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J. Biol. Chem., Vol. 277, Issue 10, 7945-7954, March 8, 2002

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AP-2 and the Homeodomain Protein Distal-less 3 Are Required for Placental-specific Expression of the Murine 3-Hydroxysteroid Dehydrogenase VI Gene, Hsd3b6^*

Lihong Peng and Anita H. Payne

From the Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California 94305

Received for publication, July 18, 2001, and in revised form, December 20, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The enzyme 3-hydroxysteroid dehydrogenase/isomerase (3-HSD) is essential for the biosynthesis of all active steroid hormones. It exists as multiple isoforms in humans and rodents, each the product of a distinct gene. Human 3-HSD I in placenta is essential for placental progesterone biosynthesis and thus is essential for the maintenance of pregnancy. The murine ortholog, 3-HSD VI, is the only isoform expressed in giant trophoblast cells during the first half of mouse pregnancy. This study was designed to identify the cis-acting element(s) and the associated transcription factors required for trophoblast-specific expression of 3-HSD VI. Transfection studies in placental and nonplacental cells identified a novel 66-bp trophoblast-specific enhancer element located between 2896 and 2831 of the 3-HSD VI promoter. DNase protection analysis of the enhancer element identified three trophoblast-specific binding sites, FPI, FPII, and FPIII. Electrophoretic mobility shift assays with oligonucleotides representing the protected sequences, FPI and FPIII, and nuclear extracts isolated from human JEG-3 cells and from mouse trophoblast cells, demonstrated the same binding pattern that was distinct from the binding pattern with mouse Leydig cell nuclear proteins. Further electrophoretic mobility shift assays identified AP-2 and the homeodomain protein, Dlx 3, as the transcription factors that specifically bind to FPI and FPIII, respectively. Site-specific mutations in each of the binding sites eliminated enhancer activity indicating that AP-2 and Dlx 3, together with an additional transcription factor(s) that are conserved between humans and mice, are required for trophoblast-specific expression of 3-HSD VI.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The enzyme 3-hydroxysteroid dehydrogenase/isomerase (3-HSD)¹ is essential for the biosynthesis of all active steroid hormones: the adrenal steroid hormones, cortisol, corticosterone, and aldosterone; the testicular steroid hormone, testosterone; and the ovarian and placental hormones, progesterone and estradiol. The 3-HSD enzyme exists as multiple isoforms in humans and rodents, each the product of a distinct gene (1). To date, six isoforms have been identified in mouse and two in human. These isoforms are expressed in a tissue- and temporal-specific manner (1-5). In the mouse, the two major isoforms involved in steroid hormone biosynthesis are 3-HSD I and 3-HSD VI. 3-HSD I is the major or only isoform expressed in gonads and adrenal glands, whereas 3-HSD VI is the only isoform expressed in giant trophoblast cells during mid-pregnancy (3). The orthologous isoforms in human are human 3-HSD II, the isoform expressed in the gonads and adrenal glands (6), and human 3-HSD I (7, 8), the only isoform expressed in placenta throughout pregnancy. The expression of human 3-HSD I in placenta is essential for placental progesterone biosynthesis and, thus, is vital for maintenance of pregnancy (9). Consistent with the role of human 3-HSD I in placental progesterone biosynthesis, we have shown that mouse 3-HSD VI is required for progesterone biosynthesis in giant trophoblast cells between embryonic day (E) 9.5 and E10.5 (10).

Progesterone biosynthesis from cholesterol requires the activity of two enzymes, cholesterol side chain cleavage cytochrome P450 (P450scc) which catalyzes the conversion of cholesterol to pregnenolone and 3-HSD which catalyzes the conversion of pregnenolone to progesterone. This latter step is brought about by distinct tissue-specific isoforms of 3-HSD. Previous studies designed to identify placental-specific regulatory elements in the human placental-specific 3-HSD promoter were unsuccessful (8). Moreover, identity of transcription factors essential for placental-specific expression of P450scc remains to be resolved (11). Therefore, the question is whether there is a unique tissue-specific transcription factor or factors required for the expression of the trophoblast-specific isoform of 3-HSD as well as the other enzyme required for progesterone biosynthesis, P450scc, in human placenta and mouse giant trophoblast cells.

In the present study, we identify a 66-bp trophoblast-specific enhancer element located between 2896 and 2831 5' of the transcription start site of the murine 3-HSD VI promoter and demonstrate the requirement for two transcription factors, AP-2, and the homeodomain protein, Distal-less 3 (Dlx 3), in determining trophoblast-specific expression of the 3-HSD VI gene. Dlx 3 is a member of a family comprising at least six Dlx genes sharing a homeobox sequence similar to that found in the Drosophila Distal-less gene (12). Dlx proteins are transcriptional activators which play an essential role during vertebrate development (12). AP-2 is a member of a family of three closely related and evolutionary conserved sequence-specific DNA-binding proteins which include AP-2, AP-2, and AP-2 (13). Dlx 3 and AP-2 are expressed in both murine and human placental trophoblast cells and are required for the placental-specific expression of a number of genes (14-18). Our studies demonstrate that these two transcription factors regulating the trophoblast-specific expression of the steroidogenic enzyme, 3-HSD VI, are conserved in both murine and human trophoblast cells. This is the first report on the identification of at least two of the transcription factors required for placental-specific expression of 3-HSD in humans and mice.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Isolation of Genomic Clones-- A  phage 129/SvJ mouse genomic library from Stratagene (La Jolla, CA) was screened with an 180-bp probe labeled with [-³²P]dCTP, which includes 18 bp of coding region and 167 bp of 3'-untranslated region of the 3-HSD VI cDNA (3). Prehybridization and hydribization were performed as described (19). Two clones (402 and 602) were identified to represent 3-HSD VI. The clones were subjected to restriction enzyme and Southern blot analysis using exon-specific oligonucleotide probes (exon 2, 5'-CCAGAGGATTGTCCAGTTG-3'; exon 3, 5'-GACATCTAGGATGGTCTG-3'; exon 4, 5'-AGGAAGCTCACAGTTTCCA-3'). Restriction fragments from clone 402 were subcloned into the pBluescript-KS vector and subjected to further analysis to identify the location of exon 1 and to establish the start site of transcription.

Rapid Amplification of 5'-cDNA Ends (5'-RACE)-- 5'-RACE was carried out using a Marathon cDNA library from CLONTECH (Palo Alto, CA) prepared from a 7-day pregnant mouse implantation site. The primary amplification was performed as described by the manufacturer using touchdown PCR techniques with adapter primer AP1 (5'-CCATCCTAATACGACTCACTATAGGGC-3') and 3-HSD VI-specific primer GSP1 (5'-GCCCGTACAACCGAGAATATT-3', 628 bp 3' of ATG in exon 4) (20). Secondary amplification was performed using nested adapter primer AP2 (5'-ACTCACTATAGGGCTCGAGCGGC-3') and 3-HSD-specific primer GSP2 (5'-CAGACCATCCTAGATGTC-3', 283 bp 3' of ATG in exon 3). The secondary PCR product was subject to sequencing using primers AP2 or GSP2.

Preparation of Giant Trophoblast Cells-- Timed pregnant C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) were killed by CO₂ followed by cervical dislocation at E9.5 and E10.5, and the uterine horns were removed and placed in phosphate-buffered saline for further isolation of giant trophoblast cells as described (5). E9.5 and E10.5 implantation sites were separated from the myometrium and the embryos were removed. Giant trophoblast cells were gently removed by scraping the inner face of the hemi-implantation site and collected for preparation of RNA or proteins.

Primer Extension-- The transcription initiation site of 3-HSD VI was determined using the primer extension kit from Promega (Madison, MI). Messenger RNA was isolated from E10.5 mouse giant trophoblast cells and adult mouse testes using the QuickPrep Micro mRNA Purification Kit (Pharmacia, Piscataway, NJ). Primer GSP3 (5'-GGTTCTGATCTCTGCAAAGGAACCAG-3', 132 bp 5' of exon 1 end), which is specific for 3-HSD VI, was end-labeled with [-³²P]ATP using T4 polynucleotide kinase. Approximately 100 fmol of labeled primer and 3-5 µg of mRNA were hybridized at 65 °C for 20 min, followed by gradual cooling to room temperature. The reactions were extended by avian myeloblastosis virus reverse transcriptase at 42 °C for 1 h and 15 min and followed by heating at 99 °C for 5 min. The extended DNA fragments were precipitated with ethanol and subjected to electrophoresis in a 6% denaturing polyacrylamide gel.

Construction of Plasmids-- To characterize the promoter region of the mouse 3-HSD VI gene, a series of 5' deletions spanning between 4700 and 40 (Fig. 3) were subcloned into a promoterless luciferase reporter vector pA3LUC at the SmaI-HindIII sites (21). To make heterologous constructs, different fragments, including 3004/1989, 3004/2500, 3004/2723, and 2722/2500, were subcloned into a heterologous thymidine kinase promoter-driven luciferase reporter vector TK164LUC at the SmaI site. Further deletions within the 3004/2723 fragment, including sequences 2896/2725, 2830/2725, and 2896/2831, were generated by PCR using primers with SmaI site added at the 5' end and subcloned into TK164LUC/SmaI. The sequences 2896/2857 and 2867/2831 were synthesized with a half SmaI site added in both 5' and 3' ends and subcloned into TK164LUC/SmaI. The PCR fidelity and orientations of inserts of each construct were verified by restriction enzyme digestion and sequencing.

Mutagenesis in the Enhancer Region-- Mutations in the potential AP-2, TEF, or Dlx 3-binding sites in each footprint (FPI, FPII, and FPIII) (Table I) that were identified by DNase I footprinting were introduced by the ExSite PCR-based site-directed mutagenesis kit (Stratagene) following the instructions given in the manual. Positive clones were identified and verified by sequencing in the DNA Sequencing Facility at Stanford University.

Cell Culture and Transient Transfections-- Both human choriocarcinoma cells, JEG-3 (ATCC HTB-36) and monkey kidney tumor cells, COS-7 (ATCC CRL-1651), were cultured in Dulbecco's modified Eagle's medium with 50 µg/ml gentamycin supplemented with 10% fetal bovine serum (Invitrogen, Gaithersburg, MD). MA-10 cells, a mouse Leydig tumor cell line (a gift of Dr. Mario Ascoli), were grown in Waymouth's MB752/1 medium containing 15% horse serum. JEG-3 and MA-10 cells were transfected by calcium phosphate-DNA precipitation (22). COS-7 cells were transfected using the FuGENE 6 reagent (Roche Molecular Biochemicals, Indianapolis, IN). Cells were plated at a density of 0.5-1 × 10⁵ cells/20-mm well. After 20 h of culture, the testing plasmid (0.5 µg DNA) and pSV₂-Gal (23) (0.1 µg of DNA) were co-transfected in triplicate. Following transfection (42 h), cells were lysed using the Reporter Lysis Buffer (Promega). The activities of luciferase and -galactosidase were measured according to the manufacturer's description. The luciferase activity of each construct was normalized to the co-transfected -galactosidase activity.

Preparation of Nuclear Extracts-- Crude nuclear extracts from JEG-3 and MA-10 cells were prepared as described (24). Nuclear extracts from E10.5 giant trophoblast cells and E15.5 placental tissues were isolated using the 1-h minipreparation techniques (25). Total protein was quantitated by Bradford assay and normalized against extraction buffer. The extracts were aliquoted and stored at 70 °C until use for footprinting or EMSA.

DNase I Footprinting Assay-- DNase I footprinting was carried out using Promega's core footprinting kit with modifications. To generate the probe, the 120-bp fragment (2916/2800) amplified from PCR was subcloned into the pBluescript-KS vector at the EcoRI-HindIII sites. The insert was sequenced for verification. A 167-bp/XbaI-XhoI fragment released from the vector was labeled with T4 polynucleotide kinase and [-³²P]ATP. Digestion with AccI or SpeI resulted in formation of a sense or antisense probe, respectively. Increasing amounts of crude nuclear extract were incubated with the labeled probe (10,000 cpm) at room temperature for 10 min, followed by digestion with 6 units of DNase I at room temperature for 3 min. The digested products were extracted with phenol/chloroform (1:1), ethanol-precipitated, and analyzed using 5% denaturing polyacrylamide gel electrophoresis.

Electrophoretic Mobility Shift Assay (EMSA)-- EMSAs were performed as described previously (26). The 20-µl binding reaction containing 5-10 µg of crude nuclear extracts and 0.05-0.25 ng of radiolabeled probes (50,000 cpm/reaction) was incubated for 20 min at room temperature. The sequences of the double-stranded oligonucleotides used for EMSAs are listed in Table I. For competition assays, a 50-500-fold molar excess of unlabeled oligonucleotides was added to the binding reaction mixture. For supershift assays, 2 µg of polyclonal antisera to AP-2, AP-2, Nkx2-5 (Santa Cruz Biotechnology, Santa Cruz, CA), Dlx 3 (a gift of Dr. Mark Roberson), or preimmune sera were added to the mixture prior to the addition of the probe. The binding reactions were resolved on a 5% nondenaturing polyacrylamide gel.

Western Blot Analysis-- Extracts of MA-10 cells, transfected COS-7 cells, E9.5 or E10.5 giant trophoblast cells, or adrenal glands or testes from 50-day-old mice were prepared by homogenization in extraction buffer followed by centrifugation as described previously (3). The supernatant was subjected to SDS-PAGE and Western blot analysis. Membranes were first incubated with antiserum generated against human placental 3-HSD and then with the horseradish peroxidase-labeled secondary antibody and exposed using the Enhanced ChemiLuminescence kit (Amersham Biosciences, Inc., Arlington Heights, IL).

In Situ Hybridization-- Cryosections (8 µm) of implantation sites from E9.5 and E10.5 were subjected to in situ hybridization as described (22) using a 359-bp ³⁵S-labeled sense or antisense cRNA probe representing 45 bp from the 3' end of the coding region and 314 bp from the 3'-untranslated region of 3-HSD VI cDNA (3). Slides were exposed at 4 °C and developed after 4 days. The slides were stained with hematoxylin and eosin and examined under light microscopy with bright- and dark-field illumination.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Genomic Structure of the 3-HSD VI Gene-- To characterize the 3-HSD VI gene, a  phage genomic DNA library was screened with a 3-HSD VI cDNA probe. Two phage clones, 402 and 602, were identified as encompassing the entire 3-HSD VI gene. Restriction mapping and Southern blot analysis revealed that clone 402 contains ~10 kb of 5'-flanking region, and exon 2 through exon 4 ending at the SacI site in the 3'-untranslated region (3), whereas clone 602 contains a portion of intron 2, exon 3, exon 4, and 9 kb 3'-flanking region (Fig. 1A).

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Genomic structure and transcriptional start site of the mouse 3-HSD VI gene. A, the horizontal line represents the structure derived from the two genomic clones, 402 and 602. Exons are indicated by the numbered boxes; solid boxes, coding region; open boxes, noncoding region. ATG, start site of translation; TGA, stop codon. Restriction enzymes used in mapping and subcloning are shown as vertical lines. B, BamHI; Bg, BglII; C, ClaI; EV, EcoRV; S, SacI; Sp, SpeI; St, StuI. B, primer extension analysis was performed as described under "Experimental Procedures." The arrow indicates the extended band. A single 145-nucleotide band was detected with mRNA from E10.5 mouse giant trophoblast cells (GC) and from adult mouse testes (Te), but not from adult mouse spleen (Sp). Lanes C, T, A, and G are dideoxy termination sequence reactions of the 3-HSD VI with primer GSP3.

Identification of Exon 1 Sequence and Mapping of the Transcription Start Site of the 3-HSD VI Gene-- 5'-RACE analysis was performed to identify the exon 1 sequence. The 3' 340-bp sequences from the 5'-RACE PCR product matched with known cDNA sequences including 206 bp of exon 2 and 134 bp of exon 3 (3). To confirm that the new sequence from the 5'-RACE represents the exon 1 sequence, reverse transcriptase-PCR was performed using RNA isolated from E10.5 mouse giant trophoblast cells and from mouse testicular tissues with a forward primer designed from the 5'-RACE exon 1 sequence and a backward primer designed from the known sequence of exon 2 (3). A fragment of the expected size was obtained. Thus, exon 1 was identified and the size of intron 1 was established to be 3.1 kb, which differs from human 3-HSD I or mouse 3-HSD I (1).

The transcription start site for 3-HSD VI was confirmed by primer extension (Fig. 1B). Using mRNA from adult mouse testes and from E10.5 mouse giant trophoblast cells, a single transcription start site was identified at a guanosine residue 315 bp 5' of the translation initiation codon in exon 2 which defines the size of exon 1 as 252 bp.

Mouse Trophoblast-specific Expression of 3-HSD VI mRNA and Protein-- We previously reported the expression of 3-HSD mRNA and protein in mouse giant trophoblast cells at E9.5 (5). However, the earlier study did not use isoform-specific probes for the identification of 3-HSD. To determine the isoform-specific expression of 3-HSD VI, in situ hybridization of sections of E9.5 and E10.5 implantation sites were analyzed using a 3-HSD VI-specific antisense probe (see "Experimental Procedures"). Fig. 2A shows exclusive expression of 3-HSD VI mRNA in giant trophoblast cells at E9.5 and E10.5 with considerably greater expression at E10.5. No expression of 3-HSD VI mRNA was observed in decidua or embryo. The increase in 3-HSD VI mRNA in giant trophoblast cells at E10.5 is accompanied by a parallel increase in 3-HSD VI protein as determined by Western blot analysis (Fig. 2B).

View larger version (90K): [in this window] [in a new window]   Fig. 2.   Expression of 3-HSD VI mRNA and protein in mouse giant trophoblast cells at E9.5 and E10.5. A, implantation sites from E9.5 (panels A and B) and E10.5 (panels C and D) were sectioned and hybridized with 35S-labeled antisense RNA to 3-HSD VI. Panels A and B are light and dark field views of E9.5. The dark field exposure shows hybridization only in giant trophoblast cells surrounding the embryonic cavity. Panels C and D are light and dark field views of E10.5. Panel c shows intense silver grains in the light field exposure exclusively in giant trophoblast cells. The considerably greater expression of 3-HSD VI in giant trophoblast cells in E10.5 compared with E9.5 is observed in the dark field exposure (compare B and D). Bar, 10 µm. Data with the sense probe not shown. B, Western blot analysis of 3-HSD proteins was carried out with protein extracts from E9.5 and E10.5 giant trophoblast cells (10 µg of protein); rVI, pCMV5.3-HSD VI transfected COS cells; and rI, pCMV5.3-HSD I transfected COS cells (30 µg of protein, each); T, testis from 50-day-old mouse (75 µg of protein); A, adrenal from 50-day-old mouse (1.4 µg of protein); O, ovary from 50-day-old mouse (1.5 µg of protein).

SF-1 Is Not Required for Expression of 3-HSD VI in JEG-3 Cells-- In gonads and adrenal glands expression of steroidogenic enzymes is dependent on steroidogenic factor 1 (SF-1) (27, 28). SF-1 null mice develop normal placental trophoblast cells that express steroidogenic enzymes (29) indicating that this factor is not involved in placental-specific expression of steroidogenic enzymes. Our studies (Table II) demonstrate that the transcriptional activity of the murine 3-HSD VI proximal promoter transfected into JEG-3 cells that do not contain SF-1, is 22-fold greater than the transcriptional activity of the gonadal- and adrenal-specific 3-HSD I promoter. Furthermore, co-transfection with an SF-1 expression vector resulted in a dose-dependent increase in 3-HSD I transcriptional activity, but had no effect on 3-HSD VI transcriptional activity. Thus, SF-1 is not required for expression of the 3-HSD VI enzyme in the placenta.

View this table: [in this window] [in a new window]   Table II Effect of SF-1 on transcriptional activity of the mouse 3-HSD I and VI promoters in JEG-3 cells The sequences 359/+35 of 3-HSD I and 443/+33 of 3-HSD VI were subcloned into a promoterless luciferase reporter vector pGL3-Basic (Promega), respectively, and then transfected into JEG-3 cells as described under "Experimental Procedures." Data from one experiment carried out in triplicates.

Promoter Activity and Cell-specific Expression of the 5'-Flanking Region of the 3-HSD VI Gene-- Because of the lack of a mouse trophoblast cell line and the difficulty in obtaining mouse primary giant trophoblast cells for transfection studies, the human placental choriocarcinoma cell line, JEG-3, was chosen for the transfection studies described herein. JEG-3 cells express human 3-HSD I (30), the tissue-specific ortholog to mouse 3-HSD VI. To establish trophoblast-specific expression, nonplacental cell lines, both mouse Leydig tumor cells (MA-10) and monkey kidney cells (COS-7), were also used for the promoter and enhancer analyses of the mouse 3-HSD VI gene.

To characterize the promoter sequences involved in transcriptional regulation of the 3-HSD VI gene in trophoblast cells, a series of 5' deletions of the 3-HSD VI gene, spanning from 4700 to 40 5' of exon 1 (Fig. 3), were subcloned into a promoterless luciferase reporter vector, pA3LUC, and transiently transfected into JEG-3, MA-10, or COS-7 cells. As shown in Fig. 3, a marked increase in basal transcription activity was observed in all three cell lines when the promoter sequence was increased from 40 to 91, suggesting that a common positive cis-acting element is located between 40 and 91, although the luciferase activity was ~10-fold greater in JEG-3 and MA-10 cells compared with COS-7 cells. A database analysis of this sequence shows that there are several potential binding sites for transcription factors CREB and Sp1 in the segment between 40 and 91. No further analysis was performed with this proximal sequence.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Transcriptional activity of the 3-HSD VI promoter. A series of 5' deletions of the 3-HSD VI promoter-luciferase reporter constructs, as indicated on the top, were transiently transfected into JEG-3 (JEG), MA-10 (MA), and COS-7 (COS) cells. Luciferase activity was normalized to -galactosidase activity and expressed relative to the promoterless vector pA3LUC, whose activity is set as 1. Each value represents the mean ± S.E. of three separate transfections, each performed in triplicate. The inset illustrates the relative luciferase activity between 40 and 91 in the three cell lines (×20).

As larger fragments from 91 to 4700 were included, the basal transcription activities remained essentially the same in both MA-10 and COS-7 cells. In contrast, a large increase in basal activity was observed in JEG-3 cells with fragments greater than 1989. The results suggest that the sequence between 3004 and 1989 may contain a trophoblast-specific enhancer element.

Analysis of Enhancer Activity and Characterization of the Enhancer Region-- To determine whether the sequence between 3004 and 1989 has the properties of an enhancer, this region was subcloned 5' of the heterologous thymidine kinase (tk) promoter either in the sense or antisense direction and transfected into JEG-3, MA-10, and COS-7 cells. Results of the transfections are shown in Fig. 4. The sequence between 3004 and 1989 increased tk promoter activity over 8-fold in either the sense or antisense directions in JEG-3 cells, but not in COS-7 or MA-10 cells. These data indicate that the sequence between 3004 and 1989 has the characteristics of a trophoblast-specific enhancer element. Because a further increase in transcriptional activity was observed between 4700 and 3004 (Fig. 3), this 1700-bp fragment was also subcloned 5' of the tk promoter and transfected into JEG-3 cells. No increase in the promoter activity was observed with this fragment (data not shown).

View larger version (27K): [in this window] [in a new window]   Fig. 4.   The sequence between 3004 and 1989 of the 3-HSD VI promoter contains the JEG cell-specific transcriptional element. The fragment comprising the sequence between 3004 and 1989 was subcloned in either sense or antisense orientation 5' of the heterologous tk promoter-driven luciferase reporter vector (TK164LUC). The reporter constructs were transfected into the three cell lines and luciferase activity of each construct was expressed relative to the vector TK164LUC. Each value represents the average plus the range of two separate transfections, each performed in triplicate.

To further define the regulatory domains within the 3004/1989 fragment of the 3-HSD VI promoter, subsequent deletions were made and subcloned 5' of the tk promoter. Fig. 5A shows the results of the deletion constructs transfected into JEG-3, MA-10, and COS-7 cells. No enhancer activity with any of the deletion constructs was observed in MA-10 or COS-7 cells. In contrast, in JEG-3 cells, deletions of the 3' sequence from 1989 to 2500, did not change enhancer activity compared with the 3004/1989 fragment. Further deletion from 2500 to 2723 displayed similar enhancer activity. The data suggest that the 282-bp fragment between 3004 and 2723 contains the trophoblast-specific enhancer element.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Identification of the trophoblast-specific minimal enhancer element. A, 5' or 3' deletions within the sequence between 3004 and 1989 (as illustrated on the left) were subcloned 5' of the tk promoter in the TK164LUC vector and transfected into the three cell lines. Luciferase activity of each construct is expressed relative to the vector TK164LUC. B, further 5' or 3' deletion mutants were constructed using the 282-bp fragment (3004/2723) identified in A as a backbone. The constructs were transfected into JEG-3 cells. Luciferase activity of each construct is expressed relative to the 3004/2723 construct which displayed full enhancer activity. The activity of the 3004/2723 construct was set at 1. Each value represents the average plus the range of two separate transfections, each performed in triplicate.

To characterize the minimal enhancer region, additional 5' deletions within 3004/2723 were made and subcloned 5' of the tk heterologous promoter. The transfection results are shown in Fig. 5B. When the 5' sequence was deleted from 3004 to 2896, the activity remained the same as the 3004/2723 construct. However, further 5' deletion to 2830 reduced transcriptional activity by 73%, suggesting that the enhancer element is located between 2896 and 2830. To confirm that this 66-bp fragment between 2896 and 2831 has trophoblast-specific enhancer activity, it was directly subcloned 5' of the tk heterologous promoter. Surprisingly, this 66-bp fragment increased tk promoter activity ~3-fold relative to the 3004/2723 fragment (Fig. 5B). This finding not only provides conclusive evidence that the sequence (2896/2831) contains the enhancer element, but also suggests that an inhibitory element is located between 2830 and 2723 as removal of this 108-bp fragment from the 3004/2723 region resulted in a significant increase in enhancer activity. Further 3' or 5' deletions of the 2896/2831 fragment resulted in complete loss of the enhancer activity. The data suggest that the entire sequence between 2896 and 2831 is required for trophoblast-specific enhancer activity.

Potential Transcription Factor-binding Sites-- To determine the corresponding protein-binding nucleotides within the 66-bp enhancer, DNase I footprinting assay was performed using an end-labeled fragment containing the sequence 2916/2872 (including the 66-bp fragment identified above as the trophoblast-specific enhancer element). Three protected regions, FPI, FPII, and FPIII, were observed on both antisense (data not shown) and sense strands in JEG-3 cells (Fig. 6A), but not in MA-10 cells (data not shown). A database search (TRANSFAC) for potential transcription factors revealed that the first region (FPI) protected a sequence between 2885 and 2876 which contains a potential Ker1-binding site (GGCTGTAGCC) (31) (Fig. 6B). Footprint FPII between 2869 and 2857 encompassed a predicted TEF site (CGCATTCTA) (32). A perfect binding site for GATA (WGATAR) between 2853 and 2841 (33) or a potential binding site for Nkx2-5 (TAATT) between 2850 and 2846 (34) was protected in the third footprint (FPIII).

View larger version (48K): [in this window] [in a new window]   Fig. 6.   DNase I protection analysis of the trophoblast-specific minimal enhancer element. A, a fragment of the 3-HSD VI gene from 2916 to 2800 was end-labeled on the sense strand, incubated with increasing amounts of crude nuclear extract of JEG-3 cells (from left to right, 30-120 µg) and subjected to DNase I digestion. The extent of each footprint is indicated on the right. B, nucleotide sequences of the minimal enhancer region with the nucleotides comprising each footprint are indicated by the bar above the sequence. The putative transcription factor binding sites found within each footprint are marked by a line above or below the appropriate sequence. The letters in italic indicate mismatches from the consensus sequences.

To determine whether the sequence representing each footprint interacts with the predicted factor present in trophoblast cells, EMSAs were performed using double-stranded oligonucleotides corresponding to each of these footprints as probes and nuclear extracts isolated from JEG-3 cells, E10.5 giant trophoblast cells, or MA-10 cells. The probe representing FPI or the probe representing FPIII, each gave rise to a specific DNA-protein complex. No binding activity was observed with the probe representing FPII (data not shown).

Identification of AP-2 as the Trophoblast-specific Transcription Factor That Binds to FPI-- The FPI element forms a specific DNA-protein complex (complex I) with identical mobility with nuclear extracts of either JEG-3 or giant trophoblast cells (Fig. 7B, lanes 2 and 5) which was not observed with the nuclear extract of MA-10 cells (lanes 8-10). Five hundred-fold molar excess of unlabeled FPI competed for binding of the labeled FPI probe (Fig. 7B, lanes 3 and 6), whereas an unlabeled FPI oligonucleotide containing mutations within the potential Ker1 site (mFPI) could not compete (lanes 4 and 7). This observation suggests that the FPI element may interact with the Ker1-binding protein. To test this hypothesis, labeled probes representing FPI and Ker1 were subjected to EMSAs using JEG-3 cell nuclear extract. Fig. 7C demonstrates that each of these probes yielded a DNA-protein complex with identical mobility and, furthermore, 500-fold molar excess of either unlabeled FPI or unlabeled Ker1 competed for binding of each of the probes. Binding of the JEG-3 nuclear protein to the Ker1 probe is greater than to the FPI probe (Fig. 7C, lanes 1 and 4) and competition with unlabeled FPI for binding to the Ker1 probe is somewhat less than the competition observed with unlabeled Ker1 (Fig. 7C, lanes 5 and 6). These results suggest that FPI and Ker1 bind the same trophoblast nuclear protein with different affinities.

View larger version (42K): [in this window] [in a new window]   Fig. 7.   Specific binding activity of FPI with trophoblast cell nuclear proteins. A, sequences in the oligonucleotides representing FPI, Ker1, an AP-2 binding element in the human k14 keratin gene (31) and the mutated FPI (mFPI) which represents a five-nucleotide substitution mutation in the potential Ker1 site within the FPI sequence. The consensus nucleotides for AP-2 binding are illustrated in bold. B, EMSA with 10 µg of nuclear protein isolated from JEG-3 cells, or E10.5 mouse giant trophoblast cells (GTC), or MA-10 cells and radiolabeled probe FPI. Lane 1 is free of nuclear extract. C, EMSA with JEG-3 nuclear extract and with probes FPI or Ker1.

The protein that specifically binds to Ker1 has previously been identified as AP-2 (31). The AP-2 family consists of three distinct proteins, AP-2, AP-2, and AP-2. AP-2 is the predominant AP-2 family member expressed in mouse placenta and giant trophoblast cells (15). Both AP-2 and AP-2 are expressed in JEG-3 cells and in human placenta (16, 17). To identify whether the protein that specifically binds to FPI is a member of the AP-2 transcription factor family and, furthermore, to distinguish between AP-2 and AP-2, EMSAs were performed with the FPI probe and nuclear extracts of JEG-3 cells, of E10.5 giant trophoblast cells, or of E15.5 mouse placentas in the presence or absence of polyclonal antisera to AP-2 or AP-2. As shown in Fig. 8A, mouse placental nuclear proteins formed a DNA-protein complex (lane 4) with identical mobility to complex I as shown in Fig. 7 for JEG-3 cell and mouse giant trophoblast cell nuclear extracts. This complex was displaced by the addition of antiserum to AP-2 (lanes 3, 6, and 10) but not by the addition of AP-2 antiserum (lanes 7 and 11) or by normal rabbit serum (NRS, lane 8). The results shown in Fig. 8B illustrate that the JEG-3 cell nuclear protein that specifically binds to the Ker1 probe also is AP-2 and not AP-2. The data presented in Fig. 8 establish that the human and murine trophoblast-specific protein that interacts with FPI is the transcription factor AP-2.

View larger version (36K): [in this window] [in a new window]   Fig. 8.   Identification of the FPI-binding protein in trophoblast cells as AP-2. EMSA was performed with 10 µg of nuclear extracts of JEG-3 cells, E15.5 mouse placenta, or E10.5 mouse giant trophoblast cells (GTC) and specific antisera to AP-2 or AP-2 or normal rabbit antiserum (NRS). A, FPI as the probe (see Fig. 7A). Lanes 2 and 5 represent 500-fold molar excess of cold competitor FPI. B, Ker as the probe (see Fig. 7A). Lane 2 represents 500-fold molar excess of Ker1.

Identification of Distal-less 3 (Dlx 3) as the Trophoblast-specific Transcription Factor That Binds to FPIII-- As described above, a database search for transcription factors involved in producing FPIII in the DNase I protection assay (Fig. 6) indicated two potential binding sites, GATA or Nkx2-5. These two potential sites overlapped. To characterize binding of JEG-3 and giant trophoblast nuclear proteins to the FPIII sequence, EMSAs were performed using a radiolabeled probe comprising the entire protected sequence as well as an FPIII mutant as competitor (Fig. 9A). A specific DNA-protein complex (complex III) with identical mobility was observed with nuclear extracts of both JEG-3 and giant trophoblast cells (Fig. 9A, lanes 2 and 5), which was distinct from the complex formed with MA-10 cell nuclear extract (Fig. 9A, lane 8). This finding indicates that the FPIII-binding nuclear protein is trophoblast-specific. The competition assay with mFPIII which contains mutations comprising the entire GATA site, as well as the 5' nucleotides of the Nkx2-5-binding site, resulted in loss of competition with the FPIII probe (Fig. 9A, lanes 4 and 7). To delineate the recognition sequence for the trophoblast nuclear protein within the FPIII element, a series of FPIII oligonucleotides with sequential double mutations were tested for their ability to compete with the FPIII probe in an EMSA (Fig. 9B). As illustrated in Fig. 9B, oligonucleotides containing mutations involving the nucleotides as represented by m3, m4, m5, m7, m8, and m9 lost or markedly reduced the capacity of these mutants to compete with the wild type FPIII probe indicating that the sequence TAATTG from 2848 to 2843 was the critical binding site for the FPIII-binding protein. The TAATT sequence is identical to the binding site for the murine homeodomain protein, Nkx-2.5, or the current nomenclature, Nkx2-5 (34, 35), and to the binding site of another homeodomain protein Dlx 3. Roberson et al. (18) recently demonstrated that Dlx 3 is the transcription factor that binds to the junctional regulatory element (JRE) of the human glycoprotein hormone  (hCG) subunit gene and is required for basal placental-specific expression of this gene (18) (Fig. 10A). This observation suggests that Dlx 3 may be the human and murine trophoblast-specific nuclear protein that binds to FPIII. To examine whether Dlx 3 is the FPIII-binding protein, EMSAs were carried out with radiolabeled probes FPIII and JRE (18) and JEG-3 cell nuclear extract (Fig. 10B). The FPIII-protein complex (lane 1) displayed identical mobility to the JRE-protein complex with JEG-3 cell nuclear extract (lane 8). In addition, both unlabeled FPIII and JRE oligonucleotides had similar self- and cross-competition with each of the probes for binding to the trophoblast nuclear protein (lanes 2-7 and 9-14). These results are consistent with Dlx 3 being the transcription factor that forms complex III. To establish that the trophoblast-specific protein that binds to FPIII is Dlx 3, EMSAs were performed with the FPIII probe and the JER probe, nuclear extracts of JEG-3 cells (Fig. 10C) or of mouse placentas, (Fig. 10D), specific antisera to Dlx 3 or to Nkx2-5 or preimmune antiserum. The results illustrated in Fig. 10, C and D, demonstrate that Dlx 3 is the nuclear protein in both human trophoblast cells (JEG-3) and mouse placentas that forms a DNA-protein complex with the FPIII or the JER element. Although, incubation with JEG-3 cell nuclear extract and a radiolabeled oligonucleotide containing an Nkx2-5-binding site yielded a DNA-protein complex with identical mobility to the DNA-protein complex formed with the FPIII probe (data not shown), the addition of antiserum to Nkx2-5 did not result in either a supershift or disruption of the DNA-protein complex (Fig. 10, C and D, lanes 2 and 6).

View larger version (38K): [in this window] [in a new window]   Fig. 9.   Characterization of recognition sequence for trophoblast nuclear protein within the FPIII element. A, specific binding of trophoblast cell nuclear proteins to FPIII. EMSA was performed with 4 µg of nuclear extract of JEG-3 cells, or GTC or MA-10 cells and FPIII as a probe. Lane 1 is absence of nuclear extract. Indicated competitors were added at 500-fold molar excess. B, identification of nucleotides within the FPIII element recognized by the trophoblast protein. A series of FPIII oligonucleotides with sequential double mutations as underlined (m1-m5) and as illustrated on top of the figure (m6-m10) were incubated with JEG-3 cell nuclear extract and subjected to EMSA. Lane 1 represents the absence of nuclear extract. Indicated competitors were added at 500-fold molar excess.

View larger version (50K): [in this window] [in a new window]   Fig. 10.   Identification of the FPIII-binding protein in placental cells as Distal-less 3 (Dlx 3). A, similarity between the core binding nucleotides (bold) of the FPIII element and the JRE element, which contains a Dlx 3-binding site characterized in the hCG promoter (18). B, EMSA was performed with nuclear extract of JEG-3 cells (5 µg) and radiolabeled probe FPIII (lanes 1-7) or JRE (lanes 8-14). Indicated unlabeled competitors were added in increasing amounts (50-, 200-, and 500-fold molar excess). C, radiolabeled FPIII (lanes 1-4) or JRE (lanes 5-8) probes were incubated with 5 µg of JEG-3 cell nuclear extract in the absence or presence of specific antisera against Nkx2-5 (lanes 2 and 6) or Dlx 3 (lanes 3 and 7) or preimmune rabbit sera (lanes 4 and 8). D, identical to C except the probes were incubated with 10 µg of mouse placental nuclear extract.

All Three FP Elements within the 66-bp Enhancer Are Essential for 3-HSD VI Transcriptional Activity in JEG-3 Trophoblast Cells-- To determine whether the binding sites within each FP are functional, site-directed mutations were introduced into the heterologous enhancer-tk construct 2896/2831 (Fig. 5B). To determine the requirement for the AP-2-binding site identified in FPI, the mutation, TAGGCA  GGTACC (mFPI), which had been shown to disrupt binding of AP-2, was introduced (Fig. 7A). For disruption of the Dlx 3-binding site in FPIII, TGATAA  AAGCTT (mFPIII) was introduced into the enhancer construct and the potential TEF-binding site in FPII was mutated (GCATTC  AAGCTT) (Table I). Transfection into JEG-3 cells of each of the mutated 66-bp enhancer-tk constructs resulted in complete loss of enhancer activity (Fig. 11). These results indicate that all three proteins, AP-2, FPII-binding protein, and Dlx 3 homeodomain protein, are essential for determining trophoblast-specific enhancer activity. The results do not eliminate the possible requirement for additional proteins.

View larger version (20K): [in this window] [in a new window]   Fig. 11.   Effect of mutations in core nucleotides within each footprint on trophoblast-specific enhancer activity. The binding sites for AP-2, Dlx 3, or the potential TEF protein within the sequence representing each footprint, FPI, FPIII, or FPII, respectively, were mutated as described under "Experimental Procedures." The black symbols signify mutations within the indicated FP. Both mutated and wild-type 2896/2831/TK164LUC constructs were transiently transfected into JEG-3 and MA-10 cells. Luciferase activity of each construct is expressed relative to the vector TK164LUC. Each value represents the average plus the range of two separate transfections, each performed in triplicate.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Progesterone biosynthesis in the human placenta is essential for maintenance of pregnancy (9). Although progesterone is required for maintenance of pregnancy in the mouse, the source has not been unequivocally established. The biosynthesis of progesterone from cholesterol requires two steroidogenic enzymes, P450scc and 3-HSD. P450scc is the product of a single gene that is expressed in human gonads and adrenal glands (36), the human placenta (37), mouse decidua, and giant trophoblast cells (5). Unlike P450scc, 3-HSD exists in multiple isoforms and the isoform expressed in human and mouse trophoblast cells is distinct from the isoform expressed in the gonads and adrenal glands, with one exception, mature mouse Leydig cells express 3-HSD VI in addition to the major gonadal isoform, murine 3-HSD I (Fig. 2B and Refs. 3 and 4). Both P450scc and 3-HSD VI are expressed in a tissue- and temporal-specific manner during the first half of mouse pregnancy (Fig. 2) (5). Several investigations have been undertaken during the past few years in the search for a factor or factors which determine placental-specific expression of steroidogenic enzymes. Guerin et al. (8) were unsuccessful in their attempt to identify a placental-specific element in the promoter of human 3-HSD I. They did identify a strong positive regulatory element in the first intron which functioned in a ubiquitous manner. This element had sequence similarities to the positive nontissue-specific element identified in the murine 3-HSD VI promoter between 40 and 90. The inability by Guerin et al. (8) to identify a placental-specific element in human 3-HSD I, the ortholog of murine 3-HSD VI, may have been due to the limited length of the promoter examined. A recent report by Huang and Miller (11) described two transcription factors related to human immunodeficiency virus-inducible LBP proteins, LBP-1B and LBP-9, that had the capacity to modulate placental-specific expression of human P450scc but were not involved in determining the placental-specific expression of this enzyme. Thus, the identification of the factors that regulate the tissue-specific expression of the steroidogenic enzymes necessary for progesterone biosynthesis in placenta and giant trophoblast cells remains to be established.

In the current study, we identify a 66-bp placental/trophoblast-specific enhancer element located between 2896 and 2831 of the murine 3-HSD VI promoter. DNase I protection analysis of the enhancer element identified three trophoblast-specific binding sites, referred to as FPI, FPII, and FPIII. The protein that binds to FPI was established to be a member of the AP-2 family of transcription factors. EMSA studies using specific antisera to AP-2 and AP-2 demonstrated that the protein binding to FPI is AP-2 and not AP-2. Furthermore, identical results were obtained with nuclear extracts of human placental JEG-3 cells, of mouse placentas or of mouse giant trophoblast cells, which demonstrate that the transcription factor that binds to FPI and is involved in trophoblast-specific expression of murine 3-HSD VI is the same in humans and mice. Human placenta and JEG-3 cells express both AP-2 and AP-2 (15, 16), while AP-2 is the predominant member of the AP-2 family expressed in mouse trophoblast cells and its expression is restricted to the trophoblast lineage (15). Shi and Kellems (15) suggest that AP-2 may be one of the key transcription factors regulating gene expression in trophoblast cells. Thus, the finding in this study demonstrating that AP-2 is required for trophoblast-specific expression of 3-HSD identifies one of the important target genes whose product is required for progesterone biosynthesis and thus essential for maintenance of pregnancy.

The protein that specifically binds to FP III was identified as the homeodomain protein, Dlx 3. Dlx 3 was recently identified as a placental-specific transcriptional activator of the hCG subunit gene (18). This transcription factor is expressed in human placenta as well as in JEG-3 cells (18) and in the trophoblast cell lineage that forms the placenta in mice (14). Targeted deletion of the Dlx 3 gene resulted in embryonic death between E9.5 and E10.5 due to placental defects (14). Whether mutation in the human Dlx 3 gene would lead to loss of the fetus during human pregnancy is not known at present. Immunocytochemical studies on sections of first trimester human chorionic villus samples showed that expression of Dlx 3 was found primarily in the trophoblast cell layer surrounding the villus and expression was restricted to the nucleus (18).

EMSA studies using nuclear extracts of either JEG-3 or giant trophoblast cells and an oligonucleotide representing the FPII sequence identified by DNase I protection analysis did not result in the formation of a DNA-protein complex, suggesting that the binding of a nuclear protein to FPII requires the prior binding of either AP-2 or Dlx 3 or both. Although we were unable to show binding of a nuclear protein to the FPII oligonuclotide within the 66-bp enhancer element, binding of a specific protein is essential for trophoblast-specific transcriptional activity as demonstrated by the loss of transcriptional activity when site-directed mutations were introduced separately into each of the FP-binding sites of the heterologous enhancer-tk construct (Fig. 11). This study also demonstrates that AP-2, FPII-binding protein, and Dlx 3 are required for trophoblast-specific expression of the murine 3-HSD VI gene.

A GenBank^TM search of the human 3-HSD I promoter sequence (accession number AL121995) identified an AP-2-binding site at 2857/2848 identical to the FPI core-binding site of murine 3-HSD VI and a binding site for Dlx 3 at 2495/2489 identical to the FPIII-binding site of murine 3-HSD VI (Table III). The placental-specific function of these potential binding sites for AP-2 and Dlx 3 in the human 3-HSD I promoter needs to be established.

View this table: [in this window] [in a new window]   Table III Potential binding sites for AP-2 and Dlx 3 in the human 3-HSD I promoter GenBankTM accession number AL121995.

The identification of a trophoblast-specific enhancer in the murine 3-HSD VI promoter and the demonstration that the two transcription factors, AP-2 and Dlx 3, which are required for the cell-specific expression of this gene, are expressed in both human placenta and mouse trophoblast cells, and the identification of binding sites for these two transcription factors in the human 3-HSD I promoter, suggests that AP-2 and Dlx 3 are the placental nuclear proteins that determine the cell-specific expression of human 3-HSD I whose product is required for placental progesterone production. It is of interest to note that these two transcription factors have been shown to be required for placental-specific expression of the hCG subunit gene (16, 18). Another similarity between the cell-specific expression of the hCG subunit and the expression of 3-HSD is the fact that the nuclear factor SF-1 determines pituitary-specific expression of the LH/CG subunit and adrenal- and gonad-specific expression of 3-HSD (27). It is intriguing to speculate that AP-2 and Dlx 3 are the SF-1 analogous nuclear transcription factors that coordinate trophoblast-specific expression of the placental hormones and enzymes required for maintenance of pregnancy.

	ACKNOWLEDGEMENTS

We thank Yu Ni for assistance with the in situ hybridization. We thank Dr. Mario Ascoli (University of Iowa) for providing the MA-10 Leydig tumor cells, Dr. J. Ian Mason (The Royal Infirmary of Edinburgh, United Kingdom) for providing the human placental 3-HSD antiserum, and Dr. Keith Parker (University of Texas, Southwestern Medical Center) for the mouse SF-1 expression vector. Particular thanks go to Dr. Mark Roberson (Cornell University, Ithaca, NY) for providing the specific antiserum to Dlx 3 and the preimmune rabbit antiserum. We thank Dr. Marco Conti (Stanford University Medical Center) for support and critical reading of the manuscript. We are grateful to Dong Qing Hu for expert technical assistance and Caren Spencer for editorial assistance.

	FOOTNOTES

^* This work was supported by NICHD, National Institutes of Health cooperative agreement U54 HD 31398 as part of the Specialized Cooperative Centers Program in Reproductive Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper for the mouse 3-HSD VI gene has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY046511 (3256 bp promoter and exon 1) and AY046512 (1792 bp complete cDNA).

To whom correspondence should be addressed: Div. of Reproductive Biology, Dept. of Gynecology and Obstetrics, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, CA 94305-5317. Tel.: 650-725-6802; Fax: 650-725-7102; E-mail: anita.payne@stanford.edu.

Published, JBC Papers in Press, December 31, 2001, DOI 10.1074/jbc.M106765200

	ABBREVIATIONS

The abbreviations used are: 3-HSD, 3-hydroxysteroid dehydrogenase/isomerase; P450scc, cholesterol side chain cleavage cytochrome P450; AP-2, activator protein-2; Dlx 3, distal-less 3; TEF, transcription enhancer factor; JRE, junctional regulatory element; hCG, human chorionic gonadotropin; RACE, rapid amplification of cDNA ends; EMSA, electrophoretic mobility shift assay; tk, thymidine kinase; LUC, luciferase; SF-1, steroidogenic factor 1.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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