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Testis |
a Department of Zoology, University of Hong Kong, Hong Kong, China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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early development, gene regulation, Sertoli cells, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In addition to testosterone and germ cells, the cAMP/protein kinase A (PKA) pathway regulates testin gene expression in the Sertoli cells [9]. In this study, a biphasic change of testin expression was observed following treatment with forskolin, and high concentrations of cAMP analogues rapidly reduced testin mRNA transcript levels. However, this cAMP-induced downregulation of testin gene expression could be abolished by lonidamine, suggesting a possible cross-talk between the PKA signal transduction pathway and lonidamine-induced testin expression. Nevertheless, the molecular mechanism(s) that controls the transcription of the testin gene remains poorly understood. Equally unknown is the expression of the testin gene in other species and the actual biological roles it plays. To address these questions, we cloned the mouse testin cDNA and determined its complete genomic organization. We also isolated the mouse testin 5' flanking region and characterized its proximal promoter region. These studies provide valuable information for future gene knockout experiments aimed at defining the physiological roles of testin and should facilitate our understanding of the transcriptional regulation of the testin gene.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Total RNA was extracted from testes of 20-day-old BALB/c mice using an RNA isolation kit (Stratagene, La Jolla, CA), and poly(A)+ RNA was isolated using the Poly(A)+ Quick mRNA isolation kit (Stratagene) following the manufacturer's suggested protocols. The concentration and quality of the RNA were determined by measuring absorbance at 260 nm and by agarose gel electrophoresis, respectively. A mouse cDNA library was constructed from 5 µg of testis poly(A)+ RNA by the ZAP-cDNA Gigapack II Gold Cloning Kit (Stratagene) according to the suggested protocol. The probe used for library screening was a 267-base pair (bp) mouse testin polymerase chain reaction (PCR) fragment amplified by primers that were designed based on the known rat testin cDNA sequence [6]. The forward primer was 5'-GTGAAAATGATGACAGGCTTTCAAAGG-3' (nucleotides [nt] 217–243), and the reverse primer was 5'-ACCATGGGTAACATTAGATCCCATG-3' (nt 483–459). The PCR was carried out for 30 cycles, with denaturation at 94°C for 1 min, annealing at 63°C for 1 min, and extension at 72°C for 2 min, and a final extension at 72°C for 15 min. Library screening of the full-length mouse testin cDNA was performed as previously described [11]. Clones (2 x 106) were screened by plaque hybridization using the 
-32P-labeled mouse testin cDNA probe. Positive clones were isolated, excised from the ZAP vector, and subjected to Southern blot analysis and nucleotide sequencing (ALF DNA sequencer; Amersham Pharmacia Biotech, Uppsala, Sweden).
RNA Extraction, Reverse Transcription PCR, and Southern Blot Analysis
Total RNA was extracted from adult tissues or primary cultures of Sertoli cells from 20-day-old mice using Trizol Reagent (Life Technologies, Burlington, ON, Canada) and reverse transcription (RT)-PCR was performed using the Access RT-PCR System (Promega, Madison, WI) following the manufacturer's suggested protocols. Primers specific for mouse testin (GenBank accession AY146988) and ß-actin (GenBank accession M12481) genes were designed based on the published sequence. The forward and reverse primers for the mouse testin gene were 5'-ATGATCGCTGTTCTCTTCCTAGCC-3' (nt 107–130) and 5'-CATTGTCATGGTGAAGTCATG-3' (nt 337–317), respectively. The forward and reverse primers for the mouse ß-actin gene were 5'-TCACCGAGGCCCCCCTGAACCCTA-3' (nt 236–259) and 5'-GGCAGTAATCTCCTTCTGCATCCT-3' (nt 879–846), respectively. Amplification was performed with 1 µg first-strand cDNA, 50 pmol each of the testin primers, and 5 pmol each of the ß-actin primers. The cycling parameters for the PCR were essentially the same as described in the previous section. The identities of the PCR products were confirmed by Southern blot analysis. For the Southern blot analysis, the PCR products were separated by agarose gel electrophoresis and then transferred onto a nylon membrane (Hybond-N; Amersham Pharmacia Biotech), followed by hybridization with the corresponding -32P-labeled mouse testin (a 762-bp fragment corresponding to nt 1–762 of the mouse testin cDNA) or ß-actin cDNA probe at 65°C overnight. Radioactivity of the hybridized products was visualized by autoradiography at -80°C with X-OMAT AR films (Eastman Kodak, Rochester, NY).
Cloning and Sequence Analysis of Mouse Testin Gene
Primers corresponding to various positions of the mouse testin cDNA were used to amplify introns using mouse genomic DNA (Clontech Laboratories, Palo Alto, CA) as template. The PCR was carried out for 30 cycles, with denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 2 min, and a final extension at 72°C for 15 min. The PCR products were cloned into pUC18 and sequenced with universal vector primers. The locations of introns 1 and 5 were determined by genome walking from exons 1 and 2, and exons 5 and 6, respectively using the Universal GenomeWalker Kit (Clontech) according to the suggested protocol. Five separate walker libraries were constructed by ligating a specifically designed adaptor to mouse genomic DNA digested with different restriction enzymes. The primers used for walking were 5'-GCTGAAACAGATCTGGAGAACCCA-3' (outer primer, walking from exon 1, nt 17–40), 5'-TGAGGAAATCGTCTGAGGAGCAGC-3' (nested primer, walking from exon 1, nt 62–85), 5'-CCCCTCAAGGTATTCCCAATTATG-3' (outer primer, walking from exon 2, nt 313–290), 5'-CTCAATCATTTTAAAATTCTTTTC-3' (nested primer, walking from exon 2, nt 286–263), 5'-GGTGGCTTCATGCAGAATGCCTTC-3' (outer primer, walking from exon 5, nt 644–667), 5'-AATGGCGGCCTCGCAACTGAGGAA-3' (nested primer, walking from exon 5, nt 683–706), 5'-AACTGCAACAGAGATGGGCCCCAC-3' (outer primer, walking from exon 6, nt 853–830), and 5'-TTGCACAAAATCTCTGACATTAGC-3' (nested primer, walking from exon 6, nt 787–764). Major PCR products were cloned into pUC18 and sequenced. The identities of the deduced intron positions were confirmed by direct PCR amplification of the mouse genomic DNA using corresponding gene-specific primers.
Primer Extension Analysis
Primer extension analysis was performed as previously described [12]. A synthetic oligonucleotide (5'-CTCCTCAGACGATTTCCTCAGGCTT-3', nt 81–57) was end-labeled with [
-32P]ATP using T4 polynucleotide kinase (Life Technologies), and the radiolabeled primer was hybridized with 100 µg of RNA at 42°C overnight. The RNA was then reverse transcribed at 42°C for 2 h with 20 U SuperScript RNaseH reverse transcriptase (Life Technologies), and the reaction was stopped by the addition of ribonuclease A (20 µg/ml). The extended products were purified by phenol-chloroform extraction and analyzed on a 6% polyacrylamide/7.0 M urea gel. A sequencing ladder (A, C, G, T) was generated by the same primer using the T7 Sequencing Kit (Amersham Pharmacia Biotech) for use as a size standard.
5' Rapid Amplification of cDNA Ends
5' Rapid amplification of cDNA ends (5'-RACE) was performed using the Marathon cDNA Amplification Kit (Clontech) according to the manufacturer's suggested protocol. Second-strand cDNA was constructed from 1 µg testis poly(A)+ RNA. An antisense mouse testin exon 5-specific primer (5'-TTGTCCTTCTAGGGATCCAGTTGC-3', nt 556–533) and an antisense exon 3-specific primer (5'-CATTGTCATGGTGAAGTCATGC-3', nt 337–316) were used as the outer and nested primers, respectively. The PCR product was cloned into pUC18 and sequenced.
Preparation of Mouse Testin Promoter-Luciferase Constructs
The 5' flanking region of the mouse testin gene was isolated using the Universal GenomeWalker Kit (Clontech) as described in the previous section. The outer (5'-TGGGTGTCTGCTGCTCCTCA-3', nt 94–75) and nested (5'GGCTTGAGTTTCAGAG3', nt 61–46) primers were designed from the mouse testin exon 1 sequence. Major PCR products were cloned into pUC18 and sequenced. The identities of the 5' flanking sequences were further confirmed by direct PCR amplification of mouse genomic DNA using corresponding gene-specific primers. Forward primers used were 5'-ATACGCGTCCTCCTTCTTCTATATTCTCCA-3' (nt -2253 to -2232), 5'-ATACGCGTGTGCACACTATGACTGTCTTCA3' (nt -864 to -843), 5'-ATACGCGTTGCAGATGACTCATATTTCATG-3' (nt -614 to -593), 5'-ATACGCGTACTCTTAGACATCTATCTGTGT-3' (nt -425 to -404), and 5'-ATACGCGTTATCTTAACTGAAACCACTTTC-3' (nt -290 to -269). The reverse primer used was 5'-ATAGATCTGTTTCAGCTGCATCTC-3' (nt 58–11). Italic sequences represent the introduced MluI and BglII sites. The cycling parameters for the PCR were essentially the same as described in a previous section (Cloning and Sequence Analysis of Mouse Testin Gene). The verified PCR products were cloned into the MluI and BglII sites of the promoterless pGL3-Basic vector. Plasmid DNA for transient transfection was prepared using Plasmid Midi Kits (Qiagen, Chatsworth, CA) following the manufacturer's suggested procedure. The concentration and quality of DNA were determined by measuring absorbance at 260 nm and by agarose gel electrophoresis, respectively.
Cell Culture, Transient Transfection, and Reporter Gene Assay
All culture media and reagents were purchased from Life Technologies. Primary cultures of Sertoli cells were prepared essentially as previously described [5]. Mouse Sertoli cells (MSC-1) were provided by Dr. G.L. Hammond (Department of Obstetrics and Gynecology, University of Western Ontario, London, ON, Canada). Mouse hepatoma Hepa1-6 and mouse embryonic liver TIB-73 cells were obtained from the American Type Culture Collection (Manassas, VA). All these cells were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum (FBS). Cultures were maintained at 37°C in humidified atmosphere of 5% CO2 in air. Cells were passaged or harvested for RNA extraction when they reached about 80% confluence using trypsin/EDTA solution (0.05% trypsin and 0.53 mM EDTA).
Transient transfection was carried out using LIPOFECTAMINE Reagent (Life Technologies) following the manufacturer's suggested protocol. To correct for different transfection efficiencies of various luciferase constructs, the pRL-TK vector was cotransfected into cells with the testin promoter-luciferase construct. Cells (2.5 x 105) were seeded onto six-well tissue culture plates before the day of transfection. Two micrograms of the testin promoter-luciferase construct and 0.03 µg of pRL-TK were cotransfected into cells under serum-free conditions. After 5 h of transfection, 1 ml of medium containing 20% FBS was added, and the cells were incubated overnight (18 h). Following incubation, the old medium was removed and the cells were cultured with normal fresh medium containing 10% FBS for 24 h before harvest. Cellular lysates were collected with 200 µl passive lysis buffer (Promega), and luciferase activities were measured with the Dual-Luciferase Reporter Assay System (Promega) using a Lumat LB 9507 luminometer (E.G&G, Berthold, Germany). Promoter activity was calculated as Firefly luciferase activity/Renilla luciferase activity.
Northern Blot Analysis
Northern blot analysis for mouse testin was performed as previously described [13]. Twenty micrograms of denatured RNA from MSC-1, Hepa1-6, and TIB-73 cells were resolved on a 1% agarose-formaldehyde gel and then transferred onto a Hybond-N nylon membrane (Amersham Pharmacia Biotech), followed by hybridization with an -32P-labeled mouse testin cDNA probe (as used in Southern blot analysis) at 42°C overnight. Radioactivity of the hybridized products was visualized by autoradiography at -80°C with X-OMAT AR films (Eastman Kodak).
In Vitro DNase I Footprinting
In vitro DNase I footprinting was performed essentially as previously described [14]. Regions to be footprinted were amplified by appropriate pairs of primers, of which the sense primers were end-labeled with [-32P]ATP using T4 polynucleotide kinase (Life Technologies). The DNA fragment from nt -425 to -182 was amplified by 5'-ACTCTTAGACATCTATCTGTGT-3' and 5'-ATTGTGTTTGGAGGGTTTATCAAG-3', whereas the fragment from nt -235 to +58 was amplified by 5'-CAGAGAATTATCTGTAAACACTA-3' and 5'-AGATCTGTTTCAGCTGTATCTC-3'. The radiolabeled PCR fragments were analyzed on a 1.5% agarose gel and were purified by the Sephaglas BandPrep Kit (Amersham Pharmacia Biotech). Nuclear extracts were prepared from MSC-1 cells as previously described [15], and protein concentration was measured by the Bradford assay (BioRad Laboratories, Richmond, CA). Approximately 30 000 cpm of end-radiolabeled DNA was digested with 0.45 units DNase I (fast protein liquid chromatography pure; Amersham Pharmacia Biotech) for 3 min at room temperature in the presence of various amounts of nuclear extracts. Following DNase I digestions, the products were treated with proteinase K and purified by phenol/chloroform extraction. The purified products were then analyzed on an 8% urea-acrylamide sequencing gel next to a Maxam-Gilbert sequencing reaction of the corresponding DNA fragments [16]. The resulting gel was examined by autoradiography at -80°C with X-OMAT AR films (Eastman Kodak).
Site-Directed Mutagenesis
Mutagenesis was performed with the Altered Sites II In Vitro Mutagenesis System (Promega) following the manufacturer's suggested protocol. The proximal mouse testin promoter region (nt -425 to +58) was cloned into the pALTER-1 vector, and single-stranded DNA was prepared. The DNA was then annealed to a tetracycline knockout primer, an ampicillin repair primer, and a mutagenic primer. The mutagenic primers used were GATA-a mut: 5'-ATTTTTTTCTT
ATAAACCCTCC-3'; GATA-b mut: 5'-AGCCACGCCAGATATCTTAACT-3'; SRY-a mut: 5'-CTTGATAAACCCTCC
ACACAATGTCAGGAC-3'; SRY-b mut: 5'-AGGATATCTTAACTGACCACTTTCTCTAAA-3'; SRY-c mut: 5'-GTCCAACCTTAGCTTACGAGAGACACTGCA-3'; and SF-1 mut: 5'-TGTCAGGACTCAA
TCTCAAGGATGAA3'. Mutated nucleotides are underlined. The mutated strand was synthesized and ligated with T4 DNA polymerase and T4 DNA ligase. The mutant vector was transformed into ES1301 mutS cells, and the plasmid DNA was extracted and then transformed into JM109. Desired mutations were confirmed by DNA sequence analysis.
Data Analysis
The nucleotide sequence of the mouse testin 5' flanking region was generated by three independent amplifications from either adaptor-ligated genomic DNA fragments or mouse genomic DNA and at least five separate sequencing reactions. For all transfection assays, data are shown as the mean ± SEM of triplicate assays in three independent experiments. For the mutational study, data were analyzed by one-way ANOVA followed by Tukey multiple comparison tests using the computer software PRISM (GraphPad Software, San Diego, CA). Differences were considered significant at P < 0.05. For the primer extension analysis, 5'-RACE, and in vitro DNase I footprinting, all experiments were performed at least twice, and consistent results were obtained between experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A full-length mouse testin cDNA of 1390 bp was isolated by screening a mouse testis cDNA library. The cDNA contains a 5' untranslational region of 106 bp, an open reading frame encoding a 333-amino acid protein, and a 3' untranslational region of 282 bp. A termination codon TGA is located 240 bp upstream of the polyadenylation signal (5'-AATAAA-3') (Fig. 1A ) . Comparison of the entire coding region showed that mouse testin shared 90.1%, 58.9%, 62.2%, and 64.6% identity with rat testin [6] and cathepsin L of mouse [17], rat [18], and human [17, 19], respectively, at the amino acid level (Fig. 1B). The deduced amino acid sequence revealed that mouse testin possesses a 16-amino acid signal peptide, and the positions of all seven cysteine residues and the potential N-linked glycosylation site are conserved with respect to those present in rat testin. As in the rat, the cysteine residue in the predicted active site of the cathepsin L is replaced with serine in mouse testin.
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RT-PCR and Southern blot analysis confirmed that mouse testin mRNA transcripts are present in primary cultured Sertoli cells and revealed that the gene is predominantly expressed in the testis and ovary, although much lower expression levels of its transcripts were also detected in the spleen, epididymis, kidney, and uterus. No signal was obtained from the brain, liver, small intestine, and the negative control, which contained no cDNA template (Fig. 2).
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Genomic Organization of Mouse Testin Gene
Because repeated attempts to clone the mouse testin gene by genomic library screening were not successful, we cloned this gene by direct PCR amplification of the mouse genomic DNA with different pairs of primers designed from the known mouse testin cDNA sequence. However, because we failed to locate introns 1 and 5 by this conventional PCR method (probably because of their large sizes), we identified their positions by genome walking. A schematic diagram of the genomic organization of the mouse testin gene is shown in Figure 3A. The nucleotide sequence of the mouse testin gene was established by overlapping genomic fragments. The gene spans over 20 kilobases (kb) and contains eight exons with sizes ranging from 95 to 365 bp, whereas introns are 93 bp to >9 kb in length. The exact sizes of the exons, introns (except intron 1, which was partially sequenced), and exon-intron boundaries were determined by DNA sequencing. All exon-intron boundaries revealed no deviation from the consensus 5' donor and 3' acceptor sites [20] and followed the GT/AG rule (Fig. 3B). Except exon 1, which lies within the 5' untranslated region, all other exons are located within the coding region. The splice junctions in exons 6 and 7 occur after the first (type 1) and second (type 2) nucleotide of the amino acid codon, respectively, whereas those in exons 2 to 5 occur between codons (type 0). Exon 1 and the first 11 bp of exon 2 make up the entire 5' untranslated region, whereas exon 2 contains the translation initiation codon ATG and sequences encoding the signal peptide. Exon 8 contains the translation termination codon TGA and the 3' untranslated region where the polyadenylation signal (AATAAA) is located. Analysis of the available intron 1 sequence of the mouse testin gene by BLAST (Basic Local Alignment Search Tool) revealed that this intron contains a 3.1-kb region that shares 97% identity with the Mus musculus domesticus L1 retrotransposon.
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Transcription Start Sites for the Mouse Testin Gene
To identify the transcription start sites for the mouse testin gene, a primer extension analysis was performed using an oligonucleotide derived from the 5' untranslated region. Three extension products were obtained from the mouse testis RNA but not from the negative control (yeast tRNA) (Fig. 4A). The deduced positions of these transcription start sites were 134, 131, and 129 bp upstream from the translation start codon. Because of its stronger intensity, the most upstream site was defined as the major transcription site and designated as +1. To confirm the results obtained from primer extension analysis, a 5'-RACE with cDNA constructed from mouse testis poly(A)+ RNA was also performed. A single PCR product was obtained from the nested PCR (Fig. 4B), and sequence analysis of this RACE product revealed that the 5' ends of the testin transcripts were situated at locations consistent with those determined by primer extension analysis.
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Analysis of the Mouse Testin 5' Flanking Region
PCR amplification of mouse genomic DNA revealed a 2.3-kb mouse testin 5' flanking region (Fig. 5). Nucleotide sequencing showed that this region lacks canonical TATA and CAAT boxes and has low GC content (39%). A putative initiator (Inr) element (5'-TTACTCT-3') is located between nt -49 and -43. Computer-aided analysis of the 5' flanking region of the gene revealed a number of putative binding sites for various transcription factors, including AP-4, E2A, activating protein 1 (AP-1), Ets, octamer transcription factor 1 (Oct-1), cAMP-responsive element binding protein (CREB), estrogen receptor (ER), GATA, sex-determining factor (SRY), and steroidogenic factor 1 (SF-1).
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Mapping of the Mouse Testin Promoter in the Sertoli Cells
To localize the active promoter regions, progressive 5' deletion mutants were constructed and analyzed in MSC-1 cells (Fig. 6). The maximal promoter activity was located at a region between nt -425 and +58 (9.4-fold increase in activity vs. pGL3-Basic) in the Sertoli cells. However, this activity reduced drastically (1.8-fold vs. pGL3-Basic) when the fragment was cloned in a reverse orientation, suggesting that this promoter segment functions in a unidirectional manner. A similar result was also obtained for a larger promoter fragment (nt -614 to +58). Inclusion of upstream sequences (nt -614 to -864) reduced the promoter activity of p(-425/+58)-Luc by 66% in the Sertoli cells, indicating that negative regulatory elements are present in this distal region of the 5' flanking sequence. In contrast, the activities of the construct p(-425/+58)-Luc (and other constructs whose activities were not shown) were relatively much weaker in two mouse liver cell lines, Hepa1-6 (1.7-fold vs. pGL3-Basic) and TIB-73 (2.3-fold vs. pGL3-Basic) (Fig. 7A). These results were in agreement with those of the Northern blot analysis, which showed that testin mRNA transcripts were present in MSC-1 cells but not in the liver cells (Fig. 7B).
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Localization of DNA-Protein Interactions Within the Mouse Testin Proximal Promoter
To localize regions of DNA-protein interactions within the mouse testin proximal promoter region (nt -425 to +58), in vitro DNase I footprinting was performed with two end-radiolabeled overlapping DNA fragments (R1: nt -425 to -182; R2: nt -235 to +58) using nuclear extracts from MSC-1 cells. No footprint was observed from fragment R1 (data not shown), whereas three footprints (designated as FP-1, -2, and -3) were obtained from fragment R2 (Fig. 8A). FP-1 contains a putative SF-1 binding site (5'-CAAGGTCT-3', nt -172 to -165) that shares 88% identity with the consensus sequence [21]. FP-2 and FP-3 contain a putative SRY binding site (SRY-a; 5'-CAAACA-3', nt -191 to -186) and GATA binding site (GATA-a; 5'-TGATAA-3', nt -203 to -198) that share 100% identity with their corresponding consensus binding motifs [21, 22] (Fig. 8B).
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To assess the functional significance of these binding sites in regulating mouse testin gene transcription in the Sertoli cells, site-directed mutants were constructed and transiently transfected into the MSC-1 cells (Fig. 9). A point mutation (from GATA to CATA) of the GATA-a motif within FP-3 caused an 80% reduction of promoter activity of the construct p(-425/+58)-Luc. However, mutation of another putative GATA binding site (GATA-b; nt -294 to -289) within the proximal promoter did not alter the promoter activity significantly. Mutation of the SRY-a binding motif from CAAACA to CTCACA within FP-2 but not other putative SRY sites (SRY-b and SRY-c; nt -281 to -276 and nt -339 to -334, respectively) reduced the promoter activity by about 65%. Alteration of the putative SF-1 binding site from CAAGGTCT to CAATTTCT within FP-1 produced a 50% reduction of promoter activity. These results clearly indicate the functional importance of the putative GATA, SRY, and SF-1 binding motifs within the footprint regions in mouse testin gene transcription in Sertoli cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present results demonstrated that the transcription of the mouse testin gene is initiated from a major start site located 134 bp upstream of the translation start codon. However, we cannot exclude the possibility that the other minor start sites could be used predominantly under certain physiological conditions during different developmental stages. Sequence analysis of the mouse testin 5' flanking region indicated that the gene lacks canonical TATA and CAAT boxes. Promoters lacking a TATA box fall into two classes [27]. One class of these promoters is GC rich and is found primarily in housekeeping genes. These promoters usually contain several transcription start sites and potential binding sites for the transcription factor Sp1 [28]. The other class of TATA-less promoters is not GC rich and is regulated during differentiation or development. In addition, this type of promoter initiates gene transcription at one or a few tightly clustered sites and has been reported in the Drosophila homeotic genes [29, 30] and acrosin gene [31]. Because of its low GC content (39%), the mouse testin promoter appears to belong to the class of TATA-less promoters. Because it has been suggested that TATA-binding protein-associated factors are capable of binding Inr elements [32–34], we speculate that the putative Inr element located about 40 bp upstream of the major transcription start site may function as a TATA box to accurately direct the transcription of the testin gene. In sharp contrast to the mouse testin gene, the 5' flanking region of the rat cathepsin L gene contains a canonical CAAT box and multiple Sp1 binding sites [24]. This observation suggests that the transcriptional regulation of the cathepsin L gene is different from that of the mouse testin gene, although these genes share a high level of sequence homology at the amino acid level.
Analysis of the mouse testin 5' flanking region revealed a number of putative binding sites for different transcription factors (Fig. 5). The presence of a putative cAMP-responsive element within the proximal promoter region may help explain an earlier finding that the expression of testin mRNA transcripts was regulated by the cAMP/PKA signal transduction pathway [9]. Additionally, the identification of a number of consensus E-box binding sites within the 5' flanking region may have implications in testosterone regulation of testin expression [1–3]. Evidence supporting this idea comes from an earlier finding that the interaction of the transcription factors Myc and Max with a consensus E-box site was required for androgen-mediated upregulation of androgen receptor transcription [35]. Therefore, testosterone may regulate testin gene transcription via the putative E-box binding motifs within its 5' flanking region, which does not contain any consensus androgen-responsive element. The complexity of the mouse testin 5' flanking region is further exemplified by the presence of several putative binding sequences for ER and AP-1, suggesting that testin gene expression is potentially regulated by estrogen and the protein kinase C signaling cascade.
Because the mouse testin 5' flanking region contains 16 binding sites for the liver-specific transcription factor HNF-3ß (data not shown), we analyzed the testin promoter constructs in two mouse liver cell lines, Hepa1-6 and TIB-73. Results from both reporter gene assays and Northern blot analysis confirmed that the testin gene is transcriptionally inactive in these liver cells (Fig. 7), a finding consistent with those of our tissue distribution studies (Fig. 2). In vitro DNase I footprinting and mutational analysis demonstrated that three closely spaced putative GATA, SRY, and SF-1 binding motifs (nt -203 to -165) were indispensable for the basal activity of the mouse testin promoter. However, an additional cis-acting element(s) located between nt -425 and -290 are believed to play some roles in regulating testin gene transcription in the Sertoli cells because a 1.8-fold reduction of promoter activity was observed when this region was deleted (Fig. 6). Nevertheless, the presence of any functional regulatory element(s) within this region awaits further analysis; in the present study we failed to detect any DNA-protein interaction from this region.
The roles of GATA, SRY, and SF-1 binding motifs in regulating gene transcription in testicular Sertoli cells have been reported. For instance, GATA-1 is required for the Sertoli cell-specific transcription of the FSH receptor (FSHR) gene [36], whereas GATA-1 and GATA-4 have been shown to transactivate the inhibin/activin beta B subunit promoter in MSC-1 cells [37]. On the other hand, SF-1 was recently shown to regulate the murine FSHR gene expression in MSC-1 cells via interaction with a noncanonical SF-1 binding site [38]. Similar to the mouse testin gene, three closely spaced GATA, SRY, and SF-1 binding sites are also present in the proximal promoters of the Müllerian-inhibiting substance (MIS) genes [39–41] (Fig. 10). These genes encode a glycoprotein that is secreted by fetal Sertoli cells during mammalian male sexual differentiation to initiate the regression of the Müllerian duct. Nevertheless, whether testin and MIS genes are regulated by similar mechanisms at the promoter level awaits further investigation. The identification of a functional putative SRY binding site in the mouse testin proximal promoter suggests that testin may be a downstream target of SRY. This suggestion is consistent with our recent finding of drastic and transient expression of mouse testin at the early stage of fetal development [unpublished data]. Because SRY functions as a genetic switch in the gonadal ridge to initiate testis differentiation, testin may be one of the novel genes involved in the testicular determination pathway and may play an essential role in the development of the testis during embryogenesis.
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In summary, we have isolated the mouse testin cDNA and elucidated its genomic organization. This fundamental information is useful for future gene knockout studies aimed at determining the physiological roles of testin in the testis and other organs. Also, preliminary characterization of the mouse testin 5' flanking region reveals the concomitant requirement of similar cis-acting DNA elements for the transcriptions of the testin and MIS genes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 852 25599114; hrszlwm{at}hku.hk
Received: 10 September 2002.
First decision: 1 October 2002.
Accepted: 30 October 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2-macroglobulin, and testins: biosynthesis and differential regulation by germ cells. Mol Cell Endocrinol 1992 89:127-140[CrossRef][Medline]This article has been cited by other articles:
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  R. S. Viger, S. M. Guittot, M. Anttonen, D. B. Wilson, and M. Heikinheimo Role of the GATA Family of Transcription Factors in Endocrine Development, Function, and Disease Mol. Endocrinol., April 1, 2008; 22(4): 781 - 798. [Abstract] [Full Text] [PDF]
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 N. P Y Lee, D. D Mruk, W. Xia, and C Y. Cheng Cellular localization of sphingomyelin synthase 2 in the seminiferous epithelium of adult rat testes J. Endocrinol., January 1, 2007; 192(1): 17 - 32. [Abstract] [Full Text] [PDF]
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 K.-M. Ng, M.-T. So, and W. M. Lee Expression of Rabbit Sex Hormone-Binding Globulin during Pregnancy and Prenatal Development and Identification of a Novel Isoform Endocrinology, April 1, 2005; 146(4): 1965 - 1972. [Abstract] [Full Text] [PDF]
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ATA to 
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TCT to CAA