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NF45 and NF90 in Murine Seminiferous Epithelium: Potential Role in SP-10 Gene Transcription

UMESH DESHMUKH

From the ^* Department of Pathology and the Department of Medicine, University of Virginia, Charlottesville, Virginia.

Correspondence to: Prabhakara P. Reddi, 415 Lane Road, P.O. Box 800904, Charlottesville, VA 22908 (e-mail: ppr5s{at}virginia.edu).

Received for publication July 27, 2007; accepted for publication October 16, 2007.

	Abstract

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Identification of transcription factors involved in the progression of spermatogenic cell differentiation is important for understanding the molecular mechanisms controlling spermatogenesis. To this end, we utilized the mouse SP-10 gene encoding a conserved acrosomal protein as an experimental model. Promoter analysis in transgenic mice had previously shown that the –186/–91 region of the SP-10 promoter was critical for spermatid-specific expression. Here, we focus on a purine (Pu) box (-agaaaa) located at –154, which is conserved in the mouse, monkey, and human SP-10 gene promoters. NF45 and NF90, which belong to the family of nuclear factor of activated T cells (NFAT), are known as Pu-box–binding proteins. We tested the potential of NF45 and NF90 to activate the SP-10 promoter via the Pu-box element. Immunohistochemistry showed the presence of NF45 and NF90 in the nuclei of pachytene spermatocytes, round spermatids, and Sertoli cells. In gel shift assays, recombinant NF45 bound to the mouse SP-10 promoter in an AGAAAA site–specific manner. Cotransfection of NF45 and NF90 up-regulated SP-10 promoter–driven luciferase expression in transiently transfected spermatogenic GC2 cell line; this up-regulation required the -AGAAAA- site. Furthermore, stimulation of the endogenous NF45-NF90 complex in Jurkat cells by phorbol myristate acetate + ionomycin up-regulated the SP-10 promoter activity in plasmid-based assays. In the context of chromatin, however, stimulation of NF45-NF90 alone was not sufficient to activate an SP-10 promoter–driven green fluorescent protein transgene. Based on these results, we propose that NF45 and NF90 have the potential to activate SP-10 gene transcription, and that a chromatin modification event must occur first in order to provide access to these transcription factors.

     Key words: Fertility, reproductive tract, sperm, spermatogenesis, testis, promoter, transcriptional regulation, transgenic mice

Our laboratory utilizes the mouse SP-10 gene, which is expressed exclusively in round spermatids, as a candidate gene to understand how cell type–specific gene transcription is regulated during spermiogenesis. The SP-10 gene codes for an acrosomal protein conserved in all mammals, including humans. The mouse and human SP-10 genes share 60% similarity at the amino acid level and 75% similarity within the proximal promoter region (Reddi et al, 1999). Promoter analysis performed to date established that the –186/+28 region of the mouse SP-10 gene promoter was sufficient to drive round spermatid-specific transcription in transgenic mice, whereas the –91/+28 portion did not support transcription (Acharya et al, 2006). This suggests that cis-elements within the –186/–91 region must be important for recruiting the transcription factors necessary for the SP-10 gene transcription.

Within the –186/–91 region is a conserved Pu-box element consisting of adenine and guanine nucleotides. A Pu-box–like sequence has been identified to be a part of the 5' regulatory regions of many lymphokine genes and of the long terminal repeat sequences of human immunodeficiency virus (HIV). Characterization of the Pu box located within the 275-bp enhancer region of the interleukin-2 (IL-2) gene led to the identification of its cognate factor (Randak et al, 1990). It was observed that cyclosporin A (CsA), a powerful immunosuppressive drug, inhibited the synthesis of IL-2 at the level of gene transcription in Jurkat cells. A prominent CsA-sensitive factor of 45 kd was identified to bind the Pu box. This 45-kd factor was later purified and cloned as nuclear factor NF45 and was shown to be in association with NF90 in activated Jurkat cells (Kao et al, 1994).

NF45 contains an RGG-rich single-stranded RNA-binding motif (amino acids 2–22), a DZF zinc-finger nucleic acid–binding domain (amino acids 104–338), and a highly acidic glutamic acid–rich carboxy-terminal domain (amino acids 365–390). NF90 contains 2 double-stranded RNA-binding domains and a zinc-finger nucleic acid–binding motif (Zhao et al, 2005). Besides transcription, NF45 and NF90 have been shown to play roles in the splicing, export, and translation of RNA (Tian et al, 2004). Though they are largely characterized in the lymphocyte system, earlier studies reported high expression of NF45 and NF90 in the murine testis (Buaas et al, 1999; Zhao et al, 2005).

Here, we investigated the spatial and temporal expression of NF45 and NF90 in the murine seminiferous epithelium and tested the hypothesis that NF45 and NF90 could activate the SP-10 gene promoter via the conserved Pu-box sequence. The results reported here argue in favor of a potential role for NF45/NF90 in SP-10 transcription.

	Materials and Methods

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Preparation of Testis Nuclear Extract

Animals were handled as per the Animal Use and Care Committee of the University of Virginia. Swiss Webster mice were purchased from Harlan (Indianapolis, Ind). Testes from 3-month-old mice were removed, decapsulated, and cut into small pieces using a razor. The tubules were washed once with plain Dulbecco's Modified Egale's Medium (DMEM) and treated with collagenase and trypsin at 1 mg/mL concentration at 37°C for 20 minutes. Treatment was continued until the tubules were well separated. Following 1 wash with plain DMEM, the tubules were incubated at 37°C with collagenase, hyaluronidase, and trypsin at a concentration of 1 mg/mL. At regular intervals, the release of germ cells was monitored by microscopic examination. Enzymatic treatment was terminated by adding ice-cold DMEM medium and allowing the tubules to stand for 10 minutes. Supernatant was removed and spun at 1000 x g for 10 minutes to get the germ cell pellet. Nuclear extract was prepared essentially as described by Dignam et al (1983). Briefly, the cell pellet was washed twice with buffered saline (PBS), pH 7.5, and suspended in 10 mL of buffer H (20 mM Tris HCl pH 7.5, 10 mM KCl, 1.5 mM MgCl₂, 1 mM dithiothreitol [DTT], 1 mM phenylmethylsulphonylfluoride [PMSF], 1 mM benzamidine HCl, protease inhibitor) for 15 minutes on ice. Swollen cells were ruptured by Dounce homogenizer and centrifuged at 600xg for 10 minutes at 4°C to get the nuclear pellet. The nuclear pellet was resuspended in 2 mL of buffer D (50 mM Tris HCl, pH 7.5, 420 mM KCl, 5 mM MgCl₂, 20% glycerol, 10% sucrose, 2 mM DTT, 1 mM PMSF, 1 mM benzamidine HCl, 0.1 mM EDTA, protease inhibitor) for 1 hour at 4°C. Following this extraction, the samples were centrifuged at 14 000xg at 4°C for 20 minutes, and the resulting supernatant contained the nuclear extract. Protein quantity was estimated by Bradford assay.

Immunohistochemistry

Three-month-old C57 black mice were perfused with Bouin fixative. Testes were removed and further fixed in Bouin solution for 24 hours at 4°C. Tissues were then processed for paraffin embedding and 5-µm-thick sections were obtained. Tissue sections were rehydrated through alcohol grades and equilibrated in PBS for 10 minutes. Quenching was done for 30 minutes in PBS containing 1% H₂O₂. Slides were washed and blocked in 5% nonfat dried milk for 1 hour. Primary antibodies NF45 (1:100) or NF90 (1:100) were prepared in 2.5% nonfat dry milk and added to slides, and incubated overnight at 4°C in a moist chamber. The next day slides were washed 3 times with PBS for 10 minutes. Secondary anti-rabbit antibody (1:500) was added and incubated for 2 hours at room temperature (RT) in a moist chamber. Slides were washed 3 times with PBS for 10 minutes, developed with a Nova red staining kit (Vector Laboratories, Burlingame, Calif), counterstained with hematoxylin, dehydrated, and mounted using Vectamount.

Western Blot

Purified recombinant NF45 (200 ng) or Cos7 cell extract (25 µg) was resolved on 10% SDS-polyacrylamide gel and transferred to nitrocellulose. Membrane was blocked in 5% dried milk powder for 1 hour and then incubated overnight at 4°C in primary antibody (NF45 or NF90) or anti-His at a dilution of 1:1000 prepared in 2.5% dried milk powder. Membrane was washed 5 times with PBS-T20 for 5 minutes each. Secondary anti-rabbit antibody (Sigma, St Louis, Mo) (1:5000) was then added for 1 hour at RT. Membrane was washed as earlier and developed with TMB reagent (Biorad, Hercules, Calif).

Recombinant Protein Expression and Purification

We generated recombinant NF45 protein with a histidine tag. The entire 390-amino-acid open reading frame of NF45 was polymerase chain reaction (PCR)-amplified from NF45-pcDNA3.1 (gift from Dr Michael B. Mathews, University of Medicine and Dentistry of New Jersey) and inserted into pET22b+ bacterial expression vector. BL21 E coli cells were transformed with NF45-pET22b plasmid. Histidine-tagged NF45 protein was induced with 0.4 mM isopropyl-thiogalactopyranoside at 37°C for 6 hours. At the end of the incubation, a cell pellet was obtained by centrifugation at 4000 x g for 10 minutes at 4°C. The cell pellet was treated with lysozyme and DNase for 20 minutes and then sonicated for 5 minutes to release the inclusion body pellet. The sample was spun at 14 000xg for 15 minutes at 4°C. The inclusion body pellet was washed 3 times with 50 mM Tris-HCl pH8.0, 10 mM EDTA pH8.0, 100 mM NaCl, and 0.5% (v/v) Triton X-100 and spun at 13 000 rpm for 10 minutes at 4°C. Inclusion bodies were then dissolved in 100 mM NaH₂PO₄, 10 mM Tris-HCl, and 6 M guanidium HCl (pH 8.0), by gentle vortexing. The sample was cleared by centrifugation at 13 000 rpm for 10 minutes at 4°C. Slurry of Ni-NTA resin (Qiagen, Valencia, Calif) was added to the supernatant and incubated for 30 minutes at RT. It was then centrifuged for 10 seconds at 14 000xg. Resin was washed 3 times with 100 mM NaH₂PO₄, 10 mM Tris-HCl, and 6 M guanidium HCl (pH 6.3), and then eluted 3 times with 100 mM NaH₂PO₄, 10 mM Tris-HCl, and 6 M guanidium HCl (pH 4.5). The eluted sample was dialyzed against 25 mM HEPES (pH 7.6), 0.1 mM EDTA, 10% glycerol, 50 mM KCl, and 0.05 mM DTT.

View larger version (22K): [in this window] [in a new window]   Figure 1. Conservation of Pu box in the SP-10 gene promoter. Alignment of the –186/–91 region of human, monkey, and mouse SP-10 gene promoters shows that the Pu-box sequence AGAAAA, located at the –154 position in the mouse, is conserved among all the species. NF45 and NF90 were shown to bind to the Pu-box in the IL2 gene promoter (Kao et al, 1994). In the present study, we investigated the role of NF45 and NF90 in SP-10 gene transcriptional activation. Color figure available online at www.andrologyjournal.org.

SP-10P (–166/–139): att cta cac aca gaa aat gct ctt cac t; SP-10M (–166/–139): att cta cac aca tag cct gct ctt cac t (Pu-box mutated); IL-2P (–255/–285): gat cgg agg aaa aac tgt ttc ata cag aag gcg t; IL-2M (–255/–285): gat cgg act tag cct tgt ttc ata cag aag gcg t (Pu-box mutated). Double-stranded oligonucleotides were obtained by incubating an equimolar concentration of sense and antisense oligonucleotide in tris hydrochloride: ethylene diaminete traacetic acid buffer at 95°C for 5 minutes and then allowing it to slowly cool at RT. Double-stranded DNA (5 pmol) was end labeled by incorporating ³²-ATP using T4 polynucleotide kinase (Promega, Madison, Wis) as per manufacturer's instructions. DNA binding assay was carried out in 20-µl reaction with 5 fmol of end-labeled DNA oligonucleotide in a final buffer consisting of 25 mM HEPES (pH 7.6), 0.1 mM EDTA, 10% glycerol, 50 mM KCl, and 0.05 mM DTT, in the presence of 100 ng of poly (dI-dC) (GE Healthcare, Buckinghamshire, United Kingdom) as a nonspecific competitor for DNA binding proteins. For recombinant NF45, 300 ng protein was used per binding reaction, whereas for testis nuclear extract (TNE), 10 µg was used. First, the nonspecific interaction between protein and DNA was allowed to take place at room temperature for 20 minutes. Labeled probe was then added and incubated for 30 minutes. Following the incubation, the reaction mixture was then resolved on 4% polyacrylamide gel in 0.5x tris borate EDTA buffer at 150 V for 3 hours. Gel was dried under vacuum and exposed to x-ray for autoradiography.

Transient Transfection Assay

Transient transfection assays were performed in GC-2 cell line using Mirus (Invitrogen, Carlsbad, Calif) as the fusogen according to the manufacturer's instructions. Cells at a density of 10⁵ were seeded in 6-well plates for 24 hours prior to transfection. Luciferase reporter constructs were built in the pGL3 basic vector (Promega). The –186/+28 SP-10 promoter fragment was PCR-amplified from –408SP-10-gfp (Reddi et al, 1999) and cloned into the XhoI, HindIII site of pGL3 to obtain –186/+28Luc plasmids. To generate –186/+28Luc with a mutant Pu-box site, a forward primer was designed with the mutant site (5'-cctcgaggaagctacccctaacacactattctacacaca tagcctgctcttca ct-3') and used for PCR. Cells were harvested 48 hours after transfection. Luciferase activity was measured per 10 µg protein extract using the Luciferase Reporter Assay System (Promega) according to instruction provided in the kit. Student's t-test was performed for calculating the P value. Results are means of 3 independent experiments, each performed in duplicate, and error bars represent ± SE.

Indirect Immunofluorescence

Cos7 cells were seeded on a coverslip at a density of 10⁵ cells per well in a 6-well plate 24 hours prior to transfection. Full-length NF45 or its c-terminal glutamic acid region deletion construct were transiently transfected using Mirus reagent as per the manufacturer's instructions. After 48 hours, coverslips were fixed with 4% paraformaldehyde prepared in PBS for 10 minutes. At the end of the incubation, the coverslips were washed 2 times with PBS for 5 minutes. Cells were then permeabilized in 0.2% Triton X-100 in 10% normal goat serum (NGS) for 10 minutes and washed 2 times in PBS for 5 minutes. The coverslips were then incubated with 1:500 anti-myc monoclonal antibody prepared in PBS with 10% NGS for 35 minutes at RT. After 4 washes in PBS for 5 minutes each, goat anti-mouse Cy3 (Jackson ImmunoResearch Laboratories, West Grave, Pa) was added at 1:200 dilution for 30 minutes at RT. Coverslips were washed 4 times with PBS for 5 minutes each and then mounted on a slide with Slowfade reagent (Invitrogen). Slides were then viewed under an inverted fluorescent microscope.

In Vitro Spleen Cell Stimulation by Phorbol Myristate Acetate and Ionomycin

Single cell suspension of spleen cells from transgenic –408/+28 SP-10-gfp and control wild-type B6 mice were prepared in DMEM supplemented with 10% FBS, 2 mM L-glutamine, nonessential amino acids, sodium pyruvate, 100 U/mL penicillin, 100 mg/mL streptomycin, and 5 x 10^–5 M 2-mercaptoethanol. Cells (2 x 10⁵/mL) were stimulated with 20 ng/mL phorbol myristate acetate (PMA) (Sigma) and 2 µM ionomycin (Sigma) for 48 hours, and IL-2 production was measured by enzyme-linked immunosorbent assay (ELISA) following manufacturer's instructions (BD Biosciences, San Jose, Calif). Cells were stained with PE-conjugated hamster anti-mouse CD3 antibody (BD Biosciences). GFP expression in CD3+ cells was analyzed by flow cytometry. Single-cell suspension of testis cells from –408/+28 SP-10-gfp mice was used as a positive control for GFP expression.

In Vitro Jurkat Cell Stimulation by PMA + Ionomycin

Jurkat cells were stimulated with 20 ng/mL PMA and 2 µM ionomycin for 4 hours and then transfected with 500 ng of wild-type and Pu-box mutant –186/+28pGL3 reporter plasmids. pRLTK (50 ng) vector was used as an internal control for transfection efficiency. Dual luciferase assay was performed to measure reporter gene activities 48 hours after transfection. Cell supernatants were saved for IL-2 assay by ELISA. Results shown are mean of 3 separate experiments, and error bars represent ± SE.

	Results

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Pu-Box Sequence in Mouse, Monkey, and Human Sp-10 Gene Promoters

Previous studies in transgenic mice established that the –186/–91 region of the SP-10 gene promoter is important for round spermatid-specific transcription. Alignment of the corresponding region from human, monkey, and mouse SP-10 genes showed high homology in the –186/–144 region. Two ACACAC motifs located within this region at –172 and –160 in the mouse were previously shown to be dispensable for transcription in round spermatids, suggesting that cis-elements for activation must lie elsewhere. Here we focused on the adjacently located Pu-box sequence, 5'-AGAAAA, present at the –154 position in the mouse SP-10 promoter. Figure 1 shows the evolutionary conservation of this element in the mouse, human, and monkey SP-10 promoters. The SP-10 Pu-box is nearly identical to the Pu-box of the IL2 gene promoter (5'-GGAAAA). Nuclear factors of activated T-cells NF45 and NF90 were identified as the cognate transcription factors responsible for IL2 gene expression. Here, we characterized the potential role of NF45 and NF90 in SP-10 promoter activity.

NF45 and NF90 in Murine Seminiferous Epithelium

In order to begin to address the relevance of NF45 and NF90 to SP-10 gene transcription, we investigated the pattern of spatiotemporal expression in the murine seminiferous epithelium. Immunohistochemistry was performed on adult mouse testis cross-sections using antisera specific to NF45 and NF90 (Figure 2). The stage of the seminiferous cycle represented by each cross-section was identified by using established morphological criteria (Russell et al, 1990). Both NF45 and NF90 were found to be widely expressed in meiotic and postmeiotic germ cells, as well as in Sertoli cells (labeled "Se" in Figure 2A and F), at various stages of the cycle of seminiferous epithelium. Spermatogonia and leptotene spermatocytes did not show immunoreactivity with NF45 and NF90 antibodies (Sg and Lp, respectively; Figure 2). Based on immunoreactivity, the highest levels of expression of NF45 and NF90 were found in pachytene spermatocytes (Pa in Figure 2 A through C and E through G), suggesting a role for these proteins in meiotic divisions. Both transcription factors, however, persisted in the round spermatids at stages I through VII (Rs in Figure 2A through B and E through F), but no expression was observed in the elongated spermatozoa (Es, Figure 2). Thus, the presence within transcriptionally active round spermatids is indicative of a role for NF45 and NF90 in the regulation of gene expression during early spermiogenesis when the SP-10 gene is transcribed. The presence of RNA binding domains suggests that NF45 and NF90 could also play a role in posttranscriptional regulation. Some testis-specific transcripts are in fact stored for translation in condensing spermatids (Yang et al, 2005). In all cell types, the immunostaining was confined to the nuclei, in agreement with the proposed role for these factors in transcriptional and/or posttranscriptional regulation. Only in the case of spermatocytes undergoing division at stage XII of the cycle was the staining seen diffusely in the cytoplasmic region (arrows in Figure 2C and G). Overall, the spatiotemporal expression pattern of NF45 and NF90 was consistent with a specific role for these proteins in male germ cell differentiation, beginning with meiosis.

View larger version (122K): [in this window] [in a new window]   Figure 2. Immunolocalization of NF45 and NF90 in mouse testis. Adult mouse testis cross sections were probed with NF45 (A–D) or NF90 (E–H) antibodies. The upper 6 panels represent antibody staining and the lower 2 panels (D and H) represent controls lacking the primary antibody (no 1° antibody control). The Roman numerals indicate the stage of the cycle of seminiferous epithelium represented in each cross section. In general, NF45 and NF90 first appeared in the pachytene spermatocytes (Pa) and persisted in the round spermatids (Rs). There was no staining of NF45 or NF90 in spermatogonia (Sg) or elongating spermatids (Es). Both proteins were present in the Sertoli cells (Se). The spatial and temporal expression data suggest that NF45 and NF90 must play a role in the meiotic and postmeiotic phases of male germ cell differentiation. Black bar = 50 µm.

Recombinant NF45 Protein Binds to the SP-10 Promoter:

NF45 and NF90 exist as a complex, and there has been a suggestion that NF45 acts as a potential regulator of IL-2 gene expression (Zhao et al, 2005). This suggests that NF45 has potential to bind DNA. We investigated whether NF45 binds to the SP-10 promoter in vitro by performing gel shift assays. Figure 3a shows the size and immunoreactivity of the purified recombinant NF45 protein. Western blotting with anti-His as well as anti-NF45 antibodies confirmed the identity and purity of the recombinant NF45. Double stranded oligonucleotide corresponding to the (–166/–139) SP-10 promoter region encompassing the Pu-box element (SP-10P) was used in gel shift assays to test NF45 binding. A double-stranded oligonucleotide corresponding to the –255/–285 region of the IL2 gene promoter, which contains a canonical Pu box, was used as a positive control (IL-2P). The SP-10P oligomer showed a prominent gel shift with the NF45 protein (thick arrow in Figure 3b, lane 2). Specificity of this binding was confirmed by competition experiments. The addition of 50- and 100-fold excess of unlabeled SP-10 oligomer abolished the gel shift in a dose-dependent fashion (Figure 3b, lanes 3 and 4). Importantly, when used as a cold competitor, the IL-2P oligomer was also able to abolish NF45 binding to the SP-10P (Figure 3b, lanes 7 and 8). To address the requirement of the -agaaaa- site for NF45 binding, unlabeled mutant SP-10M and IL-2M oligonucleotides, wherein the -agaaaa- site was mutated to -atagcct-, were used at 50- and 100-fold excess concentration in the binding reaction. Both the mutant oligonucleotides failed to compete for NF45 binding (Figure 3b, lanes 5, 6, 9, and 10), thus implicating the -agaaaa- site in NF45 binding. This is the first report that defines a cis-element for NF45 binding to double-stranded DNA. This in vitro assay suggested that NF45 protein could potentially bind to the –166/–139 region of the SP-10 gene promoter in a -agaaaa- site–specific manner. When TNE was used in gel shift assays (Figure 3c), the SP-10P oligomer showed 1 strong upper gel shift complex (thick arrow), and weaker complexes of lower molecular size (hollow arrows) (Figure 3c, lane 2). The upper complex showed a dose dependent reduction when competed with unlabeled wild-type SP-10P (lanes 3 and 4) and IL-2P oligo (lanes 7 and 8), but not with the mutant SP-10M oligo (lanes 5 and 6) or mutant IL-2M oligo (lanes 9 and 10). The major gel shift band obtained with the TNE migrated at a higher position compared to that obtained with recombinant NF45. One interpretation of this result is that the TNE-gel shift complex may include NF45 in association with its binding partners. Competition with a bona fide NF45-NF90–binding IL2-P oligo (Figure 3c, lanes 7 and 8), and the presence of NF45 and NF90 in the germ cell nuclei (Figure 2), suggest that NF45 may be a part of the protein complex in TNE interacting with the –166/–139 SP-10 promoter in a Pu-box–specific manner.

View larger version (41K): [in this window] [in a new window]   Figure 3. SP-10 promoter binds to a protein complex containing NF45 protein. (A) Histidine tagged recombinant NF45 (rNF45) used in electrophoretic mobility shift assay (EMSA). Lane A: Coomassie stained rNF45 protein profile. Immunoblot showing reactivity with lane B: antihistidine tag and lane C: anti-NF45 antibodies. (B) Recombinant NF45 protein binds to the SP-10 promoter in a -agaaaa- site (Pu-box)–specific manner. EMSA was performed using rNF45 and the double stranded –166/–139 SP-10 region (SP-10P) or a Pu-box mutant version(SP-10M). As positive control, we used the IL2 promoter fragment (IL-2P) or its Pu-box mutant version (IL-2M). Lane 1, free SP-10P oligo; lane 2, with rNF45; lanes 3 and 4. competition with SP-10P oligo; lanes 5 and 6, competition with SP-10M oligo; lanes 7 and 8, competition with IL-2P oligo; lanes 9 and 10, competition with IL-2M oligo. Lane 1 shows migration of the free probe at the bottom of the gel (thin arrow) and lane 2 shows the SP-10P and rNF45 complex (thick arrow). The specificity of this interaction was challenged by competition with the denoted oligos at 50x and 200x molar concentrations (lanes 3–10). The wild-type SP-10P and IL-2P oligos, but not their Pu-box mutants, competed for rNF45 binding to the SP-10 promoter. (C) The SP-10 promoter interacts with a protein(s) from testis nuclear extract (TNE) in a Pu-box specific manner. The lane description is identical to that of (B) except that TNE was used in place of rNF45. The thick arrow points to the SP-10P + TNE complex that was sensitive to Pu-box mutation. Based on the specificity of binding, we predict NF45 to be a part of this complex. The hollow arrows show minor, nonspecific gel shift complexes.

View larger version (19K): [in this window] [in a new window]   Figure 4. NF45 and NF90 together, but not alone, activate the SP-10 promoter. NF45 and NF90 expression plasmids were cotransfected into the mouse GC2 cells with luciferase reporter plasmids driven by either the wild-type –186/+28 SP-10 promoter (–186/+28 pGL3) or its Pu-box mutant (m-186/+28 pGL3). Reporter activities were normalized against those obtained with the empty expression vector (pcDNA) and fold differences plotted on the y-axis. The expression and reporter plasmids were used as indicated. The 1.8-fold increase in transcriptional activity seen only when NF45 and NF90 were used together suggests that they may act as a complex. This effect was abolished upon mutation of the Pu box and the difference was statistically significant. These data suggest that NF45 and NF90 complex has the potential to activate SP-10 gene transcription.

Stimulation of Endogenous NF45-NF90 Complex Activates the SP-10 Promoter

Next, we addressed whether stimulation of the endogenous NF45-NF90 complex could activate the SP-10 promoter. The stimulation of Jurkat cells (human T-cell lineage) using PMA + ionomycin is known to induce NF45-NF90–mediated transcription of the IL-2 gene (Kao et al, 1994). Jurkat cells were stimulated by PMA + ionomycin to activate the NF45-NF90 and, 4 hours later, were transfected with an SP-10 promoter–bearing reporter plasmid. The wild-type –186/+28 SP-10 promoter showed a 2.4-fold increase over the Pu-box mutant plasmid (Figure 5), thus indicating the potential of the –186/+28 Pu-box to respond to the activated NF45-NF90 complex in an endogenous context.

View larger version (11K): [in this window] [in a new window]   Figure 5. Stimulation of the endogenous NF45 and NF90 complex supports the SP-10 promoter–driven transcription in a Pu-box–dependent manner. Jurkat cells were treated with PMA and ionomycin to stimulate the NF45 and NF90 complex prior to transfection with SP-10 promoter–based luciferase reporter plasmids. Luciferase values were normalized against those obtained with empty reporter vector (pGL3). The resulting activation of the SP-10 promoter activity was Pu-box–dependent. The difference was statistically significant.

View larger version (18K): [in this window] [in a new window]   Figure 6. The glutamic acid–rich domain of NF45 plays a role in transcriptional activation. (A) The schematic diagrams show the location of the glutamic acid–rich domain at the carboxy terminal of NF45 and the lack of it in EE. The corresponding cDNAs were expressed in the pcDNA 3.1 vector. Western blot with anti-myc antibody confirms their expression and shows the expected difference in the molecular weight of the 2 proteins. (B) Indirect immunofluorescence of Cos7 cells transfected with NF45 and EE constructs. Staining of anti-myc antibody or 4',6'-diamidino-2-phenylindole is shown in the top 2 panels with the accompanying phase contrast image. Both NF45 and EE localized in the nuclei as expected. Magnification 40x. (C) The effect of deletion of the glutamic acid domain was measured by transient transfection in GC2 cells. The wild-type and mutant –186/+28 SP-10 luciferase reporter plasmids were cotransfected with full-length or EE-NF45 in the presence of NF90 expression plasmid. There was a decrease in transcriptional activity as a result of deletion of the Glu domain of NF45. Although the Pu-box mutant also showed a similar decrease, this was to a lesser extent compared to the effect on the wild-type SP-10 promoter. Color figure available online at www.andrologyjournal.org.

View larger version (14K): [in this window] [in a new window]   Figure 7. NF45 and NF90 complex alone is not sufficient for SP-10 promoter activation in the context of native chromatin. (A) Spleen cells from the SP-10 promoter GFP transgenic mouse (Reddi et al, 1999) were stimulated with PMA and ionomycin. Secreted IL-2, measured by ELISA, confirmed the presence of transcriptionally active NF45-NF90 complex. Unstimulated spleen cells did not have detectable level of IL2 (not shown). (B) To determine whether this activated NF45-NF90 complex was sufficient to activate the integrated –408/+28 SP-10-GFP transgene in spleen cells, we measured GFP reporter gene expression by flow cytometry. The straight and dotted lines represent the –408/+28 transgenic and wild-type mice, respectively. No difference in fluorescence intensity was observed between the sorted CD3+ T-cells from the transgenic or wild-type mice, indicating lack of SP-10 promoter activation. This experiment shows that additional factors must act on the chromatin prior to NF45 and NF90 in order to activate the SP-10 promoter.

	Discussion

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The utilization of the mouse SP-10 gene promoter as a model to study spermatid-specific gene transcription has led us to investigate NF45 and NF90 in mouse testis. The present study characterizes the developmental stage- and cell-specific expression patterns of NF45 and NF90 within the murine seminiferous epithelium and shows that together, NF45 and NF90 have the potential to activate SP-10 gene expression.

Previous promoter studies in transgenic mice established that the –186/–91 region of the SP-10 promoter must recruit transcription factors responsible for its activation in round spermatids (Reddi et al, 1999; Acharya et al, 2006). Here, we focused on an evolutionarily conserved Pu-box, -AGAAAA- located at -154 in the mouse SP-10 promoter, which is also conserved in human and monkey SP-10 promoters. Pu-box sequences have been identified in the 5' regulatory regions of many lymphokine genes and in the LTR sequences of HIV (Randak et al, 1990). Pu-box binding proteins belong to the family of nuclear factor of activated T cells (NFAT), which includes the NF45-NF90 complex (Kao et al, 1994) as well as the calcium/calcineurin-dependent NFATc1, NFATc2, NFATc3, and NFATc4 (Rao et al, 1997). A recent bioinformatics study identified the NFAT binding element as an abundant cis-recognition site present in testis-specific gene promoters (Lee et al, 2006). Our immunohistochemical data support the idea that NF45 and NF90 may play regulatory roles during spermatogenesis (Figure 2). Expression within the round spermatids is indicative of a role for NF45 and NF90 in the regulation of gene expression during early spermiogenesis, where the SP-10 gene is expressed. Interaction between recombinant NF45 and the SP-10 promoter in an AGAAAA-site specific manner (Figure 3b) provided the rationale for investigating its functional role in transcription using reporter gene assays. Cotransfection experiments in GC-2 cells provided evidence for the potential of NF45 and NF90 together to activate the –186/+28 SP-10 promoter (Figure 4). The reciprocal approach (ie, stimulation of the endogenous NF45 and NF90 with PMA + ionomycin) also resulted in similar activation of the SP-10 promoter–driven reporter construct (Figure 5). Although the level of transcriptional activation was modest, the increase was consistent whether the endogenous NF45 and NF90 were stimulated or were exogenously provided.

This prompted the experiment addressing the sufficiency of NF45 and NF90 to activate the SP-10 promoter in the context of chromatin. Stimulated NF45 and NF90, however, failed to activate SP-10 promoter–driven GFP expression in the context of chromatin (Figure 7). This can be explained by our previous demonstration that the SP-10 proximal promoter acts as a chromatin insulator in somatic cells preventing SP-10 transcription (Reddi et al, 2003). We predict that the SP-10 promoter region must first be remodeled by chromatin-modifying enzyme activity (yet to be identified), after which NF45 and NF90 can participate in SP-10 gene transcription. In this regard, Hazzouri et al (2000) showed that the treatment of spermatogenic cells in vitro with trichostatin A (histone deacetylase inhibitor) increased histone H4 acetylation in round spermatids. It is possible that the action of unique histone deacetylases at specific stages of spermatogenesis would lead to open chromatin configuration favorable for gene transcription.

It is therefore important to assess the properties of NF45 and NF90 in the appropriate cellular context. The NF45-NF90 complex has been shown to interact with the double-stranded DNA-dependent protein kinase, the translation initiation complex eIF2, and the DNA-binding Ku70 and Ku80 (Ting et al, 1998; Shi et al, 2007). Studying the expression pattern of these interacting proteins during spermatogenesis will provide new insights into the role of NF45 and NF90 in testis.

Our earlier studies identified TDP-43, Pur , and Musashi 2 (Msi2) as factors binding to the SP-10 promoter (Acharya et al, 2006). TDP-43 binds to the SP-10 promoter via 2 GTGTGT motifs located on the opposite strand. Mutation of these sites results in premature expression in the seminiferous epithelium, suggesting a role for TDP-43 in blocking SP-10 expression in spermatocytes. Pur is a sequence specific DNA- and RNA-binding protein with local helix unwinding capacity. Pur ^–/– knockout mice die by 4 weeks of age. Haploinsufficiency of Pur protein has been shown in heterozygous mice (Khalili et al, 2003). Msi2 is a cytoplasmic protein involved in translational repression. Future studies will determine whether any of the above factors functionally interact with NF45-NF90 to affect SP-10 gene expression.

Although it had been established several years ago that haploid germ cells carry out transcription, functional information regarding the actual transcription factors involved has been limited. Gene knockout mouse models have established that sequence specific transcriptional activators CREM and A-myb, as well as components of the general transcription machinery ALF, TLF, TAF7L, and TAF4b, are necessary for the completion of spermatogenesis (Kimmins et al, 2004; DeJong, 2006). CREM and -ACT is a well-characterized activator-coactivator pair known to play a significant role in postmeiotic gene transcription. In CREM knockout mice, spermatid arrest takes place at step 4; however, the SP-10 gene expression is not affected by the absence of CREM. In this regard, the SP-10 gene provides a unique postmeiotic promoter module for the identification of CREM-independent transcription factors.

The characterization of transcription factors such as NF45 and NF90 in the testis and understanding the molecular mechanisms underlying spermatid development are important in light of the use of round spermatids for assisted reproductive technology (round spermatid injection [ROSI]). In fertility clinics, overall fertilization rates achieved with ROSI are lower (45%–50%) compared to intracytoplasmic sperm injection using mature sperm or elongating spermatids (69%–74%) (Levran et al, 2000).

Overall, the study suggests that NF45 and NF90 play important roles in spermatogenesis and that this finding warrants further investigation of their biology in the testis.

	Acknowledgments

 
We thank Dr Peter N. Kao (Stanford University Medical Center, Stanford) for providing the NF90 pcDNA clone and Dr Michael B. Mathews (University of Medicine and Dentistry, New Jersey) for providing both the NF45 antibody and the NF45 pcDNA clone. We thank Harini Bagavant for her help with flow cytometry. We also thank members of our laboratory for their helpful suggestions and discussion.

	Footnotes

 
Supported by NIH HD 36239 (P.P.R.). S.A.R. received the Fogarty International fellowship for part of the duration of the study.

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