©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
An E Box Element Is Required for the Expression of the ad4bp Gene, a Mammalian Homologue of ftz-f1 Gene, Which Is Essential for Adrenal and Gonadal Development (*)

(Received for publication, December 7, 1994; and in revised form, January 17, 1995)

Masatoshi Nomura (1) Stephan Bärtsch (1)(§) Hajime Nawata (2) Tsuneo Omura (1) Ken-ichirou Morohashi (1)(¶)

From the  (1)Department of Molecular Biology, Graduate School of Medical Science and (2)The Third Department of Internal Medicine, School of Medicine, Kyushu University, Higashi-ku, Fukuoka 812, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Ad4BP, also known as SF-1, is a cell type-specific transcription factor regulating all the steroidogenic P-450 genes. Recently, the targeted disruption of the mouse ftz-f1 gene encoding Ad4BP/SF-1 has established its essential function in both adrenal and gonadal development and sexual differentiation. As an initial step toward understanding its role in the cascade of gene activations necessary for the differentiation of the steroidogenic tissues and the sex differentiation of the gonads, we isolated and characterized the rat ad4bp gene. A sequence analysis of the ad4bp gene revealed that another nuclear factor ELP was also transcribed from the same gene by alternative promoter usage and splicing. The promoter of the ad4bp gene showed activities in the steroidogenic cells such as Y-1 adrenocortical cells and I-10 testicular Leydig cells when examined by transient transfection assays. Using deletion analysis and site-directed mutagenesis, we identified a cis-element at the position from -82 bp to -77 bp in the 5`-upstream region. The cis-element was identical to the consensus E box element, which is the binding site for the basic-helix-loop-helix proteins. Gel mobility shift analyses revealed the amount of a binding factor to this E box in the nuclear extract prepared from the rat testes attained a maximal level 1 week after birth and then decreased dramatically thereafter, and only trace amounts were detected in adult rats. In contrast, the binding factor in the ovaries attained a maximal level just after birth and kept its level thereafter. These dimorphic expressions of the binding factor to the E box correlated well with those of Ad4BP, and thus suggested that the expression of Ad4BP is transcriptionally regulated through this E box element.


INTRODUCTION

Steroid hormones play an important role in the maintenance of body homeostasis and sexual differentiation. The biosynthesis of steroid hormones requires six distinct forms of cytochrome P-450, cholesterol side chain cleavage P-450, 21-hydroxylase P-450, 17alpha-hydroxylase P-450, 11beta-hydroxylase P-450, aldosterone synthase P-450, and aromatase P-450(1) . The expression of these steroidogenic P-450 genes is tissue-specific and regulated by peptide hormones secreted from the pituitary gland, such as adrenocorticotropic hormone, luteinizing hormone, and follicle-stimulating hormone(2, 3, 4) . These peptide hormones bind to their receptors on the target cell membrane, resulting in the increase of the intracellular concentration of cAMP as a second messenger. Our initial studies were focused on the mechanisms of the cAMP-dependent gene regulation and the steroidogenic cell-specific gene regulation of a bovine 11beta-hydroxylase P-450 gene (cyp11b). Seven cis-elements, Ad1, Ad2, Ad3, Ad4, Ad5, Ad6, and Ad7, were identified in the promoter region(5, 6) . Interestingly, among these elements, the Ad4 site was present in the promoter regions of all the steroidogenic P-450 genes and was found to be essential for their full response to cAMP(7) . Next, we purified a binding protein to the Ad4 site, Ad4BP, also designated as SF-1(8) , from bovine adrenal cortex nuclear extract and isolated the corresponding cDNA(7, 9) . The nucleotide sequence of the cDNA revealed that Ad4BP had Zn-finger motifs for the DNA-binding domain and was a new member of the steroid hormone/thyroid hormone receptor supergene family(10) . A functional study using an Ad4BP expression vector confirmed that this transcription factor governs the tissue-specific expression of the steroidogenic P-450 genes(11) .

We investigated the expression of Ad4BP in the steroidogenic tissues including the adrenal glands, testes, and ovaries throughout the prenatal and postnatal life of the rats by immunohistochemical techniques using the antiserum specific to Ad4BP(12) . Consistent with its role in regulating steroidogenic P-450s, Ad4BP was expressed in the cells of all three zones of the adrenal cortex, the granulosa, and theca cells of the ovary, and the Leydig cells of the testis in adult rats. Remarkably, Ad4BP was expressed even in the primordial cells of the adrenal glands and gonads of the 13.5-day postcoitum fetal rats. In the fetal gonads, a significant amount of Ad4BP was expressed in the somatic cells of the testicular Sertoli cells and Leydig cells, whereas only a trace amount was expressed in the ovaries. Drastic alterations in the amount of Ad4BP occurred during the first to the third week after birth. The expression of Ad4BP in the testes attained a maximal level 1 week after birth and decreased markedly thereafter, whereas that in the ovaries increased after the first week. A similar observation in the change of the expression of SF-1 mRNA was reported by Shen et al.(13) . It is generally accepted that sexual differentiation is initiated by the transient expression of SRY in the somatic cells in the urogenital ridge(14, 15, 16) . The sexually dimorphic expression of Ad4BP observed in the gonads just after sexual differentiation suggested that the ad4bp gene might be one of the genes located downstream to SRY. The expression profile of Ad4BP in the Sertoli cells of fetal rats showed a coincident pattern with that of the Müllerian inhibitory substance which causes Müllerian duct regression in developing males(17, 18, 19) . In fact, recent studies have indicated that Ad4BP is involved in the regulation of the Müllerian inhibitory substance gene(12, 13) . These findings suggested that Ad4BP regulates the genes essential for gonadal development and sexual differentiation in addition to the steroidogenic P-450 genes. Such possible functions of Ad4BP were also strongly supported by the targeted disruption of the gene(20) . A transcriptional analysis of the ad4bp gene, a mammalian homologue of the Drosophilaftz-f1 gene, will pave the way to the identification of the genes involved in the cascade of gene activations necessary for the differentiation of the steroidogenic tissue and the sex differentiation of the gonads. As an initial step toward this goal, the rat ad4bp gene was isolated and characterized.


EXPERIMENTAL PROCEDURES

Construction of the Rat Genomic Library and Cloning Procedures

Total DNA was prepared from the liver of a single male Sprague-Dawley rat weighing 280 g as described by Sambrook et al.(21) , and a genomic library was constructed in a cosmid vector as described before(22) . The library, containing 3 times 10^5 colonies, was screened using the bovine Ad4BP cDNA as a probe which contained a nucleotide sequence for the whole protein coding region (1.4 kb) (^1)followed by a portion of the 3`-untranslated region (0.3 kb), and three positive clones were obtained. The recombinant DNAs prepared from the positive clones were digested with various restriction enzymes to create the restriction enzyme maps. The digests were then separated by agarose gel electrophoresis, and blotted onto a nitrocellulose filter. The blot was hybridized with the bovine Ad4BP cDNA and a 5`-untranslated region generated from rat adrenal total RNA by a 5`-rapid amplification of cDNA ends (5`-RACE) as the probes. A 5`-RACE was carried out with the reverse primer, 5`-GAGCTGCAAAATCGACAAGAC-3`, corresponding to the sequence from the third nucleotide of the 53rd amino acid to the second nucleotide of the 60th amino acid of rat Ad4BP, to prime the reverse transcriptase reaction using the 5`-RACE System for Rapid Amplification of cDNA Ends (Life Technologies, Inc., Grand Island, NY). The restriction fragments from the isolated genomic clones were subcloned into pUC vectors. Nucleotide sequences were determined by the dideoxy chain termination method using a Sequenase kit (U. S. Biochemical Corp.).

PCR Amplification of RNA

Complementary DNAs were synthesized with 1 µg each of the total RNAs prepared from the rat adrenal glands, testes, and ovaries in a 10-µl reaction mixture containing 50 mM Tris-HCl (pH 8.6), 40 mM KCl, 1 mM MgCl(2), 1 mM dithiothreitol, 0.5 mM dNTPs, 150 ng of random hexanucleotides, and 200 units of Molony murine leukemia virus reverse transcriptase (U. S. Biochemical Corp.). After incubation at 37 °C for 1 h, the reaction mixtures were boiled for 5 min and chilled on ice, then added to a 90-µl PCR mixture containing 50 mM KCl, 10 mM Tris-HCl (pH 8.8), 1.5 mM MgCl(2), 0.1% Triton X-100, 200 µM dNTPs, and 400 ng each of the primers described below. PCR was carried out for 30 cycles (92 °C, 1 min; 58 °C, 2 min; 72 °C, 1.5 min) with Taq DNA polymerase (Takara Shuzo Co., Ltd., Kyoto, Japan). 19-mer oligonucleotides, 5`-GACCAGATGACACTGCTGC-3` corresponding to the sequence from the first nucleotide of the 371st amino acid to the first nucleotide of the 377th amino acid, and 5`-ATTCAGGGACATGCACTAG-3` corresponding to the sequence from the third nucleotide of the 428th amino acid to the third nucleotide of the termination codon of rat ELP, were synthesized and used as the primers. After the reaction, the products were subcloned into pUC vectors and used to determine the nucleotide sequences.

S1 Nuclease Protection Assay

S1 nuclease protection assays were carried out as described previously(23) . In brief, adrenal poly(A) RNA was prepared from 8-week-old male Sprague-Dawley rats. The probe for the 5`-end mRNA coding for Ad4BP was prepared by kination with [-P]ATP (222 TBq/mmol, Amersham, United Kingdom) at the EcoRI site of the genomic Eco81I (-264)-EcoRI (+136) fragment (400 bp) containing the first exon and the 5`-flanking region. After 20 µg of the poly(A) RNA had been hybridized with the probe the mixture was treated with 80 units of S1 nuclease at 37 °C for 30 min. The S1 nuclease-resistant products were analyzed on a 6% denaturing polyacrylamide gel followed by autoradiography. As a marker, the DNA fragment used as the probe was chemically modified by a method previously described (24) and electrophoresed on the same gel.

Plasmid Constructions for Transient Transfection

The 4.8-kb long fragment containing the 5`-upstream region from the EcoRI site located in the first exon of ad4bp gene was ligated with the CAT structural gene of pSV00CAT (25) to produce Ad4CAT4.8K. Various recombinant plasmids were constructed from Ad4CAT4.8K as shown in Fig. 5A. For the construction of the recombinant plasmids, Ad4CAT2.0K, Ad4CAT1.2K, Ad4CAT0.8K, Ad4CAT0.4K, and Ad4CAT92, we used restriction endonuclease sites in the upstream region, EcoRI site at 2.0 kb, HindIII site at 1.2 kb, BamHI site at 0.8 kb, Eco81I site at 264 bp, and StuI site at 92 bp upstream from the transcription initiation site, respectively. Bal31 nuclease digestion was performed from the StuI site to produce the deletion mutants, Ad4CAT60 and Ad4CAT26, which have the 60- and 26-bp upstream, respectively. The 2.6-kb long XbaI-XbaI fragment containing the 5`-upstream region from the XbaI site located in the first exon of ELP gene was ligated with the CAT structural gene of pSV00CAT to produce ELPCAT2.6K. For the construction of the recombinant plasmids, ELPCAT1.4K, ELPCAT0.8K, and ELPCAT0.4K, we used restriction endonuclease sites, HindIII site at 1.4 kb, SmaI site at 0.85 kb, and SacI site at 0.45 kb upstream from the XbaI site, respectively.


Figure 5: Promoter activities of the ad4bp gene. A, transient transfection assays with a series of the deletion mutant plasmids carrying the promoter regions of ad4bp and elp using Y-1 adrenocortical cells. The construction of the recombinant plasmids are indicated below the map of the ad4bp gene. The plasmids and the mock-expression plasmid, pSV00CAT, were transfected into the Y-1 cells and then the CAT assays were performed as described under ``Experimental Procedures.'' The autoradiographs of the CAT products are shown. A closed, open, and shaded box indicate the ad4bp first exon, elp first exon, and shared exons, respectively. The separated two XbaI sites, connected with a broken line shown in the map, are identical sites in the gene. B, cell type-specific expression of ad4bp gene promoter. Transient transfection assays with Ad4CAT plasmids were performed using I-10 cells and CV-1 cells in addition to Y-1 cells. The relative CAT activities shown in the figure indicate the percentages of the CAT activities to that of Ad4CAT92. In the case of CV-1 cells, the CAT activities are shown relative to the CAT activity of pSV00CAT in I-10 cells. The closed, hatched, and open bars indicate the results with Y-1, I-10, and CV-1 cells, respectively.



Site-directed Mutagenesis

Site-directed mutagenesis within the ad4bp gene 5`-upstream sequence was performed by the method of Kunkel et al.(26) . The 0.9-kb long BamHI-EcoRI fragment containing the first exon and the 5`-flanking region was subcloned into the BamHI/EcoRI sites of the pUC119 vector. Single strand DNA was used as the template for the mutations. The oligonucleotides containing the mutations were synthesized and used as primers. All mutant clones were selected and confirmed by DNA sequencing, and the mutated fragments were subcloned back to the Ad4CAT0.8K plasmid.

Cell Culture and Transient Transfection

Y-1 cells, I-10 cells, and CV-1 cells were supplied by Japanese Cell Research Bank (Tokyo, Japan). Y-1 cells and CV-1 cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Whittaker, Walkersville, MD) at 37 °C in a humidified atmosphere containing 10% CO(2) and 90% air. I-10 cells were maintained in Ham's F-10 medium (Life Technologies, Inc.) supplemented with 2.5% fetal bovine serum and 15% horse serum (Hazleton Biologics, Inc., Lenexa, KS) at 37 °C in a humidified atmosphere containing 5% CO(2) and 95% air. Transient transfections by the lipofection method were carried out when cultured cells reached 80% confluency in 60-mm dish. Three µg of the recombinant plasmids and 0.2 µg of RSV/luciferase were introduced into the cultured cells. The efficiencies of the transfection were normalized by luciferase activities derived from the luciferase expression vector, RSV/luciferase, as described previously(11) . The cells were harvested 42 h after the lipofection, and CAT assays were performed (27) using 1-deoxy-[dichloroacetyl-1-^14C]chloramphenicol (56 mCi/mmol, Amersham). All transfection experiments were performed at least 3 times.

Gel Mobility Shift Assays

Double stranded oligonucleotides, dEbox, containing the E box element (5`-gTGCAGAGTCACGTGGGGGCAGAG-3`/3`-ACGTCTCAGTGCACCCCCGTCTCg-5`) and dAd4, containing the consensus Ad4 site (5`-ggACATACCCAAGGTCCCCTTT-3`/3`-TGTATGGGTTCCAGGGGAAAgg-5`) were used as probes. One or two nucleotides (g) were added to the 5` end of the synthetic nucleotides and used for labeling reaction with Klenow fragment and [alpha-P]dCTP (110TBq/mmol, Amersham). Nuclear extracts were prepared from various rat tissues according to the method previously described(28) . For the competition assays, 25 or 50-fold molar excess of the nonradiolabeled double stranded 24- or 20-mer nucleotides, dEbox or dM1-Ebox (5`-CTGCAGAGTCTGTAGGGGGCAGAG-3`/3`-GACGTCTCAGACATCCCCCGTCTC-5`), dM2-Ebox (5`-gCAGAGTATCGTGGGGGCAG-3`/ 3`-GTCTCATAGCACCCCCGTCg-5`), dM3-Ebox (5`-gCAGAGTCACGATGGGGCAG-3`/3`-GTCTCAGTGCTACCCCGTCg-5`), or dM4-Ebox (5`-gCAGAGTCACCTGGGGGCAG-3`/3`-GTCTCAGTGGACCCCCGTCg-5`) were added prior to the addition of the probe. The dM1, dM2, dM3, and dM4-Ebox's have various nucleotide substitutions in the consensus Ebox sequence emphasized with underlines. Double stranded 20-mer oligonucleotides, dM2, dM3, and dM4-Ebox, were also labeled with [alpha-P]dCTP and used as probes. The procedure for the gel mobility shift assay has been previously described(7) .


RESULTS

Ad4BP and ELP Are Generated from a Single Structural Gene

We constructed a rat genomic library using a cosmid vector and screened it with a bovine Ad4BP cDNA as the probe(5) . Three positive clones were obtained and analyzed by restriction mapping. Since the insert DNAs of the cosmid clones were too long to characterize, the restriction fragments of the clones with appropriate lengths were subcloned into pUC vectors for restriction mapping and DNA sequencing. The restriction endonuclease maps of these three clones showed that they were derived from the same gene. A comparison of the nucleotide sequences between the bovine cDNA and the rat gene revealed that the Ad4BP cDNA had a 5`-untranslated region which was encoded by the distinct exon. Since the bovine cDNA did not contain a sufficient length of the 5`-untranslated sequence to use as a probe for the mapping of the exon, another cDNA clone carrying a longer 5`-upstream was isolated from a rat adrenal cDNA library prepared from the 5`-RACE products. As shown in Fig. 1, the ad4bp gene consists of 7 exons divided by 6 introns and spans about 15 kb. Exon-intron junctions were identified by comparison with the bovine cDNA sequence. All exon-intron junctions follow the ``GT-AG rule'' for the splice donor and acceptor. The nucleotide sequence revealed that the gene encoding Ad4BP also encoded a rat homologue of ELP, which was cloned as a murine homologue of Drosophila FTZ-F1 (29) from the undifferentiated murine embryonal carcinoma cells (EC cells)(30) .


Figure 1: Restriction enzyme map of the rat ad4bp gene. The closed boxes with numbers indicate the locations and sizes of the rat ad4bp gene exons. The shaded boxes indicate those of ELP. The initiation methionines and termination codons for Ad4BP and ELP are also indicated. Since the precise transcription initiation site for ELP has not been defined, the putative first exon of ELP is shown according to the structure of ELP cDNA(30) .



The transcription initiation site for Ad4BP in the adrenal glands of adult rats was determined by an S1 nuclease protection assay as shown in Fig. 2. By comparison with the cDNAs isolated by the 5`-RACE using total RNAs from adrenal glands, testes, and ovaries of adult rats, the transcription initiation sites were mapped around the adenosine nucleotide as indicated by an arrow in Fig. 2. Since the precise transcription initiation site for ELP had not been defined, we performed an S1 nuclease protection assay with the elp upstream region. However, the transcription initiation site was not determined precisely, because of the appearance of several protected signals; one major and several notable signals (data not shown).


Figure 2: Determination of the transcription initiation site of the ad4bp gene by an S1 nuclease protection assay and 5`-RACE. An Eco81I/EcoRI fragment containing the first exon and the 5`-flanking region was P-end labeled by kination at the EcoRI site and used as a probe. After the probe was hybridized with adrenal poly(A) RNA, the mixture was digested with S1 nuclease as described under ``Experimental Procedures.'' To estimate the 5` nucleotide of the DNA fragment protected from S1 nuclease digestion, a marker was prepared using the same end-labeled probe by a modification according to Maxam and Gilbert(24) . The nucleotide sequence including the protected signals is shown. The arrowheads indicate the transcription initiation sites, and the sizes of the arrowhead correspond to the strengths of the protected bands. Almost all the cDNAs generated by the 5`-RACE using the total RNA prepared from the rat adrenal glands, testes, and ovaries started at the A as indicated by an arrow.



As shown in Fig. 3and schematically summarized in Fig. 1, ad4bp is transcribed from a further 5`-upstream promoter than elp and has a noncoding first exon. A TATA box was not found in the 5`-upstream region of the ad4bp gene. elp is transcribed from the downstream promoter within the ad4bp first intron and 77 more amino acid residues are translated preceding the Ad4BP initiation methionine. Using the 3`-splice acceptor site of the ad4bp first intron just upstream the Ad4BP initiation methionine, the shared exons are generated. An Ad4BP translation starts at the internal methionine in the elp first intron, and only 12 amino acid residues corresponding to the A and B regions precede region I, which is also called the C region(31, 32) . The second and third exons encode region I which contains two zinc finger motifs. The fourth exon encodes the long D region containing the proline-rich region between regions I and II. Region II is encoded by the fourth and fifth exons. Using the 5`-splice donor site of the ad4bp fifth intron, two additional ad4bp-specific exons, the sixth and seventh exons, are generated, by which region III is encoded. On the other hand, the splicing at this site does not occur in the case of elp, and a termination codon appears after the 24 amino acid residues. The rat elp gene has a 22-nucleotide deletion when compared with the mouse gene(30, 33) , which causes a frameshift resulting in the shift of the termination site to further upstream as shown in Fig. 3and Fig. 4.


Figure 3: Nucleotide sequence of the ad4bp gene and the deduced amino acid sequence. The nucleotide sequence of the rat ad4bp gene and the deduced amino acid sequence are shown. The nucleotide sequences of the exons of ad4bp gene are written in bold letters. The numbers on the left indicate the ad4bp gene exons. The positions indicated by the wide arrows represent the initiation methionines and the termination codons for Ad4BP and ELP. A thin arrow indicates the position where the poly(A) tail is added. The sequences that are uniquely found in the protein coding region of ELP are underlined with thin lines. Regions I, II, and III are indicated with wide lines above the nucleotide letters. An arrowhead indicates the position where a 22-nucleotide deletion is present in comparison to the mouse gene. The termination codon of mouse ELP is indicated by asterisks.




Figure 4: Genetic difference of the nucleotide sequences between rats and mice. The nucleotide sequences corresponding to the elp fourth exon are shown. The upper three lanes correspond to the EC cell cDNA(30) , the mouse gene(33) , and the rat gene, respectively. The rat gene has a 22-nucleotide deletion as indicated by the asterisks. The genetic difference between the rats and mice was confirmed by synthesizing the cDNAs corresponding to the elp fourth exon. The nucleotide sequences of the cDNAs generated from the total RNAs prepared from the rat adrenal glands, testes, and ovaries were identical to that of the rat gene as shown in the bottom lane. The hyphens indicate the identity with the nucleotides of the EC cell cDNA. The arrows indicate the synthetic oligonucleotides used as the PCR primers. The numbers indicate the positions of the nucleotides in the rat gene.



In order to confirm that the deletion was caused by the genetic difference between the animal species and not by cloning artifacts, the cDNAs corresponding to the elp fourth exon were analyzed by the reverse transcriptase-PCR as described under ``Experimental Procedures.'' As shown in Fig. 4, the nucleotide sequences of the cDNAs generated from the total RNAs prepared from rat adrenal glands, testes, and ovaries were identical with that of the rat gene, thus confirming that the deletion resulted from the genetic difference between the rats and mice.

Promoter Activity of ad4bp Gene

To investigate the promoter activities of the ad4bp and elp genes, various lengths of each promoter region were used for the construction of the recombinant plasmids for the CAT assays as shown in Fig. 5A. The recombinant plasmids were introduced into mouse adrenocortical tumor Y-1 cells. When their activities were examined, the cells transfected with ELPCAT vectors produced low levels of CAT, while the cells transfected with Ad4CAT vectors produced high levels of CAT. These promoter activities showed a good correlation with the amounts of the mRNAs expressed in vivo(34) .

The CAT activities of the ad4bp gene increased gradually when the promoter region was truncated down to -265 bp (Ad4CAT265). The truncation of the promoter region from -265 to -92 bp (Ad4CAT92) resulted in a decrease in the CAT activity by about half. A drastic decrease of the CAT activity was observed when the 5`-deletion reached to -60 bp (Ad4CAT60). A further truncation to -26 bp completely abolished the remaining weak CAT activity. In addition to Y-1 cells, I-10 cells were used as another type of steroidogenic cell. The promoter activities of the ad4bp gene in the I-10 cells corresponded well to those in the Y-1 cells and a drastic decrease of the CAT activity was also observed between -92 bp and -60 bp. On the other hand, when non-steroidogenic CV-1 cells and NIH3T3 cells (data not shown) were used, the promoter activities of the ad4bp gene were almost undetectable as shown in Fig. 5B, which indicated that the promoter of ad4bp gene is active only in the steroidogenic cells. In I-10 cells, a decrease of the CAT activity observed in Y-1 cells between Ad4CAT265 and Ad4CAT92 was not observed as shown in Fig. 5B. One reason for the decrease in Y-1 cells is the presence of a sequence involved in the transcriptional activation of ad4bp gene in Y-1 cells between -256 and -92 bp. On the other hand, a drastic decrease of the CAT activity between -92 and -60 bp was observed in I-10 cells as well as in Y-1 cells, suggesting the presence of a sequence involved in the transcriptional activation of the ad4bp gene in the steroidogenic cells.

Identification of cis-Element in the Promoter Region of ad4bp Gene

The deletion analysis of the ad4bp gene promoter revealed the presence of a functional sequence between -92 bp and -60 bp. In order to identify the cis-element, the recombinant plasmids carrying four sequential nucleotide substitutions in this particular region (Ad4CATM1-M6) were constructed from the Ad4CAT0.8K plasmid by site-directed mutagenesis as shown in Fig. 6. The mutant plasmids were transfected into Y-1 cells, and their CAT activities were examined. By comparing the CAT activity in the cells transfected with Ad4CAT0.8K, a marked decrease of the CAT activity was observed only in the cells transfected with Ad4CATM3, which had a four nucleotide substitution from -81 to -78 bp. The other substitutions at the positions, -91/-88, -86/-83, -76/-73, -71/-68, and -65/-62, did not affect the promoter activity of the ad4bp gene. We concluded that the sequence from -82 to -77 bp was a cis-element in the promoter of the ad4bp gene. Interestingly, this sequence, CACGTG, was identical with the sequence widely known as the E box element, the binding site for the basic-helix-loop-helix (bHLH) proteins.


Figure 6: Essential function of the E box for the expression of the ad4bp gene in Y-1 cells. The recombinant plasmids carrying sequencial four nucleotide substitutions between -92 bp and -60 bp (Ad4CATM1-M6) were constructed from the Ad4CAT0.8K plasmid and transfected into the Y-1 cells. The autoradiograph of the CAT products is shown. The shaded boxes and a closed box indicate the nucleotides which were substituted with the nucleotides indicated by the arrows. The indicated numbers correspond to the positions from the transcription initiation site of the ad4bp gene.



Binding Factor to E Box Element in Rat Gonads Shows Sex-dependent Expression

We next investigated the binding factor to the E box element found in the promoter region of the ad4bp gene by a gel mobility shift assay using the nuclear extracts prepared from Y-1 and CV-1 cultured cells and labeled probe, dEbox. Complex formation with the dEbox was clearly detected when Y-1 cells nuclear extract was used, whereas no complex was formed with CV-1 cells nuclear extract. The complex formation was specifically inhibited by excess amounts of unlabeled dEbox, but not by an unlabeled dM1-Ebox, which confirmed that the nuclear factors in the Y-1 cells recognized the E box consensus sequence (data not shown).

As reported previously(12) , the expression of Ad4BP in the rat gonads shows the sexually dimorphic pattern and drastic alteration during the development, while that in the adrenal glands is almost constitutive. This observation prompted us to investigate the binding factor to the E box element in the adrenal glands, testes, and ovaries through the life of rats. As shown in Fig. 7A (upper panel), dEbox formed a single complex when the nuclear extracts prepared from the rat tissues were used. The complexes showed the same mobility on the polyacrylamide gel with each other. The complex formation was also specifically inhibited by excess amounts of unlabeled dEbox, and not by an unlabeled dM1-Ebox as shown in Fig. 7B, confirming that the nuclear factors in the rat tissues also recognized the E box sequence. In the adrenal glands, the amount of the binding protein to the E box showed no significant difference between the fetal and the adult animals. In the fetal gonads of 18.5-day postcoitum rats, a significant amount of binding protein was detected in the testes, whereas only a small amount was detected in the ovaries. Dramatical alterations occurred during the perinatal and early neonatal stages. The amount of binding protein to the E box in the testes attained a maximal level 1 week after birth and decreased thereafter, and only a trace amount was detected in an adult. In contrast, the amount of binding protein in the ovaries attained a maximal level immediately after birth and kept its level until adulthood. This dimorphic change of the binding protein to the E box showed a good correlation with the expression of Ad4BP (lower panel in Fig. 7A). Moreover, the ovarian expression of the binding factor to the E box started before the expression of Ad4BP. The developmental changes in the amounts of the binding factor to the E box described above strongly indicated that the expression of the ad4bp gene was transcriptionally regulated through this E box and therefore by the binding factor.


Figure 7: Binding factor to the E box element. A, expression of the E box binding factor in the rat adrenal glands and gonads shows a good correlation with that of Ad4BP. P-End labeled oligonucleotides, dEbox (upper panel) and dAd4 (lower panel), were incubated with 5 µg each of the nuclear extracts indicated above the panels in the presence (+) and absence(-) of 25-fold molar excess of the corresponding nonradiolabeled probe. The incubation mixtures were then examined on a 4.5% polyacrylamide gel. The nuclear extracts were prepared from the rat adrenal glands, testes, and ovaries at various developmental stages (18.5 dpc indicates 18.5 days of post coitum; 1 day, 1 w, 3 w, and Adult indicate 1 day, 1 week, 3 weeks, and 8 weeks after birth, respectively). The arrowheads indicate the complexes with dEbox and dAd4. B, nuclear factor recognizes the E box sequence ``CACGTG.'' To characterize the sequence specificity of the E box in the ad4bp promoter, the oligonucleotides containing various mutations within the E box were used as competitors and probes in the gel mobility shift assays. The indicated P-end labeled probes were incubated with 5 µg of the nuclear extract prepared from the testes of 1-week-old rats. For the competition assays, 25-fold molar excess of the indicated nonradiolabeled oligonucleotides were added prior to the addition of the probes. The probes and competitors: -, C, dM1, dM2, dM3, and dM4 indicate none, dEbox (CTCGTG), dM1-Ebox (CTGTAG), dM2-Ebox (ATCGTG), dM3-Ebox (CACGAT), and dM4-Ebox (CACCGT), respectively. An arrowhead indicates the complex with dEbox.



We characterized the binding sequence by competition experiments with the oligonucleotides containing nucleotide substitutions within the E box, dM1-Ebox (CTGTAG), dM2-Ebox (ATCGTG), dM3-Ebox (CACGAT), and dM4-Ebox (CACCTG), using the nuclear extract prepared from the testes of 1-week-old rats as shown in Fig. 7B. The oligonucleotides, dM1, dM2, and dM3-Ebox did not carry a CANNGT motif, and dM4-Ebox carried a MyoD consensus binding sequence in the muscle creatine kinase gene enhancer (35) . None of the mutant oligonucleotides were able to function as competitors to the dEbox. When dM2 and dM3-Ebox were used as probes, no complex formation was observed because of the destruction of the consensus E box sequence. When dM4-Ebox was used as a probe, a single complex formation was observed. The complex with dM4-Ebox showed a slower mobility than that with dEbox on the polyacrylamide gel, which indicated that a different protein in the nuclear extract of rat testis recognized another E box consensus sequence, CACCTG. Only one nucleotide substitution in the central two nucleotides of the E box consensus sequence resulted in the discrimination of the E box binding factors. We thus concluded that the efficient complex formationwith the nuclear extracts of steroidogenic tissues required the sequence CACGTG.


DISCUSSION

Ad4BP, also known as SF-1, was first identified as a transcription factor regulating all the steroidogenic P-450 genes(7) . It was later found that Ad4BP has wider functions beyond regulating the steroidogenic P-450s; it also regulates the differentiation of such steroidogenic tissue as the adrenal gland and gonads(12, 13) . Furthermore, the sexually dimorphic expression of Ad4BP was observed in the fetus gonads just after the transient expression of SRY by which mammalian sex is known to be determined(14, 15, 16) , which suggested that the role of Ad4BP is thus to regulate the genes necessary for gonadal sex differentiation. In fact, Ad4BP was recently reported to be involved in the Müllerian inhibitory substance gene regulation (12, 13) . Collectively, Ad4BP is a crucial factor in the cascade of the gene activations necessary for the differentiation of the steroidogenic tissues and the sex differentiation of the gonads. An analysis of the transcriptional regulation of the ad4bp gene was thus considered to help identify the genes involved in the cascade of the gene activations necessary for gonadal sex differentiation as well as for the development of the steroidogenic tissue.

The sequence and structural analyses of the rat ad4bp gene revealed that another nuclear factor ELP was also transcribed from the same gene by alternative promoter usage and splicing, which has also been reported in the case of the mouse gene(33) . ELP was originally isolated as a murine homologue of Drosophila FTZ-F1 from undifferentiated murine embryonal carcinoma cells (EC cells), which suppresses the transcription of Moloney leukemia virus by binding to the recognition site in the long terminal repeat(30) . There are known cases of protein generation with different functions from a single gene by alternative splicing and/or the alternative usage of the initiation codon, by which the functions of the gene products are dramatically switched, such as erbA(36) , fosB(37) , mTFE3 (38) , CREM(39) , and Lap(40) . As in the case of the transcription factors described above, a similar mechanism of the transcriptional regulation was expected to work in the case of Ad4BP and ELP. However, the functional analysis revealed that ELP lacked activation function and showed only a faint dominant negative effect on the function of Ad4BP because of significantly lower affinity for Ad4 site than Ad4BP as described earlier(34) . In this study, we found a 22-nucleotide deletion and an earlier occurrence of the termination codon in the last exon of the rat elp gene when compared with the mouse counterpart. In general, such an alteration in the primary structure does not occur for an essential protein. Owing to the deletion, the Ad4BP and ELP of rats could be discriminated by the difference of the molecular masses (Ad4BP, 52.1 kDa; ELP, 47.9 kDa) on the SDS-polyacrylamide gel, although the mouse counterparts have almost the same molecular masses. Even careful observation with an immunoblot analysis could not detect the signal corresponding to ELP in the rat steroidogenic tissues, which correlated well with the observation that the expression of ELP mRNA was significantly lower than Ad4BP in the steroidogenic tissues(12, 34) . Taking these observations into consideration, ELP does not seem to have any significant functions in differentiated steroidogenic tissues.

The promoter region of the ad4bp gene lacks a TATA motif but does not have a G/C-rich promoter nor a Sp1 consensus binding site (41) . In contrast to housekeeping genes which have G/C-rich promoters (42) , this type of promoter has been reported for the Drosophila homeotic genes such as Ultrabithorax(43) , engrailed(44) , and Antennapedia(45) , whose expressions are not constitutive but regulated during the early developmental stages. As in the case of the genes described above, the expression of the ad4bp gene is also regulated during the development of steroidogenic tissue. Although the mechanism of the transcription initiation of such TATA less promoters is less understood than the promoters having TATA motif, the initiator (Inr) motif was demonstrated to be sufficient for basal transcription of the genes(46) . The Inr includes a transcription initiation site and has a weak consensus sequence, 5`-PyPyCAPyPyPyPyPy-3`, with transcription beginning at the A. It has been shown that TFII-I, a transcription initiation factor that activates core promoters through the Inr, interacts cooperatively with myc or USF at both Inr and upstream E box(47, 48) . The sequence around the transcription initiation site of the ad4bp gene is also pyrimidine-rich and the transcription begins at around A, which suggests the involvement of a similar mechanism in the transcription initiation of the gene.

As reported previously, Ad4BP is expressed in steroidogenic tissue such as the adrenal gland, testis, and ovary, and it is coincident with the presence of steroidogenic P-450s(12) . Moreover, Ad4BP was expressed even in the primordial cells of the adrenal gland and gonads of the 13.5-day postcoitum rats, which suggests that Ad4BP regulates the genes essential for the gonadal development and sexual differentiation in addition to the steroidogenic P-450 genes. What mechanism governs such a temporal and spatial expression of the gene? To address this issue, we analyzed the upstream region of the ad4bp gene. An investigation of the promoter activity revealed that an E box sequence at the position from -82 to -77 bp was mainly responsible for the steroidogenic cell-specific transcriptional activity of the gene. This result led us to hypothesize that the E box binding factor regulates the expression of the ad4bp gene in the developmental stages. We then examined the expression profile of the E box binding factor in the steroidogenic tissues throughout the life of rats. Interestingly, the amounts of the E box binding factor showed a developmental change which correlated well with the expression of Ad4BP. Moreover, the ovarian expression of the binding factor to the E box started before the expression of Ad4BP. It is generally accepted that a specific cascade of gene activations is required for the differentiation of each tissue. In steroidogenic tissue, the E box binding factor must be located upstream to the ad4bp gene. In the series of our studies including the present one, we have clarified two steps in the gene activation cascade; that from the E box binding factor to ad4bp, and from Ad4BP to the steroidogenic P-450s.

A number of bHLH proteins including the proto-oncogene myc(49) , MyoD(50) , TFE3(51) , TFEB(52) , USF/MTLF(53, 54, 55) , and the regulators of myc function, Max(56) , Mad(57), and Mxi1 (58) , have been reported. The proteins of the bHLH gene family bind to sequences of the general type CANNTG(59) , where the central two nucleotides contribute to a discrimination of the binding among the different family members(60, 61) . To obtain further insight into the sequence specificity of the E box element in the ad4bp promoter, competition experiments were performed with the oligonucleotides containing various mutations within the E box, and we thus concluded that the efficient complex formation with the nuclear extracts of the steroidogenic tissue required the sequence, CACGTG, which is identical to the binding site for myc, TFE3, TFEB, and USF. An interesting question which still needs to be addressed is whether the binding factor to the E box in the ad4bp gene promoter is identical to the bHLH proteins described above or some unknown protein. It is likely to be an unknown E box binding protein because of the cellular genes regulated by myc and its partner factors(62, 63, 64) , USF(65, 66, 67, 68, 69) , TFE3, and TFEB have not been reported to be expressed in the steroidogenic tissue. The bHLH protein, c-myc, was reported to be expressed in steroidogenic tissue but its expression during the developmental stages of mice is different from that of the E box binding factor examined in this study(70, 71, 72) . Judging from the expression profile during the perinatal and postnatal stages of rats, the binding factor to the E box in the ad4bp gene promoter seems to function as the main transcriptional regulator of the ad4bp gene, which encodes a crucial factor for the differentiation of the steroidogenic tissues and the sex differentiation of the gonads. The isolation and characterization of the E box binding factor seem to be a key step toward the elucidation of the mechanism of differentiation in steroidogenic tissue and the sex differentiation of the gonads.


FOOTNOTES

*
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence data reported in this paper will appear in the GSDB, DDJB, EMBL, and NCBI nucleotide sequence data bases with the accession numbers D42151[GenBank]-D42156[GenBank].

§
Present address: Institute of Toxicology, Dept. of Genetics, ETH and University of Zurich, Schorenstrasse 16 CH-8603, Schwerzenbach, Switzerland.

To whom correspondence should be addressed. Tel.: 81-92-641-1151 (ext. 3475); Fax: 81-92-632-2373.

(^1)
The abbreviations used are: kb, kilobase; P-450, cytochrome P-450; CAT, chloramphenicol acetyltransferase; PCR, polymerase chain reaction; 5`-RACE, 5`-rapid amplification of cDNA ends; bp, base pair(s); bHLH, basis-helix-loop-helix.


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