Characterization of the Mouse FTZ-F1 Gene, Which Encodes a Key Regulator of Steroid Hydroxylase Gene Expression
Yayoi Ikeda, Deepak S. Lala, Xunrong Luo, Edward Kim, Marie-Pierre Moisan, and Keith L. Parker
Departments of Medicine and Biochemistry and the Howard Hughes Medical Institute Duke University Medical Center Durham, North Carolina 27710
The cytochrome P450 steroid hydroxylases are coordinately regulated by steroidogenic factor 1 (SF-1), a protein expressed selectively in steroido- genic cells. Based on its expression in steroidogenic tissues and DNA-binding specificity, we isolated a putative SF-1 cDNA from an adrenocortical cDNA library. As evidence that this cDNA encodes SF-1, we now show that it is selectively expressed in steroidogenic cells, that an antiserum against its protein product specifically abolishes the SF-1-re- lated gel-shift complex, and that its coexpression increases promoter activity of the 21-hydroxylase 5’-flanking region in transfection experiments.
Sequence analyses of the SF-1 cDNA revealed that it is the mouse homolog of fushi tarazu factor I (FTZ-F1), a nuclear receptor that regulates the fushi tarazu homeobox gene in Drosophila. A second FTZ- F1 homolog, embryonal long terminal repeat-binding protein (ELP), was recently isolated from embryonal carcinoma cells. SF-1 and ELP cDNAs are virtually identical for 1017 base pairs, including putative DNA-binding domains, but diverge at their 5’- and 3’-ends. One genomic clone contained both SF-1- and ELP-specific sequences, confirming their origin from a single gene. Characterization of this gene defined shared exons encoding common regions and alternative promoters and 3’-exons leading to differences between the two FTZ-F1 transcripts.
We used in situ hybridization with transcript-spe- cific probes to study the ontogeny of SF-1 and ELP expression. ELP transcripts were not detected from embryonic day 8 to adult, consistent with its previous isolation from embryonal carcinoma cells and its postulated role in early embryonic development. In contrast, SF-1 transcripts were first detected at em- bryonic day 12, when steroidogenic organs begin to develop and steroidogenic enzymes are first ex- pressed, and persisted thereafter at high levels in
0888-8809/93/0852-0860$03.00/0 Molecular Endocrinology Copyright 1993 by The Endocrine Society
adrenal glands and testis. Collectively, these studies establish that the mouse homolog of the FTZ-F1 gene generates developmentally regulated transcripts, one of which encodes an essential regulator of ste- roid hydroxylase gene expression. (Molecular En- docrinology 7: 852-860, 1993)
INTRODUCTION
The regulated production of steroid hormones requires a related group of cytochrome P450 steroid hydroxyl- ases that convert cholesterol to the various steroid products (reviewed in Ref. 1). We recently isolated an orphan nuclear receptor, designated steroidogenic fac- tor 1 (SF-1), which we proposed regulates the coordi- nate expression of three mouse steroid hydroxylases by interacting with a shared promoter element (2). The putative SF-1 cDNA closely resembles a cDNA isolated from ECA2 embryonal carcinoma cells, designated em- bryonal long terminal repeat-binding protein (ELP) (3); both sequences are identical for approximately 1000 base pairs (bp), including their zinc finger DNA-binding domains, but diverge at their 5’- and 3’-ends.
Sequence comparisons further indicated that the SF- 1/ELP transcripts are homologs of an orphan receptor, designated FTZ-F1, that regulates the expression of the Drosophila fushi tarazu (ftz) homeobox gene (4, 5). Interestingly, gel mobility shift assays had discriminated two forms of the Drosophila FTZ-F1 protein with iden- tical or closely related DNA recognition specificities: an early form, which correlates with ftz expression, and a late form, which follows ftz expression and serves an unknown function (4).
In this report, we verify that the putative SF-1 cDNA actually encodes the shared regulator of steroid hy- droxylase gene expression, explore the relationship between the SF-1 and ELP transcripts, and define their sites of expression. Our results suggest that develop- mentally regulated products of the FTZ-F1 gene play
differential roles in early embryonic development and in maintaining the steroidogenic capacity of steroidogenic tissues.
RESULTS
The Putative SF-1 cDNA Encodes a Regulator of Steroid Hydroxylase Gene Expression
We originally isolated a putative SF-1-encoding cDNA by virtue of the homology between its DNA-binding domain and that of another orphan nuclear receptor, retinoid X receptor-8. As defined by Northern blotting analyses, this cDNA was only expressed in steroido- genic tissues, where it encoded a protein that bound steroidogenic regulatory elements with a recognition specificity indistinguishable from authentic SF-1 (2). These studies, however, did not absolutely exclude the possibility that this cDNA encoded another DNA-binding protein with the same recognition specificity as SF-1.
To study more rigorously the link between this cDNA and SF-1, we first performed in situ hybridization assays to identify the cells in steroidogenic tissues that ex- pressed transcripts. As shown in Fig. 1, high levels of SF-1-specific transcripts were detected in three classi- cal steroidogenic cells: adrenocortical cells, testicular Leydig cells, and theca cells of the ovary. Additional analyses of ovary showed that SF-1 was also ex- pressed at high levels in corpus luteum (Ikeda, Y., unpublished observation), consistent with our previous demonstration of SF-1 in corpus luteum RNA in North- ern blotting analyses (2). Lower levels of SF-1 tran- scripts were present in ovarian granulosa cells (Fig. 1, lower panel). Finally, although the significance of this finding remains to be determined, the SF-1-specific antisense probe also produced signals in some semi- niferous tubules (see Fig. 1, middle panel). Control hybridizations with an SF-1-specific sense probe showed no hybridization above background in any of these cell types (Ikeda, Y., unpublished observation). These results significantly extend previous Northern blotting analyses of SF-1 expression, directly linking expression of this putative SF-1-encoding cDNA and steroidogenic cell types in vivo.
Next, we examined the effect in gel mobility shift assays of polycional antisera raised against the amino- terminal region of the protein encoded by this cDNA (see Materials and Methods for a description of the sequences used for immunization). As previously re- ported (6), Y1 adrenocortical nuclear extracts formed prominent, SF-1-related shift complexes with both the aldosterone synthase -310 and 21-hydroxylase -65 elements (complex I in Fig. 2). Addition of antisera raised against the product of the putative SF-1 cDNA abolished this complex; in contrast, none of the other shift complexes seen with the 21-hydroxylase -65 element were affected by the antisera. Although incu- bation of both probes with control and immune sera also led to the appearance of larger complexes whose
precise nature remains to be defined, the specific effect of the antisera on the SF-1-related complex strongly supports the model that this cDNA encodes SF-1.
Finally, we analyzed the ability of the putative SF-1 cDNA to affect transcriptional activity. First, we deter- mined the effect of coexpression of the SF-1 transcript on expression of p21-OHaseGH, a reporter plasmid that contains 6.4 kilobases of 5’-flanking sequences from the mouse 21-hydroxylase gene upstream of the human GH (hGH) reporter gene (7). As shown in Fig. 3, cotransfection of mouse Y1 adrenocortical cells with the putative SF-1 cDNA increased by 12-fold the expression of the hGH reporter gene. Supporting the specificity of this effect, no enhancement followed co- transfection with an expression plasmid driving the expression of the ELP cDNA, which has an identical DNA-binding domain (Fig. 3).
To establish the role of SF-1 sites in this induction, we examined the effect of SF-1 coexpression on a reporter plasmid (p-65-luc) containing five copies of a known SF-1-binding site cloned upstream of the core promoter from PRL driving the expression of the luci- ferase reporter gene (7). As shown in Fig. 4, the parental plasmid pPro36-luc was expressed at low levels and showed no response upon cotransfection with SF-1. In contrast, p-65-luc, which has five copies of the 21- hydroxylase -65 element, showed a marked response to SF-1, with a 50-fold increase in luciferase activity. The finding that expression of the putative SF-1 cDNA markedly augments promoter activity of the 21-hydrox- ylase 5’-flanking region and of a reporter plasmid con- taining a known SF-1-responsive element strongly sup- ports the link between this cDNA and SF-1.
A Single Structural Gene Encodes Both SF-1 and ELP
Based on genomic blotting experiments and partial sequence of the SF-1 cDNA, we proposed that the SF- 1 and ELP transcripts arise from a single structural gene by alternative promoter usage and/or RNA splic- ing (2). We therefore screened a Balb/c mouse genomic library in the EMBL 3 bacteriophage vector with 5’- and 3’-probes derived from our SF-1 cDNA and isolated overlapping clones containing both 5’- and 3’-se- quences (see Materials and Methods for the derivation of the probes). Hybridization with ELP-specific probes showed that the SF-1 clones also contained sequences specific for the ELP transcript, establishing definitively that both cDNAs derive from a single structural gene, which we designate FTZ-F1 in keeping with its original identification in Drosophila (5).
We next determined the organization and DNA se- quence of the gene encoding the SF-1 and ELP tran- scripts. Probes corresponding to the unique 5’-ends of each cDNA were used to isolate the 5’-ends of the transcription units; the transcription start site for SF-1 in Y1 cells was identified by primer extension and RNase protection assays and by comparison with CDNAs isolated by 5’-rapid amplification of cDNA ends
ADRENAL
C
M
TESTIS
ST.
OVARY
O
G
T
Fig. 1. SF-1 Is Selectively Expressed in Steroidogenic Cell Types The patterns of expression of transcripts encoded by the putative SF-1 cDNA were assessed by in situ hybridization analyses with cRNA probes as described in Materials and Methods. Bright-field examination is shown on the left and dark-field examination on the right. Adrenal gland (magnification, x92.5): C, cortex; M, medulla. Testis (magnification, x185): ST, seminiferous tubule; I, interstitium. Ovary (magnification, ×185): O, oocyte; G, granulosa cells; T, theca cells.
(8). Although the precise transcription start site of ELP has not been defined, primer extension analyses sug- gest that transcription initiates near the most 5’-se- quences contained in the previously reported ELP CDNA (3; Kim, E., unpublished observation). Exon-in-
tron junctions were identified by comparison of genomic and cDNA sequences; in all cases (Fig. 5), canonical GT … AG splice donor/acceptor sequences were present at exon-intron junctions (9).
As listed in Fig. 5 and summarized schematically in
Control
Preimmune
anti-SF-1
Control
Preimmune
anti-SF-1
-
III
II
1
AS -310
21-OHase -65
Rabbit polyclonal antibodies against the amino terminal re- gion of the putative SF-1 cDNA were produced as described in Materials and Methods. Gel mobility shifts were performed with the aldosterone synthase -310 (AS -310) and 21-hy- droxylase -65 (21-OHase -65) elements and nuclear extracts from Y1 adrenocortical cells. Where indicated, the nuclear extract was preincubated with either preimmune or immune sera before the addition of probe.
200
hGH (1g/ml)
100
0
Control
PCMVSF-1(-) pCMVSF-1(+)
PCMVELP
Y1 adrenocortical cells were transfected with 2.5 µg p21- OHaseGH, and hGH was measured as described in Materials and Methods. Where indicated, cells were cotransfected with 5 µg pCMV expression vector directing the expression of the putative SF-1 cDNA in either the correct (+) or reverse (-) orientation or the ELP cDNA in the correct orientation. The results are the mean of four independent transfections for each condition.
80000
00000
Luciferase units
40000
20000
0
pCMV119(-)
pCMV119(+)
pCMV119(-)
pCMV119(+)
pPro36-luc
p-65-luc
Y1 adrenocortical cells were transfected with 2.5 µg pPro36- luc or with p-65-luc, lysed 48 h after transfection, and luciferase expression was measured using a kit from Promega as de- scribed in Materials and Methods. Cells were cotransfected with 5 µg SF-1 expression vectors in the correct (+) or reverse (-) orientations. The results are the means of four independent transfections for each condition.
Fig. 6, the complete SF-1 coding sequence predicts a protein with an M, of approximately 52,000, consistent with previous estimates of the size of SF-1 (2). SF-1 is transcribed from the more 5’ promoter, and its entire first exon contains 5’-untranslated sequences. Al- though we have not definitively mapped the ELP tran- scription initiation site, the most 5’ sequences in the ELP cDNA (3) fall within the first intron of SF-1 (position 228 in Fig. 5). Splicing at the 3’-splice site of the SF-1 first intron generates common ELP and SF-1 coding sequences just upstream of sequences encoding the first zinc finger. SF-1 translation initiates at an internal Met in the ELP sequence, and only 12 residues precede the shared first zinc finger region (positions 694-760). The next two shared exons encode the second zinc finger region (positions 908-1039) and a conserved region, termed the FTZ-F1 box, recently implicated in DNA binding (positions 1122-1214) (10).
After additional shared sequences that encode part of the ligand-binding domain (11), alternative splicing generates ELP-specific sequences. In contrast, two additional exons generate the 3’-SF-1-specific se- quences. The precise 3’-terminus of the ELP gene has not been mapped, as a full-length ELP cDNA has not been isolated, and sequencing of genomic clones has not identified a potential AATAAA 3’-splice signal. Nonetheless, as shown in Figs. 5 and 6, the entire organization of SF-1 and the general features of the FTZ-F1 gene that lead to the similarities and differences between the ELP and SF-1 transcripts have been elucidated.
GTCCGCCGCTCCGCCGCTGCCCGCCCGCTGCTGGGTGAAGAAGTTTCTGAGAGCCCGCTAGCCACTGCCCTACCTGAGGCCTOGGAGCCTCCCCACCAGGACCCTGGTGTCCAGTGTCCAC 121
CCTTATCCGGCTGAGAATTCTCCTTCCGTTCAGTAAGTGAAGGCCGGGGCCAGGCTCTANNNNNNNNNNAATGTCGTTGGCACCGCATTCCTGTCCCTGTCACCCAGGATCCCCTCTCCGT 242 + 3.1 kb
TGAGCAGTACTGGCTGGAGTCTCTGTCTCTTCTAGACTCTCTGCCTCAGGTCTCTGTCGGGGCCCCCAGAACAATCCACCTGTGTGECCTACTICGCCCTGCTTCCCTGGCCTCTGTCTTG 363
CCCCCCTCCGACCCTATCTGATTTTCTCAGAATCGGCCTTTTGTTCTCAGACAAACCAATCTGGATCCAAATGCATCGAATCCCAGCGTCCCCGATCGGGCGCGGCACAGGCCCCCACCAA 484
Mat CCAGCCCTGCAACGCGGCCGGTGGCTGAGCTGCAGCCCAGGGACATGGCCCACAAACCCCCCCACCCGGCCCGGGCTGGGGACCGCCCCGTGTGCACAGACCAGGGCAATCCCAAGCCAGT 605
CGCCGTCGGCCCGCGCTGACCCCATCCTCCTTCCACAGGCGGACGCCCCGGGCATGGACTACTCCTACCACGAGGACCTCGACGACCTGTGTCCAGTGTGTCCTCACAAGGTCTCGCCCTA 726
Met Asp Tyr Ser Tyr Asp Glu Asp Lou Asp Glu Lau Cys Pro Val Cys Gly Asp Lys Val Ser Gly Tyr
CCACTACGGGCTGCTCACGTGCGAGAGCTGCAAGGTGTCGGGCTACCACTAGGGCTGCTCACGTGCGAGAGCTGCAAGGTGAGCGCAAAGCGGGAGCAGGGAGGGTGTAGGCAGCGGGTGG 847
His Tyr Gly Lou Lou Thr Cys Glu Ser Cys Lys
CAAGGCTGCGGGCCGCCAACGAAGGTEGGGACETGGCGCTCAYTCCCTTCCTCTGGAGCAGGGCTTCTTCAAGCGCACAGTCCAGAACAACAAGCATTACACGTGCACCGAGAGTCAGAGC 968
Gly Phe Phe Lys Arg Thr Yal Gin Asn Asn Lys His Tyr Thr Cys Thr Glu Ser Gin Ser
TGCAAAATCGACAAGACGCAGCGTAACCGCTGTCCCTTCTGCCGCTTCCAGAACIGCCTGACGGTGGGCATGCGCCTGGAAGGTGGCGTCCTGCACGCCCGGGACCCCCTCACTCCTGGCC 1089
Cys Lys Ile Asp Lys Thr Gin Arg Lys Arg Cys Pro Phe Cys Ang Phe Gin Lys Cys Leu Thr Val Gly Met Arg Lou Glu
GNNNNNAACCTGACGACCTGCTCCTGCCCACAGCTGTGCGTGCTGATCGAATCCGGGGTCCCCCCAACAAGTTTGCCCCCATGTACAAGACAGACCGGGCCTTGAAGCAGCAGAAGAAACC 1210 2.0 kb
Ala Val Ang Ala Asp Any Net Angy Gy Gy Ang Asn Lys Phe Gly Pro Met Tyr Lys Ang Asp Ang Ao Leu Lys Cin Cin Lys Lys Ala
ACAGATTCGGGCCAATGGCTTCAAGCTGGAGACCGGACCACCGATGGGGGTGCCCCCCCCACCCCCTCCCCCACCGGACTACATGTTACCCCCTACCCTGCACGCACCGGAGCCCAAGGCC 1331 Gin Hle Arg Ala Aen Gly Phe Lys Leu Glu Thr Gly Pro Pro Met Gly Yal Pro Pro Pro Pro Pro Pro Pro Pro Asp Tyr Met Lau Pro Pro Ser Leu His Alo Pro Glu Pro Lys Ala
CTGCTCTCTGGCCCACCCAGTGGGCCGCTGGGTGACTTTGGAGCCCCATCTCTACCCATCGCTGTGCCTGGTCCCCACCGACCTCTGCCTGCCTACCTCTATCCTGCCTTCTCTAACCGCA 1452 Lou Val Sar Gly Pro Pro Ser Gly Pro Lau Gly Asp Phe Gly Ala Pro Ser Leu Pro Met Ala Val Pro Gly Pro His Gly Pro Lau Na Gly Tyr Lau Tyr Pro Ala Phe Ser Asn Arg
CCATCAAGTCTGAGTATCCAGAGCCCTATGCCAGCCCCCCACAACAGCCAGGGCCACCCTACAGCTATCCAGAGCCCTTCTCAGGAGGGCCCAATGTACCAGAGCTCATATTGCAGCTGCT 1573
The Ile Lys Ser Giu Tyr Pro Glu Pro Tyr Ala Ser Pro Pro Gin Gin Pro Gly Pro Pro Ty Sar Tyr Pro Ciu Pro Phe Ser Gly Gly Pro Aan Val Pro Glu Lou Ile Lau Gin Lou Lou
GCAACTAGAGCCAGAGGAGGACCAGGTGCGCGCTCGCATCGTGGGCTGTCTGCAGGAGCCAGCCAAAAGCCGCTCTGACCAGCCAGCGCCCTTCAGCCTCCTCTGCAGAATCCCCGACCAG 1694
Gin Lau Glu Pro Clu Clu Asp Cin Val Arg Ala Arg Ile Val Gly Cys Leu Cin Glu Pro Ala Lys Ser Ang Ser Aap Gin Pro Ala Pro Phe Ser Lau Leu Cye Arg Met Ala Amp Gin ACCTTTATCTCCATTGTCGACTGGGCACGAAGGTGCATGGTCTTTAAGGAGCTGGAGGTGGGTACCNNNNNNNCTCTGCATTATTCTTCCCTACAGGAGCTGACCAGATGACACTGCTGCA 1615
-5.8 kb-
Thr Phe le Ser Hle Val Asp Trp Na Ang Ang Cys Met Val Phe Lyt Glu Lau Glu Yal
Ala Amp Gin Met Thr Lou Lou Gin
GAACTGTTGGAGCGAGCTGCTGGTGTTGGACCACATCTACCGCCAGGTCCAGTACGCCAAGGAAGACAGCATCCTGCTGGTTACTGGACAGGAGGTAACTGAACTGGTCAMACCCCTAGTC 1936
Aan Cys Trp Sar Glu Lau Lau Val Lau Asp His Ile Tyr Arg Gin Val Gin Tyr Gly Lys Glu Aap Ser Ile Lou Lou Val Thr Gly Cin Glu CTGCATAATCCCAGGCCTCTCCAGGCTGACTCGGCACACCCCAAATTCCAAATTCAGGGACATGCACTACCCAGGCTTCTCTCTGTCCTGGGGCCATTTGAGGAGCCACACTCTCGAATGG 2057 TCAGTGGAAGTTCTTATAGGAGATAACACTGGAATTGGATCTCCCCAAAATAAGCCACAGTTGGTCAGCTGGTACTGAACTGACACGTGGCAGTCTATGCTCTCACACATGTGGATGCTGC 2178 AGGATCTAGGACTCTGAGAAGGGATTCGGAATAGGGCCAAGCAACTGTGGGGTGGCAAGTGTAAGAGATGAGGTTGCAGGCATTGCTAAGGGCTGGTGAGCAGGTGAGACCCTGTAGGTCT 2299 CCATTCTCATAAATGATGAATGAATAAATGAATGAACGAATGAACCAACCAACACAAGAGTGGGCGAAAAGTAAAGAGACCTCAGACTCAGAATTTGGCCCTTTTACATCAGAGAATGTAT 2420 TACGGGGTOGGAAGTCATTAGGAAGGCCTGGAAACTCCTGAACTCCACCTCCACCCACAGGAGGGACATTAGGGTTCCTAGTGGATGTGGCTGAAGGAGCCTAAAGTGCTGGGGTTTGGAC 2541
ACATTTAAAGAGGACAGCACAGACATGGTGAGAGGAGGTATACGGAAAGAAGTAGGAGGAAGAAAACAMAATCTCTCGAGACAGGAGAGTGGCCTCATGACGGCACACAGGTTNNNNNNNN 2662
-0.9 kb-
NNJACCIGCCCTCTGCTGGTACAGGTGGACCTGAGCACAGTGGCTGTGCAGGCTGCCTCCCTGCTGCACAGCCTCGTGCTGCCCCCCCAAGAGTTAGTGCTCCAGTTCCATCCACTCCACC 2783
Val Glu Lau Ser The Val Na Val Cin Ala Gly Ser Leu Leu His Ser Lou Val Leu Arg Ala Gin Glu Leu Val Leu Cin Leu His Ala Lou Gin
TGGACCCCCAGGAGTTCGTCTGTCTCAAGTTCCTCATCCTCTTCACCCTCGGTGAGTCCCTCTGCAGGCTNNNNNNNNNNTGGGCTGCTCCCATTGCAGATGTGAAATTCCTGAACAACCA 2904
2.5 kb
Leu Asp Arg Gin Glu Phe Val Cys Lou Lys Phe Lou Ile uru Pre Ser Lou
Asp Yat Lys Phe Lou Ann Asn His
CAGCCTCGTAAAGGACGCCCAGGAAAAGGCCAACGCTGCCCTGTTGGATTACACCTTGTGTCACTACCCACACTGCGGGGACAAATTCCAGCAGTTGCTATTGTGCCTGGTGGAGGTCCGG 3025
Ser Lou Val Lys Asp Na Gin Glu Lys Aa Aan Na Ala Lau Leu Asp Tyr Thr Lou Cys His Tyr Pro His Cys Gly Amp Lys Phe Gin Gin Lou Lou Lou Cys Lau Val Glu Val Arg
GCCCTGACCATGCACCCCAAGGAGTACCTGTACCACAAGCATTTGGGCAACGAGATGCCCCCCAACAACCTTCTCATTGAGATGCTGCAGGCCAAGCAGACTTGAGCCTGGGTGCCAGGCA 3146
Ala Lau Ser Met Gin Ala Lys Glu Tyr Lau Tyr His Lys His Lau Gly Asn Glu Met Pro Ang Aan Asn Lou Lou Ile Giu Mat Leu Cin Ala Lys Gin Thr .
GCGGGCAATAGGCAGGGATGCCACTGCCTCCAAAAGACTCCTTGCATTAGGGATCCAGGAGCCCTGTCACTAAGCCCCTGCCCCTGAGCTCCAGAGCTGTGTGTTTGGGCAAGGATGGGCG 3267
GGGATTGGCCGGGGGCAGGTTGCCTTTACTAGCCATTGGCCTGTGTCCGCCACTTGGAGTGCCCCAAAGGGCTTCTAACCATTCCTTCCTCCATCAGCCCCCAGCTTTTTTTCCTGGTATC 3388 TGAGGTCCCAGGAGGAGGCTCAGGATTCCCTGGTGGGTCTGGATGTCCCTTGGGTCAGAGGTCATCCTTTCCCTCTCTCCTGTTATCAGAGGCAAGGAAGGTCTACAGGCATCAATGAGGG 3509
CAAAGGAGGGGGTCTCCAGACTCCACTGAAGCAGGAAGTCCACTGTTGTAAACTGAGTTTGCTAAATTCGGTCCCCAGAGGATACCATGAGAGTGGGTAGGGCAAAAAGAGCCCTTTCCGC 3630
CCTCTACCCATCTAATTCTGATCCTCTACCAGTAGGAGGACTTTGGTGTGATCATCCTTCTCCCAGGGCCCGGCTACCCAGGGAGGAGGAGTCTGGTCTAGCCAACATTCCTCCCCTAACC 3751
CTGCCCATCACCAGCTGGCTCGCTGGTATTTATCTGCAAGGTTGAACTCACTGCCATTCTTTTCTTCACCTAGATAGTCCTTGGAAAGTGTGTCAGAGAGAACTGGGCAGGAGACACACTG 3872
GGGACTGAGCTGGGATATGGCGACTACCATCAAACCTTTCTCCTCACATCTCTTTCCAAGAGTCGGGGTGGCATCTGTACCCCACCTCACCCCCCACAAGTGCTATTGCTTGCCCTCTCCC 3993
TCAGCCCCACTAGGAACAACAGGAGGCCTGCTGGGGCTTAGACTCCGTGCAGGTGGGGATATGCCTAAATCTAGGAGAACTCACAGATCTTTATATGAGGACAGTGCTGAGGACTTTCTCA 4114
TGGCTCCATCTTTTGGTCCCCTCGCCACTACCCTTCAACCTGGCTTCAGTTCCCTGGCTGCTGCTTTGCCTCCTGAAAGCCACTCTGTAGGACCAAGCACTCGGGGGAGACGCCTAAGCCA 4235
TCCTCTGTTCCAGACTGGACATCCACTGTCTTTCCTGCTTTCGCGTCAGATTTACAGCTTATGCTAGGCCCACCCAACTGGACAAGGCTGICTCCTGTCTICTACTACCCTGCTCAGACCC 4356
CCACCTCTGCCCCTGAAATGCGTGCTCCCACCAAGGCCAGAGACCCACAGCCCCAGACAAGAAGTGCCCTTATAAACCCCTGCAGCCCTGAAATAAATTTTCCAATTACTTTTCCAGTAGC 4477
ATTTGTGAATTTGCATTCTGAATCGAG 4604
ELP ATG
SF-1
FIZ-F1 Box
ELP Stop
SF-1 Stop
ATG
ZF ZF
1
2
1 kb
The Two Transcripts Encoded by the Mouse FTZ-F1 Gene Have Different Patterns of Expression
Because studies with FTZ-F1 in Drosophila suggested developmentally regulated expression of the distinct forms, we studied the ontogeny of ELP and SF-1 tran- scripts. To this end, we performed in situ hybridization analyses with ELP- and SF-1-specific cRNA probes. ELP transcripts were not detected in any mouse devel- opmental stages studied (embryonic day 8 to adult; Ikeda Y., unpublished observation), consistent with the previous isolation of ELP from embryonal carcinoma cells and its postulated role in early embryonic devel- opment. In contrast, SF-1 transcripts were first de- tected in mouse adrenal gland at embryonic day 12 (Fig. 7) and persisted thereafter at high levels. SF-1 expression in the testis likewise began at embryonic day 12 and persisted at high levels throughout the rest of the prenatal period (Ikeda, Y., unpublished obser- vation). Of interest, expression of the cholesterol side-chain cleavage enzyme was first detected in the developing adrenal gland at embryonic day 12 (12), consistent with the model that SF-1 expression is tightly coupled to that of the steroid hydroxylases. Consistent with the relatively inactive state of the fetal ovary, SF- 1 expression was not detected in this organ in fetal sections (Ikeda, Y., unpublished observation).
DISCUSSION
In this report, we establish that the mouse homolog of FTZ-F1 encodes two distinct transcripts: a protein orig- inally isolated from embryonal carcinoma cells, termed
ELP, and a global regulator of steroid hydroxylase gene expression, termed SF-1. We further document that the cloned SF-1 cDNA augments promoter activity of the 5’-flanking region of steroid 21-hydroxylase and that SF-1 is expressed very early in the development of primary steroidogenic tissues, consistent with its pos- tulated key role in the function of steroidogenic cells.
To date, direct demonstration of functional effects of SF-1 has only been obtained with adrenocortical cells. However, SF-1 binding sites are found upstream of all steroidogenic cytochrome P450 genes characterized (13). Experiments with aromatase expression in rat granulosa cells implicate SF-1 in steroid hydroxylase expression in nonadrenocortical steroidogenic cells (14, 15). Moreover, preliminary experiments suggest that SF-1 also regulates Leydig cell expression of oxytocin, a noncytochrome P450 gene (Parker, K., and R. Ivell, unpublished observation), and our in situ hybridization experiments (Fig. 1) suggest that SF-1 may be ex- pressed in cells of the germ cell lineage. The emerging evidence thus points to an important role of SF-1 in proper function of all of the primary steroidogenic tissues.
The role of ligand(s) in SF-1-mediated transcriptional activation remains to be defined. Our transfection ex- periments (Figs. 3 and 4) show significant effects of coexpression of SF-1 in Y1 adrenocortical cells without the addition of any exogenous ligand. As these cells lack endogenous expression of steroid 21-hydroxylase and thus cannot produce either mineralocorticoids or glucocorticoids, it is unlikely that corticosteroid end products subserve such a role. Other candidate ligands include intermediates in the steroid biosynthetic path- way and vitamin E, which is found at very high levels in the adrenal cortex (16). Alternatively, it remains possible
The DNA sequence of the mouse FTZ-F1 gene and predicted translation of the SF-1 transcript are indicated. All positions are relative to the SF-1 transcription initiation site. The numbers below the brackets indicate the sizes of introns whose sequences are not included. Sequences common to both SF-1 and ELP transcripts are underlined with wide arrows, sequences that are uniquely found in the SF-1 transcript are underlined with arrows of intermediate width, and sequences that are uniquely found in the ELP transcript are underlined with thin arrows. The Met at position 450 denotes the initiator Met for ELP.
A
B
that SF-1 does not require a specific ligand for activa- tion. NGFI-B, another orphan receptor that regulates steroid hydroxylases (7), apparently activates transcrip- tion in a ligand-independent manner.
Recent studies have shown surprising similarities between the nuclear receptors of Drosophila and mam- mals (reviewed in Ref. 17). Among the conserved members are chicken ovalbumin upstream promoter transcription factor/seven-up (18, 19), retinoid X recep- tor-B/ultraspiracle (20, 21), and hepatocyte nuclear fac- tor 4/tailless (22, 23). Based on these similarities, it is not entirely unexpected that mice express a homolog, which we designate FTZ-F1 in keeping with its original description in Drosophila. Based on the strict mainte- nance of developmentally regulated forms of FTZ-F1, certain hypotheses regarding the function of the related transcripts in the two species seem attractive. The early form of Drosophila FTZ-F1 is proposed to regulate the expression of the ftz homeobox gene. Although the corresponding gene in mice remains to be identified, it is plausible that ELP (the early form of FTZ-F1 in mice) regulates the expression of a mouse hox gene that contributes to embryological development. Clearly, ELP does not regulate the expression of the steroid hydrox- ylases, as an ELP expression plasmid did not affect 21- hydroxylase promoter activity under the conditions where a marked response to SF-1 was obtained (see Fig. 3).
Models can likewise be formed about the late form
of FTZ-F1 in Drosophila. There are a number of similar- ities between the vertebrate steroid hormones and the ecdysteroids of invertebrates. In particular, both ecdy- sone and mammalian steroids are derived from choles- terol by the action of cytochrome P450 mixed-function oxidases (1, 24), and both act by binding to ligand- responsive nuclear receptors (25, 26). Finally, both compounds play key roles in the normal development and sexual function of the organism. It is tempting to speculate that the late form of FTZ-F1, for which no function has been elucidated, regulates the expression of enzymes involved in ecdysone biosynthesis. Ulti- mately, proof of this model will require the characteriza- tion and cloning of the proteins that synthesize ecdy- sone and the demonstration that late FTZ-F1 regulates their expression.
MATERIALS AND METHODS
Materials
Restriction and modification enzymes were obtained from New England Biolabs (Beverly, MA) or Boehringer-Mannheim (Indi- anapolis, IN). Radionuclides were purchased from New Eng- land Nuclear/E. I. DuPont (Boston, MA). Levels of hGH were measured by RIA using a kit purchased from Nichols Diagnos- tics. Expression of luciferase was measured with an LKB (Piscataway, NJ) luminometer using an assay kit purchased from Promega (Madison, WI). Sequenase Il kits for sequence analysis of double-stranded plasmids were purchased from
United States Biomedical (Cleveland, OH). A kit for 5’ rapid amplification of cDNA ends was purchased from Bethesda Research Laboratories (Gaithersburg, MD).
Preparation of Anti-SF-1 Antisera
Sequences comprising the putative zinc finger DNA-binding domain of SF-1/ELP (exonic sequences corresponding to po- sitions 136-1218 in the FTZ-F1 sequence in Fig. 5) were cloned in the prokaryotic expression plasmid pGEX-1xt and expressed in Escherichia coli as previously described (2). The crude bacterial lysate containing the glutathione S-transferase- SF-1 fusion protein was partially purified by affinity chromatog- raphy as described (27). Briefly, 12 ml bacterial extract was mixed with 500 ul of a 1:1 suspension of glutathione (GSH)- sepharose beads and PBS and gently rocked at 4 C for 30 min. The GSH-sepharose beads were centrifuged for 5 min at 1000 x g and washed three times with 5 ml of PBS + 10 mm EDTA. The protein was then resuspended in 2 ml PBS + 10 mM EDTA at a concentration of approximately 200 ug protein/ ml and stored at 4 C. Rabbits at Organon-Teknica-Cappel laboratories (West Chester, PA) were then immunized with the affinity-purified SF-1-GST/GSH-sepharose slurry as described (27).
Cell Culture
Mouse Y1 adrenocortical tumor cells were cultured and trans- fected by the CaPO. precipitation technique as described (7), using 2.5 µg of the reporter gene and 5 µg of the SF-1 expression plasmid. Levels of hGH were determined by RIA 48 h after transfection. In experiments with the firefly luciferase reporter plasmid, cells were lysed and luciferase activity de- termined according to the protocol supplied with the luciferase assay kit.
Plasmids
The plasmids used in this study included: p21-OHaseGH, which contains 6.4 kilobases of 5’-flanking sequences from the mouse 21-hydroxylase gene placed upstream of an hGH reporter gene (7); pCMV-SF-1(+), a cytomegalovirus (CMV) promoter-based eukaryotic expression plasmid, which con- tains SF-1 cDNA sequences from the EcoRI site 33 bp up- stream of the initiator Met to 604 bp downstream of the termination codon; pCMV-SF-1(-), a CMV promoter-based eukaryotic expression plasmid that contains the identical SF- 1 sequences in the reverse orientation; pCMV-ELP, a CMV promoter-based eukaryotic expression plasmid that contains the ELP coding sequences in the correct orientation; pSF-1 5’, which contains SF-1 cDNA sequences from +30 to +114; pSF-1 3’, which contains SF-1 3’-untranslated sequences (3488-3733 in Fig. 5); pELP 5’, which contains ELP sequences from +1 to +430 (3); pPro36-luc (28), which contains the core promoter from the PRL gene upstream of the firefly luciferase reporter gene; and p-65-luc, a derivative of pPro36-luc with five copies of the 21-hydroxylase -65 element inserted up- stream of the PRL promoter (17).
Gel Mobility Shift Assays
Crude nuclear extracts were prepared from Y1 cells by the method of Shapiro et al. (29). Gel mobility shift assays with the aldosterone synthase -310 and 21-hydroxylase -65 probes were performed as described (2). Where indicated, preimmune or immune anti-SF-1 antiserum was included in the binding reaction for 30 min before addition of probe in the gel shift assays.
In Situ Hybridization Analyses
Seven-micrometer-thick paraffin sections of mouse adult tis- sue (adrenal, testis, and ovary) and embryos (E8-E16) were
purchased from Novagen (Madison, WI). In situ hybridizations with [35S]cRNA probes were performed using an in situ hybrid- ization kit obtained from Novagen according to the recom- mended protocol. Probes included SF-1 3’, which contains sequences from the SF-1 3’-untranslated region (positions 3488-3733 in Fig. 5), and ELP 5’, which contains sequences from the 5’-untranslated region of ELP (+1 to +430 in Ref. 3). Exposures were for 3-4 weeks, and slides were counter- stained with methyl green. No signals over background were seen with any of the sense probes.
Note Added in Proof
After this paper was submitted, Honda et al. reported the sequence of a cDNA encoding Ad4-binding protein, the bovine homolog of SF-1 (Honda S .- I. et al., J Biol Chem 268:7494- 7502). They also concluded that their protein was the bovine homolog of FTZ-F1.
Acknowledgments
We thank Dr. Richard Garber for helpful discussions about in situ hybridization, Dr. Bernard Schimmer for helpful discus- sions and critical reading of the manuscript, Drs. Ken Moro- hashi, Jeffrey Milbrandt, and Thomas Wilson for helpful dis- cussions, Dr. Ohtsura Niwa for generously providing the ELP CDNA, Dr. William Kuziel for helpful discussions about the PCR, Jeana Meade and Andrea Mouw for superb technical assistance, and Jane Everson for secretarial assistance.
Received February 18, 1993. Revision received April 19, 1993. Accepted May 13, 1993.
Address requests for reprints to: Dr. Keith L. Parker, Box 3047, Duke University Medical Center, Durham, North Carolina 27710.
This work was supported by the Howard Hughes Medical Institute.
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