Nucleosome Formation Activity of Human Somatic Nuclear Autoantigenic Sperm Protein (sNASP)*

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.

The nucleosome is the fundamental unit of eukaryotic chromatin and is composed of a 146-base pair DNA and a histone octamer (1). The histone octamer comprises two H2A⅐H2B dimers and one H3⅐H4 tetramer (2). During the nucleosome assembly process, an H3⅐H4 tetramer is first deposited into chromatin, and H2A⅐H2B dimers are then incorporated into the H3⅐H4 tetrasome to form a complete octameric nucleosome. These nucleosome assembly processes are mediated by the histone chaperones, which directly bind to H2A⅐H2B and/or H3⅐H4 in cells (3)(4)(5)(6)(7)(8).
The N1/N2 protein family, which was originally identified in Xenopus laevis (43)(44)(45), is widely conserved among eukaryotes and is known to bind to H3⅐H4, suggesting that it is a chaperone family for H3⅐H4. In mammals, NASP (nuclear autoantigenic sperm protein) was found to share a high degree of sequence similarity with the N1/N2 family. In mice, disruption of the NASP gene caused early embryonic lethality (46), indicating that NASP is an essential protein for mammals. Two spliced isoforms of NASP are present in humans. One is testicular NASP (tNASP), which was mainly found in the testis and embryonic tissues (47). The other one is somatic NASP (sNASP), which is produced in all dividing cells (47). NASP reportedly possesses chaperone activity for the linker histone H1 (46 -50). In addition, tNASP and sNASP were found as subunits in the large histone-chaperone complex containing CAF1, HIRA, ASF1, and H3⅐H4 (26,51), suggesting their function in nucleosome assembly. However, direct nucleosome-assembly activity, which may be a common property among the H3⅐H4 chaperones, has not been reported for the human NASPs so far, although sNASP preferentially binds to H3⅐H4 over H2A⅐H2B (52).
In the present study, we purified human sNASP as a recombinant protein and found that sNASP itself promotes nucleosome assembly in vitro. sNASP promotes the assembly of nucleosomes containing human H3 variants, H3.1, H3.2, H3.3, and CENP-A, but not H3T, with different efficiencies. The differences in the sNASP-mediated nucleosome-assembly efficiency among the H3 variants found in the present study may provide important insights into the sNASP function in chromatin organization and dynamics.

EXPERIMENTAL PROCEDURES
Purification of Recombinant Human sNASP-Human sNASP and its deletion mutants were overexpressed in Escherichia coli cells as N-terminal hexahistidine (His 6 )-tagged proteins. The DNA fragments encoding sNASP were ligated into the NdeI and BamHI sites of the pET15b vector (Novagen), which harbors the His 6 tag and the thrombin protease recognition sequence (GE Healthcare) at the N terminus. Freshly transformed E. coli strain BL21(DE3) cells, which also carried an expression vector for the minor tRNAs (Codon(ϩ)RIL; Stratagene), were grown on LB plates containing ampicillin (100 g/ml) and chloramphenicol (35 g/ml) at 37°C. After a 16-h incubation, 5-20 colonies on the LB plates were collected and inoculated into LB medium (5 liters) containing ampicillin (100 g/ml) and chloramphenicol (35 g/ml), and the cultures were incubated at 30°C. When the cell density reached an A 600 ϭ 0.4, 1 mM isopropyl-␤-D-thiogalactopyranoside was added to induce the expression of sNASP, and the cultures were further incubated at 18°C for 12 h. The cells producing sNASP were harvested, resuspended in 50 mM Tris-HCl buffer (pH 7.5) containing 2 mM 2-mercaptoethanol, 10% glycerol, and 0.5 M NaCl, and disrupted by sonication. The cell debris was removed by centrifugation (27,216 ϫ g; 20 min), and the lysate was mixed gently with 4 ml (50% slurry) of nickel-nitrilotriacetic acid (Ni-NTA)-agarose resin (Qiagen) at 4°C for 1 h. The sNASP-bound Ni-NTA beads were then packed into an Econo-column (Bio-Rad) and washed with 100 ml of 50 mM Tris-HCl buffer (pH 7.5) containing 10% glycerol, 500 mM NaCl, and 5 mM imidazole at a flow rate of about 0.8 ml/min. His 6 -tagged sNASP was eluted by a 100-ml linear gradient of imidazole from 5 to 500 mM in 50 mM Tris-HCl buffer (pH 7.5) containing 10% glycerol and 500 mM NaCl. The His 6 tag was removed from the sNASP portion by thrombin protease (3 units/mg of protein). The sample was immediately dialyzed against 20 mM Tris-HCl buffer (pH 7.5) containing 100 mM NaCl, 1 mM EDTA, 10% glycerol, and 2 mM 2-mercaptoethanol. After removing the His 6 tag, sNASP was purified using a Mono Q (GE Healthcare) column by elution with a 30-ml linear gradient of 100 -600 mM NaCl in 20 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA, 10% glycerol, and 2 mM 2-mercaptoethanol. The peak Mono Q fractions were collected, and the protein was further purified by using a Superdex 200 column eluted with 1.2 column volumes of the same buffer containing 100 mM NaCl. After this step, sNASP was again purified and concentrated by Mono Q chromatography by elution with a 30-ml linear gradient of NaCl from 100 to 600 mM in 20 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA, 10% glycerol, and 2 mM 2-mercaptoethanol. The sNASP deletion mutants were prepared by the same procedure as the fulllength sNASP. The purified sNASP and sNASP deletion mutants were dialyzed against 20 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl, 1 mM DTT, 0.5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and 10% glycerol.
Preparation of Recombinant Human Histones and Human Nap1-Human H2A, H2B, H4, and all H3 variants (H3.1, H3.2, H3.3, H3T, and CENP-A) were overexpressed in E. coli cells as N-terminal His 6 -tagged proteins (53). During the protein purification, the His 6 tag was removed from the histones, and the H2A⅐H2B and H3⅐H4 complexes were prepared by the method described previously (10). Human Nap1 (hNap1) was overexpressed in E. coli cells as an N-terminal His 6 -tagged protein and was purified by a four-step purification method, including the Ni-NTA column, the His 6 tag removal, the heparin-Sepharose column, and the Mono Q column, as described previously (10).
Preparation of a DNA Fragment for Nucleosome Reconstitution-A 195-bp DNA fragment containing the Lytechinus variegates 5 S ribosomal RNA gene was amplified by PCR using following primers: 5 S rDNA forward (5Ј-CAACGAATAACT-TCCAGGGATTTATAAGCCG-3Ј; 5 S rDNA reverse (5Ј-AATTCGGTATTCCCAGGCGGTCTCC-3Ј). After the PCR reaction, the DNA fragment was extracted by phenol/chloroform, precipitated by ethanol, and further purified by Superdex 75 gel filtration chromatography to remove the primers and dNTPs. Nucleosome Assembly Assay-H2A⅐H2B (8 ng/l) and H3.1⅐H4 (8 ng/l) were preincubated with sNASP or hNap1 at 23°C for 10 min. The nucleosome assembly reaction was initiated by the addition of 5 S rDNA (8 ng/l), and the reaction was continued in 10 l of 20 mM Tris-HCl buffer (pH 8.0) containing 80 mM NaCl and 1 mM DTT at 23°C for 60 min. After the reaction, the samples were incubated at 42°C for 60 min to eliminate nonspecific DNA binding by the histones and then were analyzed by 6% PAGE in 0.2 ϫ TBE buffer (18 mM Tris base, 18 mM boric acid, and 0.4 mM EDTA). The gel was run at 6.25 V/cm for 3 h and stained with ethidium bromide.
Micrococcal Nuclease (MNase) Assay-After the nucleosome assembly reaction with sNASP or hNap1, each sample, containing 40 ng of 5 S rDNA, was treated with 0.8, 0.4, 0.2, 0.1, and 0 unit of MNase (Takara) in 10 l of 20 mM Tris-HCl buffer (pH 8.0) containing 45 mM NaCl, 5 mM CaCl 2 , and 0.5 mM DTT. After a 5-min incubation at 23°C, the reaction was stopped by the addition of 60 l of a proteinase K solution (20 mM Tris-HCl (pH 8.0), 20 mM EDTA, 0.5% SDS, and 0.5 mg/ml proteinase K (Roche Applied Science)). After a 15-min incubation at 23°C, the DNA was extracted with phenol/chloroform and precipitated with ethanol. The DNA fragments were then analyzed by 10% PAGE in 0.2 ϫ TBE buffer (21 V/cm for 1 h) and ethidium bromide staining.
Topological Assay for Nucleosome Assembly-H2A⅐H2B (10 ng/l) and H3⅐H4 (10 ng/l) were preincubated with sNASP or hNap1 at 37°C for 15 min. The nucleosome assembly reaction was initiated by the addition of relaxed X174 DNA (10 ng/l), which was previously incubated with 1.7 units of wheat germ topoisomerase I (Promega) per 100 ng of DNA at 37°C for 150 min. The reaction was continued in 10 l of 20 mM Tris-HCl buffer (pH 8.0) containing 140 mM NaCl, 2 mM MgCl 2 , and 5 mM DTT at 37°C for 60 min. After the reaction, the samples were incubated at 42°C for 60 min to eliminate nonspecific DNA binding by the histones, and the proteins were then removed by an incubation with 60 l of a proteinase K solution (20 mM Tris-HCl (pH 8.0), 20 mM EDTA, 0.5% SDS, and 0.5 mg/ml proteinase K) at 37°C for 15 min followed by phenol-chloroform extraction. The DNA samples were then analyzed by 1% agarose gel electrophoresis in 1 ϫ TAE buffer (40 mM Tris acetate and 1 mM EDTA) (3.3 V/cm for 4 h) with SYBR Gold (Invitrogen) staining.
Gel Electrophoretic Mobility Shift Assay for Binding between sNASP and H3⅐H4-sNASP (2.4 g) was mixed with H3⅐H4 (0.75-3 g) in 10 l of 20 mM Tris-HCl buffer (pH 8.0) containing 100 mM NaCl and 1 mM DTT. The samples were then incubated for 1 h at 23°C. After the incubation, the samples were analyzed by 6% PAGE in 0.5 ϫ TBE buffer (45 mM Tris base, 45 mM boric acid, and 1 mM EDTA). The gel was run at 10.4 V/cm for 110 min, and the bands were visualized by Coomassie Brilliant Blue staining.
KCl for 2 h at 4°C. The concentration of KCl was gradually reduced from 2 to 0.25 M by adding dialysis buffer containing 0.25 M KCl using a peristaltic pump (0.8 ml/min flow rate). The sample was then incubated at 55°C for 2 h. The H3T nucleosome was purified from the free DNA and histones by nondenaturing polyacrylamide gel electrophoresis using a Prepcell apparatus (Bio-Rad). The nucleosomes were analyzed by 6% PAGE in 0.2 ϫ TBE buffer (18 mM Tris base, 18 mM boric acid, and 0.4 mM EDTA) at 15.6 V/cm for 1 h followed by ethidium bromide staining. The histone contents were also analyzed by 18% SDS-PAGE.

RESULTS
Purification of Human sNASP-Human sNASP reportedly binds preferentially to histones H3⅐H4 (52). Our proteome analysis with a HeLa cell extract also showed that human sNASP bound to histones H3⅐H4 (data not shown). These facts suggest that sNASP may be a histone chaperone for H3⅐H4. We then purified human sNASP as a recombinant protein (Fig. 1). Human sNASP was overexpressed in E. coli cells as an N-terminal His 6 -tagged protein (Fig. 1A, lanes 2 and 3). The molecular mass of sNASP is 49 kDa; however, it migrated more slowly on an SDS-polyacrylamide gel, probably because of its extreme acidity (pI ϭ 4.35). The His 6 -tagged sNASP was purified by Ni-NTA-agarose chromatography (Fig. 1A, lane 4). The His 6 tag was removed from sNASP with thrombin protease (Fig. 1B). sNASP without a His 6 tag migrated more slowly than His 6tagged sNASP because of the lack of the basic His 6 tag (Fig. 1B). After removal of the His 6 tag, sNASP was further purified by Mono Q column chromatography and Superdex 200 gel filtration chromatography (Fig. 1C). A mass spectroscopic analysis revealed that the molecular mass of purified recombinant sNASP was 49 kDa, which is identical to the predicted molecular mass of sNASP.
Nucleosome Assembly by sNASP-As reported previously, purified sNASP efficiently bound to histones H3.1⅐H4 ( Fig. 2A), suggesting that sNASP may promote nucleosome assembly as the H3⅐H4 chaperone. However, the nucleosome assembly activity of sNASP has not been reported so far, although sNASP reportedly supported the nucleosome assembly reaction in the presence of a cell extract from yeast (52). We then tested the nucleosome assembly activity of sNASP by the conventional nucleosome formation assay. hNap1, which is known to have robust nucleosome assembly activity, was used for positive controls. In this assay, sNASP or hNap1 was preincubated with H2A⅐H2B and H3.1⅐H4, and then the 195-bp 5 S rDNA was added into the reaction mixture. The nucleosome formation was analyzed by a gel electrophoretic mobility shift assay. Intriguingly, we found that substantial amounts of nucleosomes were formed in the presence of sNASP as compared with the control experiments with hNap1 (Fig. 2B). To confirm nucleosome formation by sNASP, we next performed a MNase treatment. In this assay, the 147-bp DNA fragments, which were tightly wrapped around the histone octamer, were detected after the MNase treatment because MNase preferentially digests DNA free from the histone octamer surface. Therefore, if the nucleosomes were properly assembled, then the 147-bp DNA fragment would be detected after MNase treatment, as shown in the positive control experiments with hNap1 (Fig. 2C, lanes 7-11). As shown in Fig. 2C (lanes 12-16), the 147-bp protection from MNase was clearly detected, when  6 and 11). B, tetrasome assembly is shown. Reactions were conducted as described for the experiments shown in panel A, except H2A⅐H2B or H3.1⅐H4 were used instead of the four core histones, H2A⅐H2B/H3.1⅐H4. the nucleosome assembly reactions were conducted with sNASP. These results strongly supported the conclusion that sNASP actually promotes the formation of nucleosomes.
We next tested sNASP-mediated nucleosome assembly by a topological assay. When nucleosomes are formed on relaxed circular DNA, negative supercoils are introduced, and the superhelicity can be analyzed on an agarose gel. As shown in Fig. 3A, a progressive increase in the number of negative superhelical turns introduced into DNA was observed with increasing amounts of sNASP (lanes 9 -11). The nucleosome assembly efficiency of sNASP was comparable with that of hNap1, which is known to have robust nucleosome assembly activity (Fig. 3A). Like hNap1, sNASP also promoted the formation of tetrasomes, in which the DNA is wrapped around the H3⅐H4 tetramer without H2A⅐H2B (Fig. 3B). Therefore, we concluded that sNASP itself possesses the nucleosome assembly activity, as an H3⅐H4 chaperone.
To test this possibility, we performed an H3⅐H4 deposition assay (10). When the sNASP⅐H3T⅐H4 or sNASP⅐H3.1⅐H4 complex was incubated in the presence of DNA, sNASP may be released from the complex with the histones, if the histones are deposited onto DNA. As shown in Fig. 5E, sNASP was efficiently released from the complex with H3.1⅐H4 in the presence of DNA. In contrast, sNASP was not efficiently released from the sNASP⅐H3T⅐H4 complex in the presence of DNA (Fig. 5E). These results support the hypothesis that sNASP binds to H3T⅐H4, but it is defective in the H3T⅐H4 deposition onto DNA.

DISCUSSION
In mammalian cells, two spliced forms of NASP, tNASP and sNASP, were identified (47). tNASP is highly expressed in testis, suggesting its function during spermatogenesis. On the other hand, sNASP is ubiquitously found in somatic cells. Several studies suggested that the NASP proteins may be chaperones specific for the linker histone H1 (46 -50). However, significant sequence similarity has been found between sNASP and the N1/N2 family of proteins, whose members are histone H3⅐H4specific chaperones. Parthun and co-workers (52) elegantly showed that sNASP preferentially binds to both histones H1 and H3⅐H4 in vitro and in vivo. This finding suggests that sNASP may function as a chaperone for both the linker histone H1 and core histones H3⅐H4. However, the H3⅐H4 chaperone activity of sNASP alone was not reported so far, although sNASP reportedly supported nucleosome assembly in the presence of a yeast cytosolic extract (52). The requirement of the yeast cytosolic extract in this sNASP-dependent nucleosome formation implied that sNASP may not be able to generate stable nucleosomes by itself (52). Therefore, whether sNASP indirectly deposits histones onto DNA through other factors that directly assemble nucleosomes or whether sNASP is more directly involved in histone deposition remains to be determined.
In the present study we found that (i) sNASP itself promotes nucleosome assembly with four core histones, (ii) sNASP promotes tetrasome assembly with H3⅐H4 in the absence of H2A⅐H2B, and (iii) sNASP mediates nucleosome assembly with the human histone H3 variants, H3.1, H3.2, H3.3, and CENP-A, but not with H3T, with different efficiencies. (iv) The Val-71 residue, which is not conserved in H3T, is essential for the sNASP-mediated nucleosome assembly. In addition, we found that (v) the central region, amino acid residues 26 -325, of sNASP is responsible for the nucleosome assembly in vitro. These new results provide a compelling answer for the previously unsolved question of whether sNASP is able to directly promote nucleosome assembly. Therefore, we conclude that sNASP may function as a bona fide histone chaperone for H3⅐H4, in addition to the linker histone H1. It should be noted that the purified sNASP used in this study lacked the His 6 tag, whereas the sNASP used in the previous biochemical analysis (52) contained the His 6 tag. This difference between the sNASP preparations may have caused the difference in the nucleosome assembly activity, although other experimental differences, such as the composition of the reaction mixtures, may also have contributed. sNASP is an extremely acidic protein (pI ϭ 4.35). The addition of the basic His 6 tag to the sNASP sequence may affect the nucleosome assembly activity of sNASP because the binding of sNASP to the basic histones could largely depend on its extreme acidity. A yeast factor(s), which complements the negative effect of the His 6 tag, may be required to detect the nucleosome assembly activity of the His 6 -tagged sNASP protein.
In the present study we found that sNASP is inefficient in the nucleosome assembly with a testis-specific histone variant, H3T. Previously, we reported that a conventional histone chaperone, hNap1, is defective in nucleosome assembly with H3T and that the Val-111 residue of H3T is responsible for the defective H3T nucleosome assembly by hNap1 (10). These results suggest that both sNASP and hNap1 discrim-  3-12). These sNASP fragments lack the His 6 tag. Lane 1 indicates the molecular mass markers. Lane 2 represents full-length sNASP. C, nucleosome assembly by the sNASP fragments is shown. Relaxed X174 DNA (10 ng/l), which was previously treated with wheat germ topoisomerase I (lane 2), was incubated with sNASP or the sNASP fragments in the presence of core histones. Lane 3 indicates a negative control experiment without sNASP or the sNASP fragments in the presence of core histones. The reaction products were then analyzed by 1% agarose gel electrophoresis in 1 ϫ TAE buffer. The concentrations of sNASP and the sNASP fragments were 1 M.
inate H3T from other histone H3 variants. However, interestingly, our mutational analyses revealed that sNASP and hNap1 require different amino acid residues, Met-71 and Val-111, respectively, for the H3T discrimination. This difference may reflect variations in the nucleosome assembly mechanisms between sNASP and hNap1. Structural analyses of sNASP and hNap1 complexed with H3⅐H4 are awaited to address this issue.
Intriguingly, an Schizosaccharomyces pombe homologue of sNASP, Sim3, was found to be important for S. pombe CENP-A localization at the centromere region of chromosomes (54). This suggested that sNASP may also function in the formation of the centromere-specific nucleosome containing CENP-A. Sim3 reportedly interacted with non-chromosomal CENP-A (54); however, human NASPs were found only in the cytosolic H3 complex (26,51). In the present study we found that sNASP binds to the human CENP-A⅐H4 complex and promotes the assembly of the CENP-A nucleosome in vitro. Interestingly, CENP-A was one of the efficient substrates for nucleosome assembly by sNASP. These findings support the idea that sNASP may function as a chaperone for the centromere-specific nucleosome formation. A histone chaperone, HJURP, for the CENP-A nucleosome, has been identified in humans (41,42). Given that sNASP, like HJURP, functions as a CENP-A chaperone, it would be intriguing to determine how these two CENP-A chaperones participate in the formation of the centromere-specific chromatin.