Molecular Human Reproduction, Vol. 7, No. 3, 211-218,
March 2001
© 2001 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Cloning and characterization of human haspin gene encoding haploid germ cell-specific nuclear protein kinase
1 Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita City, Osaka 565-0871, Japan
Abstract
We report here the molecular cloning and characterization of human haspin cDNA and its genomic DNA construct. The haspin protein is a unique protein kinase, first isolated from mouse testis. Specifically expressed in mouse testicular germ cells, haspin is suggested to play a role in cell cycle arrest in haploid spermatids. Detection of human haspin by Northern blot analysis showed that the major transcript was 2.8 kilobases long and detected exclusively in the testis. The entire coding region of the human cDNA showed 68% identity with mouse haspin. The predicted amino acid sequence showed strong conservation of the kinase catalytic domain, leucine zipper, potential phosphorylation sites, and MEF2B homologous region, but a relatively unique N-terminal region. Human haspin protein was also demonstrated to have protein kinase activity. The human haspin gene was mapped to chromosome 17p13 by computer database cloning of human genomic DNA. Furthermore, the genomic structure of human haspin was proven to be intronless and the whole transcription unit was found to be located in an intron of the integrin 
E2 gene.
cDNA/haspin/kinase/nucleus/spermiogenesis
Introduction
Maturation of male germ cells in mammals is a complex process, which finally generates functional spermatozoa. The whole process can be subdivided into three parts: (i) a pre-meiotic phase characterized by an increase in cell number due to mitotic divisions of diploid spermatogonia; (ii) a meiotic phase, which leads to the formation of haploid round spermatids; and (iii) a post-meiotic phase, which includes the morphogenetic events required for sperm formation, i.e. spermiogenesis (Russell et al., 1990
). Spermatogonial stem cells in the seminiferous tubules of the adult testis undergo these processes and continuously provide mature spermatozoa. The precise regulation of testicular germ cell differentiation requires a strict programme of stage- and cell-specific gene expression in germ cells as well as in surrounding somatic cell types (Griswold, 1993; Hecht, 1998).
During the haploid germ cell differentiation, or spermiogenesis, the round spermatids undergo marked morphological changes to become spermatozoa: the nucleus is shaped, the mitochondria are rearranged, the flagellum is developed and the acrosome is generated (Bellve and O'brien, 1983). Over this long period of time, about 39 days in humans (Heller and Clermont, 1964; Clermont, 1972), no division of haploid germ cells occurs, indicating that some regulatory proteins localized in the nucleus must participate in the regulation.
In a previous study, we isolated many cDNA clones specifically expressed in germ cells using a subtracted cDNA library prepared from supporting cells of germ cell-less mutant testes and wild-type testes (Tanaka et al., 1994). One of these proteins, named `haspin' (haploid germ cell-specific nuclear protein kinase) (Tanaka et al., 1999), is specifically expressed in haploid germ cells, localizes in nuclei of round spermatids, binds to DNA, and has Ser/Thr-protein kinase activity. Mouse haspin contains a core part of the protein kinase sequence which is homologous to a catalytic domain of CDC2 kinase, a cell-cycle control protein (Th'ng et al., 1990). Furthermore, mouse haspin has a region homologous to MEF2B (myocyte-specific enhancer factor 2B) protein (Hidaka et al., 1995), which is a MADS box transcription factor that regulates the expression of many muscle-specific genes (Yun and Wold, 1996). Since transfection of mouse haspin cDNA encoding functional protein into cultured somatic cells causes cessation of cell proliferation, haspin protein could be involved in regulation of proliferative activity as well as specific gene expression in haploid germ cells (Tanaka et al., 1999). Here, we report the isolation and characterization of human haspin, showing that it conserves function of mouse haspin. The results suggest that haspin plays an important physiological role in spermiogenesis.
Materials and methods
Cloning of the human haspin cDNA
To isolate human haspin (h-haspin), a human cDNA library constructed with plasmid vector pAP3neo was used (Tanaka et al., 1997). More than 2x106 E.coli containing recombinant plasmid were screened by hybridization at 50°C with a probe of the mouse haspin cDNA (Tanaka et al., 1999), and washed twice in 2xSSC (0.15 mol/l NaCl/0.015 mol/l sodium citrate pH 7.6) at 50°C for 1 h each. One positive clone having significant sequence homology with the mouse haspin cDNA was isolated. Next, a human genomic sequence (accession number: AF168787) that fully matched the partial h-haspin cDNA was found in a search of the DDBJ, Genbank, EMBL, Swiss-Prot, and PIR data banks. By computer-assisted analysis of this human genomic sequence and the mouse haspin cDNA (accession number: D87326), we found a putative open reading frame (ORF) of h-haspin. To identify this novel ORF, primer A (5'-TGCGTTTGAACCTCTTGGCGGG-3') consisting of the 22 nucleotides upstream of the first methionine and primer D (5'-GATCCCAGGCTTTGAGGAGCAAG-3') consisting of 23 nucleotides from the 3' region of the partially cloned h-haspin cDNA were made for polymerase chain reaction amplification (PCR) cloning of the h-haspin cDNA (Figure 1). The 5' region of h-haspin cDNA was thus cloned from the pAP3neo human cDNA library by PCR using primers A and D. Analysis of these cloned DNA fragments identified the full-length h-haspin cDNA sequence (accession number: AB039834). Finally, h-haspin carrying full-length cDNA was made by ligating the two fragments with the restriction enzyme PstI in pBluescript II KS+.
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Dideoxy-chain-termination sequencing reactions were performed with fluorescent dye-labelled primers and thermal cycle sequencing kits purchased from Li-COR (Lincoln, NE, USA). The reaction products were analysed with a Model-4000 sequencer (Li-COR). The Genbank, EMBL, DDBJ, Swiss-Prot, and PIR databanks were searched for sequences with homology to the h-haspin cDNA or its amino acid sequence.
Northern blot analysis
To examine the tissue-specific expression of h-haspin, a Human Multiple Tissue Northern Blot (MTN Blot II provided by Clontech, Palo Alto, CA, USA) blotted with mRNA derived from various human tissues was hybridized with 0.1 µg of radiolabelled h-haspin cDNA probe, according to the manufacturer's instructions.
Western blot analysis
Testis fragments obtained from a consenting fertile patient in middle age castrated for the treatment of prostatic cancer were homogenized with a lysis buffer containing 10 mmol/l Na2HPO4(pH 7.2), 160 mmol/l NaCl, 1% Triton X-100, 1% deoxycholic acid, 0.3% sodium dodecyl sulphate (SDS) and 2 mmol/l phenylmethylsulphonyl fluoride (Sigma, St Louis, MO, USA) in ice. After centrifugation, protein concentrations of each supernatant were estimated by a Bradford Protein Assay (Bio-Rad, Richmond, CA, USA). Each extract containing ~50 µg of protein was subjected to SDS–polyacrylamide gel electrophoresis (PAGE) (Laemmli, 1970), followed by electroblotting to polyvinylidenedifluoride (PVDF) membrane filters (Millipore, Bedford, MA, USA). The filters were blocked with 5% non-fat dry milk and washed for 15 min with TBS-T [TBS (50 mmol/l Tris–HCl pH. 7.5, 150 mmol/l NaCl) and T (0.05% Tween-20)]. The filters were then reacted with polyclonal anti-haspin rabbit serum (named DDP-2; Tanaka et al., 1999) (1:200 dilution) in TBS for 1 h at 25°C and washed in TBS-T once for 3min, and then three times for 5min each. Finally, the filters were incubated with anti-rabbit immunoglobulins conjugated with peroxidase (1:500) (Amersham Pharmacia Biotech, Tokyo, Japan) for 1 h at 25°C. After further washing, reactive bands were visualized by development with the peroxidase staining kit (Wako, Osaka, Japan).
Construction of expression vectors of h-haspin and transfection to cultured cells
The expression vector carrying h-haspin was constructed by PCR cloning of the amplified h-haspin cDNA into pEGFP-C1 (Clontech). PCR of the full-length h-haspin cDNA coding region was performed using the linker (EcoRI) oligonucleotide primer for the 5'-region of h-haspin cDNA (5'-GCGAATTCGGCTTCGCTCCCGGGACCTGG-3'), and the linker (BamHI) oligonucleotide primer of the 3'-region (5'-GGGGTACCTTACTTAAACAGACTGTGCTGG-3'). Amplification products were then digested with EcoRI and BamHI, whose recognition site had been included in the linker oligonucleotide primer, and ligated at the EcoRI-BamHI site of the mammalian expression vector pEGFP-C1 (Clontech). The resultant clone was capable of expressing the enhanced green fluorescent protein (EGFP)-haspin fusion protein.
HEK-293 cells were transfected with expression vectors pEGFP-hHASP (human haspin), pEGFP-HASP (mouse haspin), and pEGFP-dHASP (deleted form of haspin) (Tanaka et al., 1999) using LipofectAmine Plus reagent (Gibco BRL, Life Technologies Oriental, Tokyo, Japan). Transfection was performed according to the manufacturer's procedures. Twenty-four hours after transfection, cells were observed with a fluorescent microscope.
In-vitro kinase assay
To examine the h-haspin kinase activity, the lysate of the HEK 293 cells transfected with h-haspin cDNA in the expression vector was treated with protein A-Sepharose beads at 4°C for 1 h to eliminate non-specific binding materials, and then preimmune normal rabbit serum or specific anti-haspin antiserum (DDP-2) was added at a dilution of 1:500 and shaken at 4°C for 2 h. The sample was incubated with protein A-Sepharose beads at 4°C for 1 h and centrifuged. The precipitated protein A-Sepharose beads were washed three times with the lysis buffer, and then two times with kinase assay buffer (40 mmol/l HEPES, pH 7.4, 10 mmol/l MgCl2, 3 mmol/l MnCl2, 5 mmol/l CaCl2, 150 mmol/l NaCl), and incubated at 37°C for 10 min in 40 µl of the kinase assay buffer with 10 µCi of [
-32P]ATP (3000 Ci/mmol) (Amersham Pharmacia Biotech, Tokyo, Japan). The samples mixed with Laemmli's sample buffer were subjected to SDS–PAGE and electrotransferred to PVDF membrane filters (Millipore, Bedford, MA, USA). 32P-labelled proteins were visualized with a Fuji Bio Imaging Analyzer (Fuji Photofilm, Tokyo, Japan).
Genomic cloning
PCR cloning of h-haspin genomic DNA was performed using primer A (5'-TGCGTTTGAACCTCTTGGCGGG-3'), B (5'-TGGA- CC AAAACCAGGGCTTCCTTC-3'), C (5'-CCGGTTTGAACATT- CTGATAGGAG-3'), D (5'-GATCCCAGGCTTTGAGGAGCAAG-3'), and E (5'-ACCAGAGGCTTCAAGACCAGTCTC-3') corresponding to h-haspin cDNA (Figure 1
). Cycling conditions were as follows: 96°C for 1 min, followed by 35 cycles of denaturing at 96°C for 45 s, annealing at 65°C for 45 s and extension at 72°C for 90 s. The PCR products were purified by PCR Kleen Spin Column (Bio Rad, Hercules, CA, USA) and the DNA sequences determined by direct sequencing using the same PCR primers.
Results
Isolation and characterization of cDNA encoding human haspin
The human haspin (h-haspin) cDNA was isolated from a human testicular cDNA library (Tanaka et al., 1994) by hybridization with mouse haspin cDNA (Tanaka et al., 1999). Analysis of the cDNA sequence revealed only one uninterrupted long ORF (nucleotide positions 1 to 2397) (Figure 1). The putative protein of 798 amino acids contained several conserved regions, such as a basic region at residues 77–83, a leucine zipper at residues 622–657 (Landschultz et al., 1988), and parts of protein kinase consensus sequences over the region of residues 484–542 (Hanks and Quinn, 1991; Knighton et al., 1991), which was particularly homologous to CDC2 kinase (Cisek and Corden, 1989) (Figure 1). It also contained many potential phosphorylation sites for protein kinase C (consensus S/T-x-R/K) (Kishimoto et al., 1985), a cAMP- and cGMP-dependent protein kinase (consensus RK-x-S/T) (Fremisco et al., 1980), and casein kinase II (consensus S/T-x-x-D/E) (Pinna, 1990). A homologous region to murine myocyte-specific enhancer factor 2 B (MEF2B) (Hidaka et al., 1995) was also conserved at residues 162–182 (Figure 1). The above regions might be related to specific functions of haspin. In addition, the amino acid sequence of the DDP-2 epitope for anti-haspin serum was also conserved in human and mouse haspin.
The ORF region of the human haspin cDNA showed 72% similarity with the previously reported mouse haspin and the amino acid sequence data showed 62% identity with mouse haspin (Tanaka et al., 1999). Mouse haspin has several deletions in the N-terminal half, namely two sets of more than 10 amino acids and one or more amino acids at more than 10 sites are missing. These deletions result in a form that is 44 amino acid residues shorter (754 residues) than h-haspin (798 residues).
The functional poly A signal of h-haspin does not correspond to that of mouse haspin, resulting in a smaller 3' untranslated terminal region (UTR) in human (364 bases) than in mouse (531 bases) haspin. There is also an Alu repetitive sequence (Weiner, 1980) in human 3' UTR (Figure 1). In contrast, the putative 5' UTR of mouse and human haspin cDNA are the same size although the mRNA starting point remains to be identified.
Expression of h-haspin mRNA and protein
Using the full-length of h-haspin cDNA as a probe, Northern blot analysis showed that h-haspin was exclusively expressed in the testis as a major transcript of 2.8 kb and a minor additional signal of 4.2 kb (Figure 2). Western blot analysis with anti-haspin rabbit antiserum (DDP-2) showed one positive signal with a molecular weight of 88 kDa exclusively in human testicular extract (Figure 3).
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Ectopic expression and localization of h-haspin in cultured cells
For characterization of h-haspin, its localization in ectopically expressed cultured cells was examined. When HEK-293 cells were transfected with the cDNA of wild-type h-haspin (pEGFP-hHASP), and traced with the fluorescence of the fused enhanced green fluorescent protein (EGFP), the protein was localized exclusively in nuclei of the cultured cells (Figure 4
), similar to the physiological localization of mouse haspin in testicular germ cells (Tanaka et al., 1999). In cultured cells, h-haspin was localized to subnuclear foci, being discretely present as a punctated form within the nuclei (Figure 4C).
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Kinase activity
Because haspin has a core kinase catalytic domain that includes consensus kinase subdomains I, II and III, but lacks the C-terminal subdomains IV-XII, we have examined h-haspin kinase activity in vitro. HEK-293 cells were transfected with the cDNA of h-haspin (pEGFP-hHASP) and, as a control, mouse haspin (pEGFP-HASP) and a haspin (pEGFP-dHASP) deleted of kinase subdomain I (Tanaka et al., 1999
). Cell lysates were immunoprecipitated with DDP-2 antiserum, and an aliquot of the immunocomplex was subjected to in-vitro kinase assay in the presence of Mg2+ and Mn2+ with [-32P]ATP. The kinase-assayed samples of immunocomplexes were subjected to SDS–PAGE, followed by Western blot analysis with DDP-2 anti-serum. By using the same PVDF filter, phosphorylated proteins labelled with 32P were detected by autoradiography. Prominent bands labelled with 32P were detected at ~116 kDa, the size of the positive immunoblotting signal of haspin-EGFP fusion protein (Figure 5). The results showed that both human and mouse haspin, but not the deleted form of mouse haspin, have kinase activity.
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Haspin has a deletion in the kinase domain (Tanaka et al., 1999Genomic localization and construction of h-haspin
On searching the DDBJ, Genbank, EMBL, Swiss-Prot, and PIR data banks for a human genomic clone having sequence homology with h-haspin cDNA, we found a genomic clone (accession number: AF168787.1) which maps to the 17p13 region and includs the h-haspin ORF as an intronless gene inside the intron of integrin
E (L25851) (Shaw et al., 1994). To confirm the localization of the h-haspin gene in this clone, we performed PCR with human genomic DNA as a template using four sets of oligonucleotide pairs (A-D, B-E, A-C, and A-E in Figure 6B) that encompassed both the 5' and the 3' ends of the h-haspin ORF. The size of each PCR product was consistent with that of the putative fragment from genomic DNA sequence and also the size expected from the cDNA sequence (Figure 6A). To further check the fragment, each PCR product was sequenced and compared with cDNA sequence. These results revealed that h-haspin was an intronless gene localized within an intron of the integrin E gene on chromosome17p13.
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Discussion
Production of a huge number of spermatozoa is usually a guarantee of successful fertilization, and infertility is often related to azoospermia. However, vertebrate haploid germ cells do not proliferate after meiosis and thus do not generate large numbers of spermatozoa, in contrast with yeast and some insects (Ross, 1993). Cell cycle arrest after meiosis and full commitment to the differentiation process in spermiogenesis are known to be coupled and constitute an important physiological event in a wide range of organisms (Douglas et al., 1998). However, it is still unclear what kind of molecular mechanisms are involved in this process. Previously, haspin encoding a unique nuclear protein kinase was isolated in mouse testicular haploid germ cells (Tanaka et al., 1994, 1999). Cell cycle arrest at G1 was induced by the expression of haspin in cultured somatic cells. We speculate that haspin plays a role in cell cycle regulation after meiosis in haploid germ cells.
Haspin protein has several interesting domains, including a basic region, a leucine zipper (L-X(6)-L-X(6)-L-X(6)-L), a region homologous to murine myocyte-specific enhancer factor 2 B (MEF2B) (Hidaka, et al., 1995) and parts of the protein kinase consensus domains (subdomains I–III) homologous to CDC2 kinase (Cisek and Corden, 1989). The MEF2B gene encodes a MADS box transcription factor which regulates the expression of many muscle-specific genes (Yun and Wold, 1996). As haspin does not have the MADS box and MEF2B does not have the protein kinase domain, haspin should differ in function from MEF2B. However, haspin has a basic domain in its N-terminus and can bind to DNA. Thus, it is possible that haspin is another type of testis-specific transcription factor or a cell cycle regulatory factor of haploid germ cells. It also contains many potential phosphorylation sites for protein kinases; protein kinase C (consensus S/T-x-R/K), cAMP- and cGMP-dependent protein kinase (consensus RK-x-S/T), and casein kinase II (consensus S/T-x-x-D/E). Human haspin (h-haspin) protein localized to the nucleus and showed kinase activity as did the mouse haspin (Tanaka et al., 1999). This conservation of unique amino acid sequence domains suggests that haspin is a prerequisite molecule for the regulation of germ cell differentiation during spermiogenesis.
By computer search of a genomic library, we compared the mouse and human haspin genome and found that the genomic constructs of human and mouse haspin are basically the same. The upstream region of the initiation position does not contain a TATA box, GC rich promoter motifs or cAMP-responsive promoter elements (Nantel et al., 1996). The whole transcription unit was located in an intron of the integrin E gene (Shaw et al., 1994), as an intronless gene, and the direction of transcription of these two genes was different. Functional intronless genes specifically expressed in haploid germ cells have been reported previously. Two genes, present on autosomes encoding phosphoglycerate kinase-2 (McCarrey and Kelwyn, 1987; McCarrey, 1987) and pyruvate dehydrogenase subunit e2a (Dahl et al., 1990), specifically expressed in male germ cells, are also known to be intronless. In this case, the two genes, both localized on autosomes, are believed to be derived from ancestral genes on the X chromosome carrying introns. To escape the effect of X chromosome inactivation during spermatogenesis, they would have had to move to an autosome. The origin of both the phosphoglycerate kinase-2 and pyruvate dehydrogenase subunit e2 genes suggested the involvement of retrotransposons. We found repetitive sequences [GAGCC(A/T)T] also conserved in human and mouse haspin genomic DNA. The cDNA of mouse haspin has two poly A additional signals (Tanaka et al., 1999). In contrast to mouse haspin, the remnant poly A sequence was not found at the end of the coding sequence in human haspin. Species-specific dynamic conversion or rearrangement, e.g. Alu insertion (Weiner, 1980) over a long period as discussed below might have erased such a sequence. These results suggest that haspin was generated by reverse transcriptase activity involving a transposon in the ancestral animal. Furthermore, comparison of the kinase domains of haspin with those of other kinases suggested that diversification of the haspin gene occurred very early. The haspin and integrin E gene construct also seems to have originated in ancient animals but is still functional since both genes are physiologically important.
The 3' region of the h-haspin genomic sequence includes three Alu repetitive sequences. One Alu is located in the 3' untranslated region (UTR) of h-haspin, the others are located between the downstream region of the coding sequence of haspin and the integrin E exon. The construction of h-haspin indicates that the original haspin gene translocated between GAGCC(A/T)T repetitive elements in the integrin E intron before human and mouse split and later, Alu-repetitive sequences and the mouse-specific repetitive sequence were inserted in this genomic region around haspin in human and in mouse, respectively. Although we do not know the meaning of these species-specific insertions of repetitive sequences, it is clear that they have induced change in the 3' UTR of two haspin genes and also in the intron of integrin E. In contrast, the coding regions of haspin genes in the two species and also the putative promoter sequences do not seem to be affected by these insertions and therefore do not compromise the function of both genes.
The mouse haspin gene has been mapped to mouse chromosome 11 (Matsui et al., 1997). Our results indicate that the h-haspin is localized on chromosome 17p13 corresponding to the haspin location on the mouse chromosome. Computer analysis of the published human chromosomal mapping data did not reveal any significant relationship between this locus and any known human diseases. However, a known mouse mutant locus close to haspin is the ovum mutant (om), which shows a notable phenotype related to fertilization (Wakasugi, 1974). Its corresponding gene acting in spermatozoa is S element. Haspin might be a kind of germ cell factor. Further studies are now in progress to elucidate the molecular function of haspin in testicular germ cell proliferation and differentiation.
These results suggest that haspin plays an important role in spermiogenesis and that the genomic construct of haspin is indispensable for haploid germ cell-specific expression.
Notes
2 To whom correspondence should be addressed. E-mail: nishimun{at}biken.osaka-u.ac.jp 
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Submitted on October 26, 2000; accepted on December 28, 2000.
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