP is responsible for nucleosome assembly in vitro. These experiments are the first demonstration that human NASP directly promotes nucleosome assembly and provide compelling evidence that sNASP is a bona fide histone chaperone for H3.H4.
核自动抗原精子蛋白 (NASP) 是 N1/N2 家族的成员，在真核生物中广泛保守。据报道，人 NASP 更喜欢与组蛋白 H3.H4 和连接组蛋白 H1 结合，而不是 H2A.H2B，预计其作为核小体组装的 H3.H4 伴侣发挥作用。然而，目前尚未报道人 NASP 的直接核小体组装活性。在人类中，鉴定出两种剪接同种型，即体细胞和睾丸 NASP (sNASP 和 tNASP)。在本研究中，我们纯化了人 sNASP，发现 sNASP 能有效地促进含有常规 H3.1、H3.2、H3.3 或着丝粒特异性 CENP-A 的核小体组装。另一方面，sNASP 不能有效地促进含有 H3T 的核小体组装，H3T 是一种睾丸特异性 H3 变体。突变分析表明，H3T 的 Met-71 残基是 sNASP 形成这种低效核小体的原因。由 sNASP 介导的核小体组装反应能有效地形成 tetrasomes，由 H3.H4 四聚体和不含 H2A.H2B 的 DNA 组成。sNASP 的缺失分析表明，sNASP 的中心区域，氨基酸残基 26-325，负责体外核小体组装。这些实验首次证明了人 NASP 直接促进核小体组装，并提供了令人信服的证据表明 sNASP 是 H3.H4 的真正组蛋白伴侣。
