(Received for publication, December 7, 1994; and in revised form, January 17, 1995)
From the
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.
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,
17-hydroxylase P-450, 11
-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 11
-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.
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.
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.
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.
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.
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.
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.
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].