The Mouse Adrenocorticotropin Receptor Gene: Cloning and Characterization of Its Promoter and Evidence for a Role for the Orphan Nuclear Receptor Steroidogenic Factor 1

Florence M. Cammas1, Gill D. Pullinger, Stewart Barker and Adrian J. L. Clark

Molecular Endocrinology Section, Department of Chemical Endocrinology, St Bartholomew’s and the Royal London School of Medicine, and Dentistry, London EC1A 7BE, United Kingdom


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To elucidate the mechanism underlying the tissue-specific expression of the ACTH receptor/MC2 receptor (ACTH-R) in the adrenal cortex, we have cloned the mouse ACTH-R promoter. The analysis of the cDNA 5'-end showed an untranslated region of 321 bp, and the ACTH-R gene was demonstrated to be composed of two exons of 113 and 112 bp lying upstream of the single coding exon. S1 nuclease protection assay showed two major transcription start sites separated by 4 bp; 1.8 kb of the 5'-flanking region inserted in a luciferase reporter vector had cell-specific promoter activity because it was functional only in mouse adrenocortical Y1 cells but not in mouse Leydig TM3 cells or fibroblast L cells. There was no dramatic change in the promoter activity in Y1 cells for all the deletions tested up to -113 bp upstream of the transcription start site. In contrast, expression in TM3 cells was switched on from deletion -908 bp, while remaining undetectable in L cells. Site-directed mutagenesis of a steroidogenic factor 1 (SF1)-like site at position -25 bp resulted in a significant reduction in luciferase expression by the promoter in Y1 cells. Gel shift analysis of this site indicated specific binding of a protein in extracts of Y1 and TM3 cells. Moreover, expression of SF1 in L cells induced promoter activity of the construct p(908). In conclusion, cell-specific expression of the mouse ACTH-R appears to be controlled by at least two factors. One of them, most probably SF1, is responsible for steroidogenic cell-specific expression. The other as yet unknown factor binding between position -1236 bp and -908 bp acts as a strong inhibitory factor in nonadrenal steroidogenic cells, resulting in the adrenal-specific expression of ACTH-R.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The production of glucocorticoids by the adrenal cortex is predominantly regulated by pituitary ACTH (for review see Refs. 1 and 2). This hormone acts via a seven-transmembrane domain receptor belonging to the G protein-coupled receptor superfamily (ACTH-R), also known as the MC2 receptor (3). The identity of this receptor as the ACTH-R was supported by association of mutations in this receptor and adrenal unresponsiveness to ACTH in familial glucocorticoid deficiency (for review see 4 . Although it is generally accepted that the ACTH-R is expressed in the adrenal cortex, a number of groups have suggested that the ACTH-R is also expressed in peripheral blood mononuclear leukocytes (5, 6), adipocytes (7, 8, 9), and skin (10) on the basis of binding studies and/or mRNA detection. There is very little evidence of ACTH-R expression in other tissues, implying that an effective mechanism exists to restrict expression of this gene.

The most probable explanation for this phenomenon could be the existence of tissue-specific regulatory elements in the ACTH-R promoter. Of particular relevance to ACTH-R expression are several factors that have been shown to be involved in steroidogenic or adrenal-specific expression. The orphan nuclear receptor steroidogenic factor 1 (SF1) is probably the best characterized (11, 12) and has been shown to be involved in steroidogenic cell-specific expression of several genes (13, 14, 15, 16, 17, 18, 19) and in the development of the adrenal and gonad (20, 21, 22). An adrenal-specific nuclear protein (ASP) has also been shown to be involved in the human CYP21B gene expression (23, 24). Disruption of the orphan nuclear receptor DAX-1 has been implicated in X-linked adrenal hypoplasia congenita, suggesting a specific role in adrenal and gonadal development (25). Furthermore, some ubiquitous factors such as NGFI-B (26) and AP1 (27) have been shown to be involved in the regulation of adrenal-specific gene expression.

We have previously reported the cloning and expression of the mouse ACTH-R (28, 29). In this report we describe the characterization of the mouse ACTH-R gene structure and the cloning of its promoter. This promoter has been functionally analyzed in the well characterized Y1 mouse adrenocortical cell line, which has been shown to express the ACTH-R and respond to ACTH (30, 31). Our data indicate the existence of two important regions that regulate tissue-specific expression of the gene. One of these regions suggests a role for SF1, or a closely related protein, in the determination of steroidogenic cell specificity of ACTH-R expression.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Characterization of the Mouse ACTH-R cDNA 5'-End
To identify the 5'-end of the ACTH-R cDNA and therefore to characterize the structure of the gene, we used the rapid amplification of cDNA ends (RACE) protocol. PCR with a sense primer complementary to the ligated adapter at the cDNA 5'-end and an antisense primer complementary to the ACTH-R coding sequence produced a single 1.1-kb fragment (Fig. 1AGo). This was subcloned, sequenced, and shown to comprise the ACTH-R coding sequence and a 5'-untranslated sequence of 241 bp (Fig. 1BGo). Comparison between this cDNA fragment and the ACTH-R genomic sequence indicated that the mouse ACTH-R gene consisted of at least two exons with an intron/exon junction 96 bp upstream of the translation start codon (Fig. 1BGo). The exonic nature of the sequence was confirmed using S1 nuclease protection assay (32) with mouse adrenal mRNA and a genomic probe spanning the putative intron/exon junction in which only the predicted exonic sequence was protected against S1 nuclease digestion. Another probe derived from the 5'-RACE cDNA product was fully protected against digestion (data not shown).



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Figure 1. Cloning of the Mouse ACTH-R cDNA 5'-Ends

A, Agarose gel analysis of the RACE product performed on 1 mg mouse adrenal mRNA with an antisense primer in the mouse ACTH-R coding sequence. Different dilutions of the final ligation mixture: 0, 1, 1:5, 1:10, 1:25, 1:50 were used as template for PCR. A predominant 1.1-kb band in all samples is indicated. B, 241-bp sequence of the 5'-untranslated region of the mouse ACTH-R gene. The underlined sequence represents the 96-bp untranslated part of the coding exon, and the translation initiation codon is indicated in bold.

 
Tissue-Specific Distribution of ACTH-R mRNA
Taking advantage of the multiexonic nature of the ACTH-R gene, we used RT-PCR with intron-skipping primers to assess ACTH-R expression in mouse adrenal gland, liver, lung, spleen, abdominal adipose tissue, whole brain, heart, testis, and kidney. The PCR products were analyzed by Southern blotting with the RACE cDNA fragment described above as the probe to obtain the maximum sensitivity. RNA quality and the efficiency of the reverse transcription step were controlled by carrying out parallel RT-PCR with ß-actin primers. As shown in Fig. 2Go, the ACTH-R mRNA was relatively abundantly expressed in adrenal tissue. However, low expression of this gene was also apparent in abdominal adipose tissue. Expression could not be detected in spleen, testis, liver, lung, heart, whole brain, or kidney.



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Figure 2. Tissue Distribution of the Mouse ACTH-R mRNA

A, Ethidium bromide-stained agarose gel analysis of RT-PCR performed with ß-actin exon-skipping primers and 10 µg total RNA obtained from the mouse tissues indicated, or a water control (-) to check the RNA quality and the reverse transcription efficiency. B, RT-PCR was performed with the mouse ACTH-R intron-skipping primers and the same RNA samples, and the product was analyzed by Southern blotting after agarose gel electrophoresis using the mouse ACTH-R 5'-RACE cDNA fragment as the probe.

 
Cloning of the Mouse ACTH-R Gene Promoter
A probe corresponding to the first 144 bp of the 5'-end of the RACE product was used to screen 106 pfu of a mouse genomic library under high stringency conditions. Five positive clones were isolated that were found to have distinct restriction patterns after digestion with Bgl2, NcoI, and PvuII and Southern blot analysis using the same probe as for the screening (Fig. 3Go). The restriction fragments marked with arrows in the figure were subcloned and sequenced. In this way the 5'-untranslated region was shown to consist of two separate exons. Exon 1 overlapped the 5'-end of the RACE clone by 33 bp. Exon 2 consisted of 112 bp, which together with the 96 bp at the 5'-end of exon 3, encode the 5'-untranslated region of this gene. The genomic clones 4 and 5 contained exon 2 while clones 1, 2, and 3 contained exon 1. The NcoI-positive fragment from clone 1, containing exon 1, was subcloned, sequenced, and shown to contain 1.8 kb upstream sequence expected to be the mouse ACTH-R promoter. This fragment was therefore analyzed further.



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Figure 3. Isolation of Genomic Clones Containing the Mouse ACTH-R 5'-End Exons

A, Agarose gel showing restriction enzyme analysis (using NcoI, PvuII, or Bgl2) of the five positive genomic clones (1–5) obtained after screening with the most 5' 144 bp of the RACE cDNA product. M represents DNA size markers of 21.6 kb, 9.4 kb, 6.6 kb, and 4.4 kb. B, Southern blot analysis of the five positive clones using the 5'-RACE cDNA product as the probe. The positive bands of 4 kb (clone 5), 2.2 kb (clone 1), and 1 kb (clone 4) marked with arrows were subcloned and sequenced.

 
The precise size of exon 1 was assessed by S1 nuclease protection assay with mouse adrenal mRNA and a probe spanning the expected transcription start site. It was demonstrated that the actual size of the first exon is 113 bp (Fig. 4Go). There was a second protected fragment corresponding to an exon of 109 bp implying that there were two major start sites for transcription separated by 4 bp. The intensity of these two protected fragments was similar, indicating that both transcription start sites are used with equivalent efficiency.



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Figure 4. ACTH-R Gene Structure and 5'-End Determination

A, Schematic representation of the ACTH-R gene, the genomic clones from which each exon was identified, the mature mRNA product, and the site and extent of the 5'-RACE cDNA clone. B, Determination of the transcription start site by S1 nuclease protection assay. A probe overlapping the putative transcription start site was obtained as described in Materials and Methods. The protected fragments of 104 bp and 100 bp marked with the bottom arrows indicate two transcription start sites corresponding to first exons of 113 bp and 109 bp, respectively. The undigested probe of 300 bp is marked with the top arrow. The (-) lane indicates the use of 10 µg of transfer RNA as template.

 
Sequencing of the 5'-untranslated region (Fig. 5Go) did not reveal any of the classic promoter features such as a TATA box, CAAT box, or GC-rich region. However, there is an initiator-like sequence overlapping the transcription start site (dashed underlining in Fig. 5Go), and there are a number of consensus response elements as depicted in Fig. 5Go including an OctB site, an Sp1 site, a glucocorticoid response element, an AP2 site, an AP1 site, and two SF1-like sites at positions -897 bp and -25 bp, respectively.



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Figure 5. Sequence of Mouse ACTH-R Gene

Lowercase letters represent intronic sequences. Only the extremities of the introns are shown separated by dots. The light-face uppercase sequence is the promoter, and the bold uppercase sequences are the three exons. Putative regulatory sequences are underlined with a solid line, and the putative initiator sequence are underlined with a dashed line. The arrows show the ends of the different deletion constructs.

 
Functional Characterization of the Mouse ACTH-R Promoter
To functionally characterize the promoter, the region between +104 bp and -1808 bp was subcloned into the pGL3-basic luciferase reporter vector. This construct was then transfected by electroporation into mouse Y1 adrenocortical cells and two other mouse cell lines derived from tissues that do not express ACTH-R: TM3 cells, derived from Leydig cells, and L cells, a fibroblast cell line. With this full length promoter construct, luciferase activity was detectable in Y1 cells, but no activity was apparent in TM3 cells. The level of this activity was equivalent to 5–10% of the activity of Rous sarcoma virus (RSV).luciferase in the same cells. L cells produced some activity, although this was extremely low and statistically indistinguishable from zero (Fig. 6AGo), showing the expected cell-specific activity of this promoter.



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Figure 6. Functional Characterization of the Mouse ACTH-R Promoter

A, Luciferase activity of the mouse ACTH-R promoter deletions in Y1 cells, TM3 cells, and L cells. The deletion constructs transfected into the three cell lines are described on the left with the numbering refering to the transcription start site of the longest transcript. The results are expressed as a mean (± SEM) arbitrary light units as described earlier for the same number of independent experiments. B, Luciferase activity obtained in three independent experiments (mean ± SEM) using the full-length promoter, the minimal promoter fragment p(113), and the SF1 site mutant of p(113). Results were compared using Student’s t test.

 
To identify the important regulatory regions for expression of the ACTH-R gene, a series of deletion constructs of the promoter were generated as indicated in Fig. 5Go. Figure 6AGo summarizes the effect of these deletions on luciferase reporter activity in the three cell lines. There was no significant difference between the full length [p(1808)] construct and all the other deletions up to -113 bp in Y1 cells. No significant luciferase activity was detected for any of the constructs in L cells. In TM3 cells no luciferase activity was detected after transfection with the full length construct or the two constructs p(1590) and p(1236). In contrast, when the deletion constructs p(908), p(717), p(641), and p(113) were transfected, they all showed luciferase activity in TM3 cells at a significantly lower level than in Y1 cells with the exception of p(113), which appeared to induce transcription at an equivalent level to the minimal promoter in Y1 cells.

Identification of a Binding Site for SF1
Of the transcription factor consensus binding motifs identified, the SF1-like sequence at position -25 bp was of particular interest in connection with ACTH-R tissue-specific expression because it is the only recognized site in the construct p(113) that showed promoter activity specifically in Y1 cells and TM3 cells. We therefore used site-directed mutagenesis to alter the -25 SF1 consensus sequence in the p(113) construct at two critical nucleotides’ SF1 binding site (AAGGTT -> ATTGTT). The results of an independent series of experiments performed in Y1 cells are shown in Fig. 6BGo and indicate a highly significant loss of promoter activity in p(113)mutant to 38% of that of p(113) itself (P < 0.01).

Further evidence for the involvement of SF1 at the -25-bp site was sought using gel shift analysis. A 46-bp fragment surrounding the SF1-like binding site at position -25 bp was used as the labeled probe (see Fig. 7AGo). A DNA/protein complex (CI) was found to be formed using cellular extracts from Y1 cells. A similar complex was apparent when a cellular extract from TM3 cells was used. However, no such complex was observed with extracts from L cells (Fig. 7BGo), although a certain amount of very slowly migrating nonspecific material was present. In Y1 cell extracts a second complex (CII) was also formed. Specificity of the interaction was confirmed by displacement of the DNA/protein complexes by a 200-fold molar excess of an unlabeled consensus SF1- binding site derived from the rat CYP17 gene (19), providing further evidence that the protein involved in these complexes was SF1 (Fig. 7BGo).



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Figure 7. Electrophoretic Mobility Gel Shift Assay with the SF1-Like Site at Position -25 bp

A, Sequence of the probe (derived from the ACTH-R sequence) and the consensus SF1 competitor (derived from the cytochrome P450 17{alpha}-hydroxylase gene) used for gel retardation assays: the SF1-binding sites are in bold. B, Thirty micrograms of Y1, TM3, and L cell cellular extracts were incubated with the end-labeled SF1-like probe without or with 200 x or 500 x molar excess of unlabeled consensus SF1 competitor. Complexes I and II are indicated on the side of the figure as CI and CII.

 
SF1 Induces ACTH-R Promoter Activity in Fibroblasts
To confirm the functional relevance of SF1 in the regulation of ACTH-R expression, an expression vector containing the SF1-coding sequence was cotransfected with the promoter construct p(908) into L cells. P(908) was chosen to avoid any influence of the putative repressor identified in TM3 cells in case it was also present in L cells. These cells do not express SF1 endogenously, as shown above (Fig. 7BGo), and do not have any ACTH-R promoter activity (Fig. 6AGo). Heterologous expression of SF1 in these cells resulted in significant induction of ACTH-R promoter activity (P < 0.05) whereas no effect was observed using a promoterless construct (Fig. 8Go), lending support to the hypothesis that SF1 is an essential factor for ACTH-R expression.



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Figure 8. Expression of SF1 in Fibroblast Cells Induces ACTH-R Promoter Activity

Five micrograms of the promoterless luciferase reporter plasmid pGL3 or the ACTH-R promoter-luciferase construct p(908) were cotransfected with or without the SF1 expression vector under the control of a cytomegalovirus promoter, and luciferase activity was measured after 72 h as described earlier. The results are the mean (±SEM) of three independent experiments. The asterisk represents a significant increase (P < 0.05) using one-tailed Student’s test.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have demonstrated that the mouse ACTH-R gene consists of at least three exons of lengths 113 bp, 112 bp and >1000 bp. The two first exons contain the 5'-untranslated region while the third exon contains 96 bp of 5'-untranslated region and the entire coding sequence. The functional relevance of the two untranslated exons is not known. Interestingly, a single exon upstream of the coding exon has been identified in the human ACTH-R gene (33), and this is highly homologous to the mouse exon 1, suggesting that any physiological role of the mouse exon 2 is not required in the human gene.

The knowledge of this structure has enabled us to use RT-PCR as a highly sensitive method of mRNA detection with intron-skipping primers followed by Southern blotting. As shown in Fig. 3Go, ACTH-R mRNA is relatively abundantly expressed in the adrenal gland, as is well recognized. However, there is also low expression in abdominal adipose tissues. This site has already been shown to express ACTH-R by binding studies, and recently this has been confirmed by Northern blot analysis (9) and has been proposed to mediate the lipolytic action of ACTH. Thus, our data confirm the tissue-specific nature of ACTH-R gene expression, and it is the molecular mechanism of this regulation, especially at the level of the adrenal cortex, that has been the focus of our further studies. It is of interest that an abstract recently presented by another group (34) indicated the existence of a fourth alternatively spliced exon lying between exons 1 and 2. This is probably an uncommon splice variant that we have been unable to identify but may be represented by the weak higher molecular weight band in the adrenal RT-PCR shown in Fig. 2BGo.

Sequence analysis of the 5'-untranslated flanking region of the ACTH-R gene indicated that the region immediately upstream of exon 1 does not show any typical characteristics of promoter regions such as a TATA box, CAAT box, or GC-rich region. However, there is an initiator-like site overlapping the transcription start site. The initiator element (Inr), first identified for viral transcriptional regulation (for review see 35 , has been shown to be involved in transcription initiation of a growing number of TATA box-containing and TATA-less promoters. In addition to this Inr-like sequence, there are several putative sites for transcription factors as shown in Fig. 5Go. Of particular interest is the presence of a partial consensus sequence at position -25 bp for binding of the mouse transcription factor SF1, which has been shown to be involved in tissue-specific expression of several steroidogenic enzymes (11, 12, 13, 20) as well as Mullerian-inhibiting substance (15), and the glycoprotein hormone {alpha}-subunit (16). SF1 has also been shown to be involved in the cAMP responsiveness of the cytochrome P450c17 enzyme expression (19). Therefore, this sequence motif represents a strong candidate for regulation of ACTH-R gene expression. Recently, Naville et al. (33) have reported the promoter sequence for the human ACTH-R gene revealing that it also contains an Inr element and an SF1-like site at position -35.

The presence of a glucocorticoid response element in the ACTH-R promoter is also important because glucocorticoids have been shown to induce a significant increase of ACTH-R expression in ovine adrenocortical cells (36). The molecular mechanism of this effect is not yet known but may occur at the transcriptional level. It is noteworthy that the ACTH-R promoter does not contain any consensus cAMP response element (CRE) despite the fact that ACTH is recognized to stimulate ACTH-R expression via cAMP (31, 37). However, this lack of a consensus CRE is a common feature for the cAMP-regulated steroidogenic enzymes and may suggest the existence of a yet uncharacterized cAMP-dependent regulatory factor. In the case of CYP17, this function has been shown to be fulfilled by SF1 (19), but that does not seem to be the case for the other steroidogenic enzymes. The functional importance of both of these regulatory elements will be addressed in our future work. In contrast, the human ACTH-R contains seven CRE-like elements in the proximal 700 bp of this promoter, although these have not yet been shown to be functional (33).

The promoter activity of the ACTH-R 5'-untranslated region was assessed after insertion upstream of the luciferase coding sequence in the pGL3 basic vector. It has been shown that this DNA fragment is necessary and sufficient to drive expression of a heterologous gene in a cell-specific manner, since the signal was detectable in Y1 cells but not in TM3 or L cells. Deletion studies allowed us to define two important regions for cell-specific expression. Promoter activity became detectable in TM3 cells when sequences upstream of position -908 bp were removed, despite remaining undetectable in L cells (Fig. 6AGo). These results suggest that the ACTH-R promoter is potentially active in steroidogenic tissues other than the adrenal, but is totally inhibited by the binding of a factor between position -1236 bp and -908 bp that would act as a repressor of expression in nonadrenal steroidogenic cells such as the Leydig cell. The existence of such transcriptional repressors or silencers responsible for tissue-specific expression of genes has been reported for several genes. However, our results are the first evidence for a repressor restricting gene expression in the adrenal gland.

In Y1 and TM3 cells, the minimal promoter extended to -113 bp, which includes the putative SF1-like binding site described earlier. This suggests that SF1 might be a strong candidate for determining steroidogenic-specific expression of the ACTH-R as it does for several other genes. Our data lend support to the notion that SF1 or an SF1-like factor is involved in ACTH-R cell-specific regulation since 1) Mutation of the SF1 site leads to significant loss of promoter activity; 2) it binds specifically to this site as shown by gel shift analysis; and 3) expression of SF1 in a cell line that normally does not have any ACTH-R promoter activity leads to an induction of its activity. However, the level of promoter activity obtained in these conditions is low, indicating that SF1 may be essential to induce the promoter in an "active conformation" whereas other factors are probably necessary for full promoter activity. In keeping with this, it is noteworthy that mutation of the SF1 site leads only to a significant reduction of activity and not complete loss of promoter function. The role of the second SF1-binding site at position -897 bp has not been extensively studied. However, it seems that SF1 binding at this site of the promoter may be more critical in TM3 cells than in Y1 cells because its absence in both constructs p(717) and p(641) resulted in a significant decrease of promoter activity in TM3 cells whereas it was almost without effect in Y1 cells. The significance of these results will need further investigation.

In conclusion, the tissue-specific expression of the ACTH-R gene appears to be mainly under the control of two factors, with SF1, or an SF1-like factor, responsible for steroidogenic cell specificity, and another as yet unknown factor binding between position -1236 bp and -908 bp silencing the promoter in nonadrenal steroidogenic cells. It might be postulated that binding of such a repressor protein upstream of the SF1-binding site prevents the formation of an SF1-dependent transcriptionally active complex.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Chemicals
All chemicals were obtained from Sigma Chemical Co. (Poole, Dorset, UK); FCS, horse serum, Ham’s F10 and F12 media were obtained from GIBCO BRL (Renfrewshire, UK). All radionucleotides were obtained from Amersham International plc (Amersham, Bucks. UK).

Rapid Amplification of cDNA Ends
The Marathon cDNA Amplification kit (Clontech, Cambridge, UK) was used following the manufacturer’s protocol. The mouse ACTH-R gene antisense primer was 5'-GACCTGGAAGAGAGACATGTAG3-'. RNA was extracted from mouse adrenal tissues using RNazolB (Biogenesis, Poole, Dorset, UK), and mRNA was purified using a Sephadex oligo-dT column (Pharmacia, Herts, UK). One microgram of this mRNA was used per reaction. The PCR product was subcloned directly in the pGEM-T vector (Promega, Southampton, UK) and sequenced using sequenase 2.0 (Amersham).

RT-PCR
Ten micrograms of total RNA from the following frozen mouse tissues were analyzed: whole brain, kidney, lung, spleen, abdominal adipose tissue, lung, adrenal, heart, and testis. One hundred nanograms of oligo-dT were used to prime the Superscriptase (GIBCO BRL) for 1 h at 42 C in a 20-µl reaction mixture. One microliter was used as template for PCR with: mouse ACTH-R exon 1 sense primer: (5'-CTTGCCGAGAAAGATCCT-3') and exon 3 antisense primer: (5'-AGCGATGTGAAGGTGAGC-3'), or the ß-actin sense primer: (5'-GTAACCAACTGGGACGAT-3') and antisense primer (5'-GACCACACCCCACTATGG-3').

Reaction conditions were 30 sec at 94 C and 30 sec at 56 C for ACTH-R or 58 C for ß-actin and 1 min at 72 C for 30 cycles.

In the case of the ACTH-R, PCR products were run on a 1% agarose gel, transferred to nitrocellulose membrane, and hybridized in 50% formamide, 5 x NaCl-sodium citrate, 0.05 M sodium phosphate buffer (pH 6.8), 0.3 mg/ml herring sperm DNA, 5 x Denharts solution, 0.05% SDS, with approximately 106cpm {alpha}-32P-labeled ACTH-R probe at 42 C for 16 h. The filters were washed under stringent conditions (1 x NaCl-sodium citrate, 0.1% SDS) at 65 C and exposed to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) at -80 C for 3 h or overnight for the long exposure.

S1 Nuclease Protection Assay
The reaction conditions were as described previously (32). PCR was performed with a biotinylated sense primer MP237S: 5'-TACAACCTTCACAATCTGC-3' designed in the putative promoter region and a phosphorylated antisense primer at the 3'-end of exon 1: 5'-CTGAAGTAGGATCTTTCTCG-3'. Fifty micrograms of total mouse adrenal RNA were hybridized with this probe overnight at 43 C.

Plasmid Constructs
For the full-length construct p(1808), PCR was performed using Vent polymerase (New England Biolabs., Beverly, MA) with a sense primer: 5'-GACCATTAACTTTGAATTAGG-3' at the 5'-end of the NcoI fragment and an antisense primer complementary to exon 1: 5'-CTGAAGTAGGATCTTTCTCG-3' using the genomic positive clone 1 as template. This was subcloned into the SmaI site of the pGL3-basic luciferase reporter vector (Promega, Madison, WI), and PCR artefacts were excluded by sequencing. For the promoter deletions, p(1808) was digested with specific enzymes as follows: -1590 bp, KpnI; -1236 bp, NheI; after separation of the insert and the backbone, the plasmid was religated on itself. For the following deletions, p(1808) was cut with KpnI and another specific enzyme, the ends were filled using Klenow fragment, and the plasmid was religated: -908 bp, Bsg1; -717 bp, TthIII.1; -641 bp, ApaI; -113 bp, BstX1. The constructs or at least their 5'- extremities were checked by sequencing.

Cell Culture and Transfection Experiments
Mouse Y1 adrenal cells were maintained in 50% DMEM and 50% Ham’s F10 supplemented with 12.5% horse serum and 2.5% FCS. Mouse TM3 cells (kindly provided by Dr Ray Iles) were grown in 50% DMEM and 50% Ham’s F12 supplemented with 5% horse serum, 2.5% FCS, and 20 mM HEPES (pH 7.4). Mouse L cells (kindly given by Prof. Tom MacDonald) were grown in RPMI containing 5% FCS. Approximately 106 cells were washed and resuspended in HEPES- buffered saline and 500 µl were used per electroporation at 300 V and 1000 milliFarads in 2-mm wide cuvettes with 5 µg of the ACTH-R gene construction and 10 µg of RSV-CAT plasmid. Cells were left 1 min at room temperature and then resuspended in their respective growth medium in 60-mm plates for 72 h. Five micrograms of SF1 expression vector were cotransfected with 5 µg of the p(908) construct in L cells in the conditions described above. Because the transfection efficiency was constant, RSV-CAT was not used systematically in all experiments.

Luciferase Activity and Chloramphenicol Acetyltransferase (CAT) Protein Measurement
The luciferase assay kit (Promega) was used for this purpose. The cells were washed twice at room temperature with PBS, covered with 400 µl lysis reporter buffer for 20 min at room temperature, scraped, recovered in Eppendorf tubes, and snap frozen at -70 C. The cell debris was pelleted and 100 µl of the supernatant were used for luciferase activity measurement with 400 µl lysis reporter buffer and 100 µl luciferase substrate solution. One hundred fifty microliters of the supernatant were used for measurement of CAT protein level according to the CAT ELISA protocol (Boehringer Mannheim, Lewes, UK). Mutagenesis of the -25 bp site in the p(113) plasmid was accomplished using the QuikChange technique (Stratagene Ltd., Cambridge, UK) with Pfu polymerase. The identity of mutated clones was determined by DNA sequencing.

Electrophoretic Mobility Shift Assay
To prepare the probe an antisense ACTH-R primer, 5'-GACAGCTACTTGTTATCC-3', was labeled with [{gamma}32P]ATP using T4 polynucleotide kinase (New England Biolabs.) and used for PCR with an unlabeled ACTH-R sense primer, 5'-CCGTTTATTTCTAGTGAC-3' producing a 46-bp fragment surrounding the SF1-like site, which was purified with microcentrifuge 5000 tubes (Sigma). Binding reactions of 10 µl were carried out in buffer containing 15 mM HEPES, 75 mM KCl, 9 mM MgCl2, 0.075 mM EDTA, 12.5% glycerol, 1 mM dithiothreitol, 0.1 mg/ml sperm DNA, approximately 5 x 103cpm of radiolabeled probe, and 30 mg of cellular extracts proteins for the three cell lines prepared as described previously (36). The reactions were incubated for 15 min at 30 C. Competitor was added 15 min before the probe and incubated at 30 C. Free and bound DNA were separated on a 4% nondenaturing polyacrylamide gel in 0.5 x Tris-borate-EDTA at 4 C. The gels were dried and analyzed by autoradiography.


    ACKNOWLEDGMENTS
 
We are grateful to Dr. Keith Parker (Duke University, Durham, NC) for providing us with the SF1 expression vector and to Dr. Joy Hinson (Queen Mary & Westfield College, London) for provision of mouse adrenal tissue. This work has benefited from helpful discussion with Dr. Paul Lavender (Wellcome CRC, Cambridge, UK) and Avtar Roopra (University College, London). F.M.C. was supported by a Studentship from the Joint Research Board of St Bartholomew’s Hospital, and G.D.P. was supported by the Medical Research Council.


    FOOTNOTES
 
Address requests for reprints to: Adrian J. L. Clark, Department of Chemical Endocrinology, St. Bartholomew’s & Royal London School of Medicine and Dentistry, West Smithfield, London ECIA 7BE, UK.

1 Present address: IGBMC, BP163, C.U. de Strasbourg, 67404 Illkirch, France. Back

Received for publication August 7, 1996. Revision received February 17, 1997. Accepted for publication February 28, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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