(Received for publication, November 27, 1995; and in revised form, January 22, 1996)
From the
The mammalian ftz-f1 (mftz-f1) gene encoding Ad4BP/SF-1 has been demonstrated to be essential for the development of adrenal and gonadal glands. In a previous study, we identified an E box as the transcriptional element in the 5`-upstream region of the rat mftz-f1 gene. In the present study, we found a steroidogenic cell-specific transcriptional element in the first intron of the gene. Gel mobility shift and DNase I footprint analyses clearly revealed that Ad4BP itself binds to the element (Ad4 site). This finding was further supported by the positive effect of an Ad4BP expression vector on the transcription and by the significant decrease in the transcription caused by nucleotide substitutions within the Ad4 site. Similar loss was also caused by substitutions in the E box, indicating that the two elements are essential for the full transcriptional activity of the gene. DNase I hypersensitivity assays revealed that the chromatin structure around the Ad4 site and the E box was ``open up'' in the adrenal glands and Y-1 cells, whereas ``closed down'' in the liver. These observations indicated that the mftz-f1 gene is controlled by the autoregulatory loop in the steroidogenic tissues. The autoregulatory mechanism seems to be necessary to keep the mftz-f1 gene activated and thus maintain the tissues differentiated.
Ad4BP (or SF-1) was identified as a tissue-specific transcription factor that regulates all the steroidogenic P-450 genes in the adrenal cortex and gonads(1, 2) . Analyses of the cDNA clone encoding Ad4BP revealed that the factor is a member of the steroid/thyroid hormone receptor superfamily(3) , in which all the members have a zinc finger motif as the DNA binding domain(4) . A functional study using an expression vector for Ad4BP confirmed that the transcription factor governs the tissue-specific expression of the steroidogenic P-450 genes(5) . Recently, wider functions of Ad4BP in addition to the regulation of the steroidogenic P-450 genes has been shown by the following investigations.
The expression of Ad4BP was examined immunohistochemically using the specific antiserum (6) and in situ hybridization(7) . Consistent with its role in the regulation of the steroidogenic P-450 genes, Ad4BP was confirmed to be expressed in the steroidogenic cells of the adrenal gland and gonads. In addition, Ad4BP was found to be expressed in the pituitary gonadotroph, which secretes follicule-stimulating hormone and luteinizing hormone, as well as in the ventromedial hypothalamic nucleus(8, 9, 10) , which controls female sexual behavior(11) . Considering the physiological functions of those tissues, it is clear that Ad4BP is one of the essential factors which control the reproductive function of animals, although the genes controlled by Ad4BP in the ventromedial hypothalamic nucleus still remain to be clarified.
Further investigation of the developing tissues revealed that Ad4BP is expressed even in the primordial cells of the adrenal gland and gonads of the fetuses(7, 12) . In the fetal gonads, a significant amount of Ad4BP was expressed in the testes, whereas only a trace amount was expressed in the ovaries. Sexual differentiation of the gonads is known to be initiated by the transient expression of SRY in the somatic cells of the urogenital ridge(13) . The sexually dimorphic expression of Ad4BP observed in the differentiating gonads suggested that the mammalian ftz-f1 (mftz-f1) gene encoding Ad4BP might be one of the genes located downstream to SRY. It is likely that the gonads express the steroidogenic P-450s and Müllerian inhibitory substance in a sex-dependent manner as a consequence of the dimorphic expression of Ad4BP(12, 14) . In the adrenal glands, however, a significant amount of Ad4BP was continuously expressed throughout the life regardless of the sexes(12) .
These postulated functions of Ad4BP have been supported by the targeted disruption of the mftz-f1 gene(10, 15, 16, 17) . Ad4BP null mice showed a complete absence of the adrenal gland and gonads, indicating that Ad4BP governs the genes essential for the adrenal and gonadal differentiation. Therefore, information about the transcriptional mechanism of the mftz-f1 gene is essential for the elucidation of the molecular basis of adrenal and gonadal differentiation. In our previous report, an E box (18) responsible for the specific expression of the gene in the steroidogenic cells was identified in the 5`-upstream region(19) . In the present study, we focused our attention on the contribution of the first intron to the gene expression and identified an Ad4 site as the transcriptional element. The function of the Ad4 site in the expression of the mftz-f1 gene clearly indicated that the gene is autoregulated by its own product, Ad4BP.
Figure 1: Identification of the regulatory region in the first intron of the rat mftz-f1 gene. Transient transfection assays using adrenocortical Y-1 cells were performed with the deletion constructs indicated below the map of the gene. Ad4CAT2.0K carries the 2.0-kb upstream region from the EcoRI site in the first exon as described previously(19) . All the other plasmids were constructed as described under ``Experimental Procedures.'' The arrows indicate the splice donor and acceptor sites. The CAT activities are shown in comparison with that of Ad4ECAT2.0K in the right panel.
To identify the transcriptional element in the
first intron, various deletion plasmids were constructed based on
Ad4ECAT2.0K as shown in Fig. 1. A drastic decrease of the CAT
activity was observed when the 5` deletion reached to the XbaI
site (5` side) (Ad4ECAT3.0K). Further truncations to the BamHI, HindIII, SacI, and XbaI (3`
side) sites (Ad4ECAT
2.2K,
1.8K,
0.8K, and
0.4K,
respectively) abolished the remaining weak CAT activity. No remarkable
change in the CAT activity was observed when Ad4ECAT
HX,
BX,
and
XX having internal deletions were transfected. A significant
decrease was, however, observed when the internal deletion reached to
-79 bp in the 5`-upstream region (Ad4ECAT
PX). The decrease
seems to be due to the lack of both the first exon and the E box
element, the latter of which had been identified as a transcriptional
element located from -82 to -77 bp(19) . Judging
from these observations, the transcriptional element in the first
intron is likely to be located from the 5` splice junction to the XbaI site (5` side) and/or from the XbaI site (3`
side) to the 3` splice junction. To locate the transcriptional element
more precisely, the transcriptional activities of Ad4ECAT
XX-A and
-B series of the CAT plasmids were investigated. In the case of the
Ad4ECAT
XX-A series, a drastic decrease of the CAT activity was
observed between Ad4ECAT
XX-A24 and -A0. In the Ad4ECAT
XX-B
series, on the other hand, a 3-fold increase of the CAT activity was
observed when the deletion reached to 82 bp from the 3` splice junction
(Ad4ECAT
XX-B82), while a decrease of the CAT activity was observed
with the plasmids from Ad4ECAT
XX-B37 to -B17. Further deletion to
1 bp (Ad4ECAT
XX-B1) abolished the CAT activity.
It is widely
accepted that the splice junction sequences for correct splicing
require not only a GT-AG rule but a pyrimidine nucleotide cluster just
upstream from the 3` splice junction and the consensus several
nucleotides at the 5` splice
junction(24, 25, 26) . Indeed, the mftz-f1 gene has a pyrimidine-rich sequence at the 3` splice
junction, (T/C)ACAG/G, and the consensus sequence,
CA/GTAAGT, at the 5` splice junction (Fig. 2). Taking the rule
into consideration, the decrease of the CAT activity between
Ad4ECAT
XX-B17 and -B1 seems to be caused by the impairment of the
consensus 3` sequence. However, since it was not clear whether the
decrease between Ad4ECAT
XX-B37 and -B17 resulted from the deletion
of a putative transcriptional element or a decrease in the splicing
efficiency, we investigated the structure of the mRNA transcribed from
Ad4ECAT
XX-B17. A significant portion of the mRNA was found to be
unspliced (data not shown). Accordingly, it is likely that the decrease
of the CAT activity mainly resulted from the decrease in the splicing
efficiency. On the other hand, there was also a possibility that a
putative transcriptional element is located in the 24-bp nucleotides
from the 5` splice junction (+154/+177). To examine the
transcriptional activity of the region, Ad4CAT0.8K-EF and -ER were
constructed by insertion of a 91-bp fragment (+146/+236)
including the 24-bp nucleotides at the Eco81I site(-265)
of Ad4CAT0.8K. The CAT activities in Y-1 cells increased by about
7-fold by the insertion of the fragment regardless of their
orientations as shown in Fig. 3. The fragment also increased the
CAT activities of the SV40 core promoter by about 5-fold (pCAT-EF and
-ER) (Fig. 3).
Figure 2: Nucleotide sequence around the first and second exons of the rat mftz-f1 gene. The shadowed regions indicate the first and second exons. The small arrows accompanied by the names of the CAT constructs indicate the truncated positions. The connecting site with the CAT gene is also indicated in the second exon. The E box, Ad4 site, and the initiation methionine are indicated by bold letters. The numbers on the right are relative to the transcription initiation site.
Figure 3: Transcriptional activity of the regulatory region in Y-1 cells. The genomic 91-bp fragment (+146/+236) was inserted into Ad4CAT0.8K or pCAT plasmid in forward (Ad4CAT0.8K-EF and pCAT-EF) and reverse (Ad4CAT0.8K-ER and pCAT-ER) directions as described under ``Experimental Procedures.'' The relative CAT activities in comparison to the original plasmids (Ad4CAT0.8K and pCAT) were averaged for three experiments (±S.E.) and are shown on the right of the figure.
Figure 4:
Binding of Ad4BP to the regulatory region. A, DNase I footprint analysis of the regulatory region. The
DNA fragment from +57 to +316 bp carrying the region was
end-labeled at +57 bp and used for footprint analysis. Increasing
amounts of the nuclear extract prepared from Y-1 cells (from 0 to 25
µg) were used. To determine the protected region, G + A and T
+ C ladders were prepared by chemical cleavage of the
probe(41) . The shaded oval indicates the portion
protected from the DNase I digestion. The corresponding nucleotide
sequence is shown in bold letters. B, the nucleotide sequences
of the probes used in the gel mobility shift assays. dENC contains the
protected region from the DNase I digestion. Each of the underlined three nucleotides is substituted to make dEM1 to dEM6. The closed box indicates the Ad4 site. C, gel mobility
shift assays with the nuclear extract prepared from Y-1 cells. The P-end labeled oligonucleotides, dENC or dAd4, were
incubated with 5 µg of the nuclear extract. For the competition
assays, a 50-fold molar excess of each nonradiolabeled oligonucleotide
was added prior to the addition of the probes. The antiserum to Ad4BP
(
Ad4BP) was added after the addition of the probes. The incubation
mixtures were then examined on a 4.5% polyacrylamide gel. The complexes
with dENC and dAd4 showing the same mobility are indicated by an arrowhead.
Figure 5: Functional analyses of the E box and the Ad4 site. The recombinant plasmids shown in the upper panel were constructed from Ad4ECAT0.8K by nucleotide substitution in either or both the E box and the Ad4 site. The crosses indicate the substitutions introduced. These constructs were transfected into Y-1 cells. The relative CAT activities to Ad4ECAT0.8K were averaged for three experiments (±S.E.) and are shown in the lower panel.
To obtain direct evidence that Ad4BP
activates the transcription of the mftz-f1 gene, an expression
vector for Ad4BP was cotransfected with Ad4ECAT2.0K or Ad4ECAT2.0KA
into CV-1 cells which lacks endogenous Ad4BP. The Ad4BP expression
vector activated the transcription of Ad4ECAT2.0K by about 3-fold as
shown in Fig. 6. Because the disruption of the Ad4 site
(Ad4ECAT2.0KA) completely abolished the activation, Ad4BP activated the
gene as a consequence of the binding to the Ad4 site. A truncated form
of Ad4BP (Ad4BP207) which lacks a putative activator domain at the
carboxyl-terminal half but has the ability to bind to the Ad4 site (3) failed to activate the transcription. An expression vector
for the catalytic subunit of protein kinase A (5) was also
cotransfected with that for Ad4BP. The protein kinase A expression
vector had, however, no effect on the transactivation function of Ad4BP
(data not shown).
Figure 6:
Activation of the mftz-f1 gene
promoter by the expression of Ad4BP. The expression vector for Ad4BP
(Ad4BP) or the truncated form, Ad4BP207 (
207), was
cotransfected with the CAT reporter plasmids, Ad4ECAT2.0K and
Ad4ECAT2.0KA, into CV-1 cells. The CAT activities relative to that of
Ad4ECAT2.0K with no effector(-) are shown. The closed and shaded bars indicate the CAT activities expressed by
Ad4ECAT2.0K and Ad4ECAT2.0KA, respectively.
Figure 7: Detection of the DNase I hypersensitive site. Digestion with the indicated concentrations of DNase I was performed with the nuclei prepared from Y-1 cells, rat adrenal glands, and a rat liver as described under ``Experimental Procedures.'' The genomic DNAs purified from the digested nuclei were subjected to HindIII digestion and then to Southern blotting. The 1.0-kb fragment indicated in the map was used as the probe. The E box and the Ad4 site are shown. The restriction enzyme sites in the region are identical between the rat and mouse mftz-f1 genes. The numbers above the autoradiographs are the concentrations of the DNase I. Size markers are shown on the left of the panel. The arrowheads indicate the digested fragments.
In the present study, we investigated the transcriptional regulatory mechanism of the rat mftz-f1 gene and obtained strong evidence for the autoregulatory loop involved in the transcriptional regulation. The significant transcriptional enhancement by the first intron gave us a clue to find the autoregulatory loop. The regulatory region was located near the splice donor site in the first intron and the presence of an Ad4 site in the region was confirmed by using the specific antiserum to Ad4BP in the gel mobility shift analyses. In a previous paper(1) , we described the purified Ad4BP bound to PuPuAGGTCA as well as PyCAAGGPyPyPu. Although the Ad4 site (GAAGGCCG) identified in the present study satisfied neither of the two consensus sequences completely, it is likely to be an active derivative of the former sequence, where TCA at the 3` side are changed to CCG. Based on our previous observation that the former consensus sequence showed a weaker binding affinity than the latter(1) , the Ad4 site identified in this study has relatively weak affinity in spite of its strong transcriptional activity. The inconsistency between the transcriptional activity and the binding affinity is probably explained by the following observation. In the mftz-f1 gene, the E box is the essential cis-element functioning cooperatively with the Ad4 site since the disruption of the E box caused a significant decrease of the transcriptional activity even in the presence of the intact Ad4 site. A similar decrease was also observed when only the Ad4 site was inactivated. These observations support the notion that the transcriptional activity of the Ad4 site is modulated by the function of the E box and thereby the Ad4 site shows the strong transcriptional activity in spite of the weak binding affinity.
The regulatory region including the Ad4 site was shown to activate both the original mftz-f1 gene promoter carrying the E box and the heterologous SV40 basal promoter. In both cases, however, the transcriptional activations by the region were insufficient to explain the significant transcriptional enhancement by the first intron. A possible reason for the discrepancy is as follow. It was reported in several genes that the splicing reaction itself seems to be critical for efficient production of the mRNA species(27, 28) . It is possible that the pre-mRNA from Ad4ECAT0.8K was efficiently spliced to produce the mature mRNA, whereas those from Ad4CAT0.8K-ES/-ER and pCAT-ES/-ER were not.
The function of the Ad4 site in the first intron of the mftz-f1 gene was further supported by the cotransfection assays with the expression vector for Ad4BP using CV-1 cells. The transcription was activated only when the CAT vector carrying the intact Ad4 site and the expression vector coding intact Ad4BP were used, although the activation by the coexpression of Ad4BP was smaller than expected. Since the transcription of the CYP11A and CYP11B genes was activated by Ad4BP only in the presence of an expression vector for the catalytic subunit of protein kinase A(5) , the effect of the protein kinase A was also examined. However, a further activation was not achieved with the mftz-f1 gene. Considering the cooperative function of the E box and the Ad4 site, the low activation might be due to the absence of the E box binding factor in CV-1 cells. In fact, when the nuclear extracts prepared from Y-1 cells and CV-1 cells were examined by the gel mobility shift analyses, the E box binding factor was detectable with the Y-1 but not with the CV-1 nuclear extract (data not shown).
Differentiated tissues originate from the primordial cells through successive events. Various sets of genes express their functions at the destined time points along the differentiation steps, and finally the tissues acquire their specific functions by expressing the final set of the tissue-specific genes. In the case of the adernal cortex, the differentiated tissue is able to synthesize steroid hormones owing to the functions of the steroidogenic tissue-specific P-450s. When the differentiation process is considered, it seems reasonable to suppose that a gene regulatory cascade functions along the process, in which the genes encoding specific transcription factors are involved as the components. In the cascade required for adrenocortical differentiation, Ad4BP should be located upstream of the adrenocortical specific genes including the steroidogenic P-450 genes, while it is located downstream of other genes regulating the mftz-f1 gene transcription. It was clarified in the present study that the mftz-f1 gene is activated by the autoregulatory loop. Therefore, it is quite interesting to suppose that once the autoregulatory loop starts to function in the particular cell types, the upstream genes essential for the initial activation of the mftz-f1 gene transcription are no longer necessary thereafter to maintain the Ad4BP expression. Such the autoregulation was reported to function in the HOX4C(29) , Hox 4.2(30) , MyoD1(31) , and pit-1(32, 33, 34) genes in mammals, and the fushi tarazu(35) , deformed(36) , even-skipped(37) , Ultrabithorax genes(38) , and sex-lethal(39) genes in Drosophila. Interestingly, all the genes listed above have been reported to play significant roles in the differentiation of particular tissues or cell types. These observations, including the present one, seem to indicate that the autoregulatory mechanism is widely adopted as the transcriptional regulation of the key transcription factors during differentiation.
The chromatin structure of the mftz-f1 gene was also investigated by detecting the hypersensitive site to DNase I. The region containing the Ad4 site and the E box was observed to be open in the steroidogenic tissue but not in the non-steroidogenic tissue. The steroidogenic tissue-specific chromatin structure is probably essential to the mftz-f1 gene transcription by making the transcription factors such as Ad4BP and E box binding factor accessible to their binding sites. Accordingly, it is supposed that the tissue-specific transcription of the gene is also guaranteed by the tissue-specific chromatin structure in addition to the tissue-specific transcription factor.
The present study demonstrated that the rat mftz-f1 gene is autoregulated through the Ad4 site in the first intron and that the E box is essential for the function of the Ad4 site. These observations, however, were made with Y-1 cells which have differentiated features as the adrenocortical cells. Because of the crucial role of Ad4BP in adrenal and gonadal differentiation, it is of importance to study the transcriptional regulation of the gene during the differentiation processes of the animal tissues.
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]-D42152[GenBank].