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Division of Reproductive Biology (L.P., Y.H., A.H.P.), Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, California 94305; and Department of Obstetrics and Gynecology (F.J., S.-W.J.), Mayo Clinic, Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Dr. Anita H. Payne, Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, California 94305-5317. E-mail: anita.payne{at}stanford.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(AP-2) and the homeodomain protein, distaless-3 (Dlx-3), that are expressed in both human and mouse trophoblast cells that were shown to be required for trophoblast-specific expression of the orthologous murine 3ßHSD, 3ßHSD VI. Although we identified specific binding sites for AP-2 and Dlx-3 in the distal promoter of the human 3ßHSD I gene, HSD3B1, it was found that these transcription factors were not involved in determining placental-specific expression of human 3ßHSD I. Instead, a 53-bp placental-specific enhancer element located between –2570 and –2518 of the HSD3B1 promoter was identified. Within this 53-bp element, two potential placental transcription factor binding sites were found. EMSAs with a 20-bp oligonucleotide containing these two potential placental-specific binding sites identified one of the binding sites specific for the transcription enhancer factor (TEF)-5, which is highly expressed in human placenta and in placental choriocarcinoma-derived JEG-3 cells and the other overlapping binding site, specific for a GATA-like protein. Site-specific mutations in either the TEF-5 binding site or in the GATA binding site, each resulted in complete loss of enhancer activity. The data indicate that TEF-5 and the GATA-like protein act in a coordinate manner to determine the placental-specific expression of the human 3ßHSD I enzyme and therefore are critical for placental progesterone production required for the maintenance of pregnancy. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(AP-2) and the homeodomain protein, distaless-3 (Dlx-3). Site-specific mutations in either of the sites to which these proteins bound eliminated enhancer activity (4). A GenBank search of the human HSD3B1 promoter sequence identified an AP-2 binding site at –2857/–2848 identical with the AP-2 binding site of the murine Hsd3b6 promoter and an identical binding site for Dlx-3 at –2495/–2489. The current study was designed to investigate whether these two transcription factors, which are expressed in the human placenta, also determine the placental-specific expression of human 3ßHSD I. |
RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and Dlx-3. A GenBank search of the human HSD3B1 promoter sequence identified an AP-2 binding site at –2857/–2848 and a binding site for Dlx-3 at –2495/–2489 (4). We therefore examined for placental-specific transcriptional activity, a series of deletions of the HSD3B1 promoter spanning from –2880 to +207, which included the AP-2 and the Dlx-3 binding sites and the first exon and first intron sequences. In addition, a fragment –492/+32 that excluded the first intron also was tested for transcriptional activity. The different HSD3B1 promoter fragments were subcloned into a promotorless reporter vector, pA3LUC (4), and transfected into human choriocarcinoma cells (JEG-3), human embryonic kidney cells (HEK293), or mouse Leydig tumor cells (MA-10). As previously reported by Guérin et al. (3), deletion of the first intron resulted in a marked decrease in transcriptional activity of the –492 promoter fragment when transfected into JEG-3 cells (Fig. 1A
). Transcriptional activity in JEG-3 cells of promoter fragments between –492 and –2880 showed a marked decrease in transcriptional activity between –492 and –2416 with a 60-fold increase observed between –2416 and –2880. Similar transfections in MA-10 and HEK293 cells of three of the reporter constructs containing –492, –1067, and –2880 bp resulted in a very different pattern of transcriptional activity than that observed in JEG-3 cells (Fig. 1B). In both of these cell types, the highest transcriptional activity was observed with the –492 promoter fragment with decreasing activity with the longer promoter sequences. In addition, the transcriptional activity relative to the pA3LUC reporter vector was 5% in MA-10 cells and 4% in HEK293 cells for the –492 fragment and considerably less for the –2861 fragment when compared with the activity in JEG-3 cells. These results suggest that the sequence between –2416 and –2861 contains the essential element(s) for determining placental-specific expression of the 3ßHSD I enzyme. Furthermore, sequences between –492 and –2416 appear to contain elements that inhibit the transcription in placental cells.
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illustrates the results obtained with this construct containing the 466-bp fragment in the sense direction. The fragment between –2861 and –2395 increased promoter activity almost 90-fold in either sense or antisense direction (antisense data not shown). No increase in transcriptional activity over the TK164 promoter construct was observed when this 466-bp fragment was transfected into HEK293 cells (data not shown). To determine whether the Dlx-3 and/or the AP-2 binding sites are the determinant sites for placental-specific expression of the enhancer element, further 5' and 3' deletions were made involving these sites, and the resulting constructs were subcloned 5' of the TK164 promoter and transfected into JEG-3 cells. Deletion of the sequence between –2518 and –2395, which eliminated the Dlx-3 binding site, yielding a fragment including sequences between –2861 and –2518 resulted in an approximately 3-fold increase in enhancer activity compared with the –2861/–2395 fragment. Further deletion 5' to the Dlx-3 binding site yielding a fragment encompassing sequences between –2861 and –2570 resulted in a complete loss of enhancer activity (Fig. 2). Deletion of the AP-2 binding site between –2861 and –2820 had no effect on enhancer activity relative to the –2861/–2518 fragment. These findings indicate that the enhancer activity was located between –2570 and –2518 (Fig. 2). To confirm that this 53-bp fragment contains the trophoblast-specific enhancer activity, a single copy and an element containing five copies of the 53-bp fragment were each cloned 5'of the TK164 heterologous promoter and transfected into JEG-3 cells. As shown in Fig. 2, the heterologous promoter construct containing one copy of the 53-bp fragment resulted in a 90-fold increase in promoter activity, whereas the promoter construct containing five copies increased promoter activity almost 450-fold. Transfection of these two plasmids containing either one or five copies of the –2570/–2518 fragment into HEK293 cells did not result in any activity above that observed with the plasmid containing only the TK164 heterologous promoter (data not shown). These results demonstrate that sequences within this 53-bp fragment determine placental-specific expression of HSD3B1. A database analysis of this sequence identified two potential binding sites, GGAATG at –2563/–2558, which represents the core of a consensus binding site (5'-(A/T) (A/G) (A/G) (A/T) ATG (C/T) (G/A)-3') for a group of four related transcription enhancer factor (TEF) transcription factors (5) and a potential GATA binding site, TGATAG at –2559/–2554 (6), which overlaps with the TEF-binding site. Boldfacerepresents consensus sequence. To examine whether JEG-3 cells contain a protein or proteins that bind specifically to either of these potential binding sites in the HSD3B1 enhancer region, we carried out EMSAs with nuclear extracts of JEG-3 cells and a 20-bp oligonucleotide (referred to as HSD-En in the text) comprising the sequence between –2567/–2548 (which included the potential binding site for TEF and GATA) and a series of sequential double mutations within this sequence (Fig. 3B). As shown in Fig. 3C, the JEG-3 nuclear extract forms two specific DNA-protein complexes, I and II (lane 2) when incubated with the 32P-labeled HSD-En probe as indicated by competition with 500-fold molar excess of the unlabeled oligonucleotides. Sequential double mutations of the oligonucleotide between –2567 and –2552 as represented by m1–m12 showed a loss of competition of complex II with nucleotides represented by m1–m6 (Fig. 3C, lanes 7–12), indicating that the sequence GGAATG from –2563 to –2558 comprised the binding site of complex II. This binding site is identical with the six core nucleotides of the placental-specific binding site, DF-4, identified in the human chorionic sommatomammotropin-B enhancer (hCS-B) (5). Loss or markedly reduced competition for protein(s) bound to nucleotides comprising complex I was observed with the mutated oligonucleotides m5 to m10 representing the sequence TGATAG (Fig. 3C, lanes 11–16). This sequence (A/T)GATA(A/G) represents a binding site for GATA transcription factors (6).
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demonstrates that 500-fold molar excess of the DF-4 oligonucleotide competed with the protein found in complex II (lane 4), whereas the same oligonucleotide with the two GGs mutated (mDF-4) in the TEF-5 binding site (ttAATG) lost the ability to compete (lane 5). Figure 4A also demonstrates that the nuclear extract from the 18-wk gestation placenta exhibited the same binding properties as observed with JEG-3 cell nuclear extract (lanes 7–10). These data indicate that the protein that forms complex II is expressed in both JEG-3 cells and human placenta and also is the protein that binds to the DF-4 binding site of the hCS-B enhancer that was previously identified as TEF-5.
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, lanes 3 and 5), but not with mHSD-En (Fig. 4B, lane 4). To compare the relative affinities of the TEF-5 protein for DF-4 and HSD-En, a competitive EMSA was performed. As seen in Fig. 4C using the HSD-En as the radiolabeled probe and TEF-5 transfected JEG-3 cell extract with increasing molar concentrations of unlabeled competitor oligonucleotides, 256-fold molar excess of unlabeled HSD-En almost completely inhibited binding of the radiolabeled oligonucleotide (lane 13), whereas DF-4 even at 1024-fold molar excess did not completely inhibit the binding of the TEF-5 protein to the HSD-En oligonucleotide (lane 7). No competition was observed with mHSD-En (lanes 16–21). These results demonstrate that in vitro-generated TEF-5 specifically binds HSD-En and furthermore under identical conditions, HSD-En appears to have a greater affinity for TEF-5 than the well-characterized TEF-5 binding element of the hCS enhancer (5, 7).
Analysis of Protein in Complex I of the HSD-En
To examine whether complex I is a specific binding site for one of the GATA-binding proteins, an initial EMSA was performed using radiolabeled HSD-En and an oligonucleotide containing two consensus GATA binding sites, GATAcw, and determined whether this oligonucleotide could compete with the radiolabeled probe HSD-En. Figure 5A demonstrates that 500-fold molar excess of the GATAcw oligonucleotide competed with the protein found in complex I (lane 4), whereas a nonlabeled HSD-En oligonucleotide with a specific mutation in the potential GATA binding site, TGcgAG (mGATA), lost the ability to compete with the protein bound in complex I, but not with the protein of complex II (lane 5). Additional EMSAs with a radiolabeled HSD-En probe and a radiolabeled GATAcw probe and JEG-3 cell nuclear extracts showed that the HSD-En /protein complex I displays identical mobility to the GATAcw/protein complex with the JEG-3 cell nuclear extract (Fig. 5B, lanes 2 and 9). Cross-competition with the unlabeled oligonucleotides for binding to the JEG-3 protein indicated that the GATAcw oligonucleotide had considerably higher affinity for the JEG-3 nuclear protein than the HSD-En oligonucleotide (Fig. 5B, lanes 3–7 and 10–15). At least part of the greater affinity could be attributed to the presence of two GATA-protein binding sites in the GATAcw probe. To assess which of the known GATA proteins is found in the HSD-En/protein complex I, EMSAs were performed using the radiolabeled HSD-En or the radiolabeled GATAcw as probes, JEG-3 nuclear extract, and specific antisera to GATA 1, 2, 3, 4, and 6. Using the consensus GATAcw as a probe, Fig. 5C demonstrates that JEG-3 nuclear extracts contain GATA 2, 3, and 4 with GATA 4 being the most abundant of the proteins and GATA 2 the least abundant (lanes 11–13). With HSD-En as probe, a weak supershift was observed with the GATA-4 antiserum (Fig. 5C, lane 6). Taken together, the results suggest that the JEG-3 nuclear protein that is found in complex I may represent GATA-4 or a GATA-like protein that shows weak cross-reactivity with a GATA-4 antiserum. We also demonstrated the presence of GATA-4 protein in first trimester chorionic villi and first and second trimester cytotrophoblast cells (Fig. 5D).
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tctATG was introduced. The mutation of these three nucleotides does not include any of the nucleotides shown to comprise the GATA binding site. For disruption of the GATA binding site, excluding any of the nucleotides shown to comprise the TEF-5 binding site, the following mutation was introduced: TGATAGTGAgAt. Transfection of each of these mutated enhancer-heterologous promoter constructs resulted in almost complete loss of enhancer activity (Fig. 6). A mutation in the 53-bp enhancer construct, which involved nucleotides in both of the protein binding sites GGAATGATAG (the boldface nucleotides represent the overlapping nucleotides) GGAAaGATAc, resulted in the complete loss of enhancer activity when transfected into JEG-3 cells (Fig. 6). These results demonstrate that both of these binding sites are required for determining placental-specific enhancer activity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In a recent study from our laboratory on the expression of the mouse orthologous 3ßHSD, 3ßHSD VI, in giant trophoblast cells during midpregnancy, two transcription factors were identified that determined the trophoblast-specific expression of 3ßHSD VI (4). The two transcription factors, AP-2 and Dlx-3, were found in both mouse giant trophoblast cells and in human placental JEG-3 cells. In addition, Ben-Zimra et al. (10) reported that AP-2 was the transcription factor required for mouse trophoblast-specific expression of P450scc. Thus, the identification of an AP-2 and Dlx-3 binding site in the promoter of the human HSD3B1 gene and the demonstration that these two transcription factor binding sites were located within an enhancer region of the HSD3B1 promoter led us to believe that the placental-specific expression of human 3ßHSD I was determined by the same transcription factors as we had found for the trophoblast-specific expression of the mouse 3ßHSD VI. Moreover, the binding sites for AP-2 at –2857/–2848 and for Dlx-3 at –2495/–2489 in the human HSD3B1 promoter were specific as determined by EMSAs using oligonucleotides representing these two binding sites, JEG-3 nuclear extracts and specific antibodies to these two transcription factors (data not shown). However, as reported in Results, neither one of these two transcription factors is involved in determining the placental-specific expression of human 3ßHSD I. Instead, a 53-bp placental-specific enhancer element was identified between –2570 and –2518 of the HSD3B1 promoter. A database search of the sequence comprising this enhancer element identified a consensus binding site for a family of transcription enhancer factors, TEF. The vertebrate genome contains four of these transcription factors, TEF-1, -3, -4, and -5, all of which bind to the same consensus sequence, 5'-(A/T) (A/G) (A/G) (A/T) ATG (C/T) (G/A)-3', containing a conserved ATG core (11). These transcription factors are expressed mostly in a tissue-specific manner. TEF-5 is predominantly expressed in the placenta. Human TEF-5 has been cloned from a human placental cDNA library (5, 7) and is specifically expressed in the differentiated syncytiotrophoblasts of the human term placenta with its expression up-regulated during the differentiation of cytotrophoblast to syncytiotrophoblast (11). Human TEF-5 is also highly expressed in cytotrophoblast-derived JEG-3 cells (5) and in human chorio-carcinoma-derived BeWo cells (7). In the current study, we demonstrate specific binding to the HSD3B1 enhancer element with a nuclear extract obtained from an 18-wk gestation placenta that is identical with the binding observed with nuclear extracts from JEG-3 cells. This observation demonstrates that the factor(s) required for expression of 3ßHSD I are present in early second trimester placenta, a time when the expression of 3ßHSD I is essential for the biosynthesis of progesterone. Previous studies have identified a placental-specific enhancer in the hCS gene referred to as hCS-B enhancer by Jacquemin et al. (5) and CSEn2 by Jiang et al. (7) as a target for TEF-5 (5, 7, 12). CSEn2 (hCS-B) is composed of multiple enhancers that act cooperatively to bring about maximal enhancer activity (5, 7).
Jacquemin et al. (13, 14) defined two distinct sites in the hCS-B enhancer, DF-3 and DF-4, which determine placental-specific expression. DF-3 contains two sites I and II arranged as a tandem repeat that specifically bind recombinant human TEF-5. Mutations in either of these binding sites that disrupt TEF-5 binding results in almost complete loss of enhancer activity when transfected into JEG-3 cells (5). DF-4 contains a single active site, TGGAATGTG, the six core nucleotides, GGAATG, are identical with the six nucleotides identified in the HSD3B1 enhancer. EMSA studies with in vitro-translated TEF-5 protein demonstrated that oligonucleotides representing the HSD-En and the hCS-B DF-4 enhancer, each formed a specific DNA-protein complex with an identical migration rate (Fig. 4C). Cross-competition studies with these two enhancer elements confirmed that HSD-En had a greater affinity for TEF-5 than the DF-4 (Fig. 4C). Point mutations in the GGAATG core sequence of HSD-En prevented the formation of the protein/DNA complex (Fig. 4B) and resulted in loss of competition with the wild-type HSD-En (Fig. 4C) and loss of enhancer activity when transfected into JEG-3 cells (Fig. 6). In addition, we demonstrated that a nuclear extract from an 18-wk gestation placenta formed a DNA/protein complex with the HSD-En oligonucleotide with identical properties as observed with nuclear extract of JEG-3 cells and the TEF-5 protein (Fig. 4, A and B). Together, these findings provide convincing evidence that TEF-5 is an essential factor in determining the placental-specific expression of 3ßHSD I.
Experiments designed to demonstrate that TEF-5 can transactivate the HSD3B1-53-bp enhancer by cotransfection of the enhancer TK164 plasmid and a TEF-5 expression vector (7) into JEG-3 cells, BeWo cells, or into HEK293 cells did not result in increased transcriptional activity (data not shown). Jacquemin et al. (5) also reported that overexpression of TEF-5 in JEG-3 or in HeLa cells did not result in increased transactivation of the hCS enhancer. However, Jiang et al. (7) reported that the coexpression of a plasmid containing the CSEn2 (hCS) enhancer element and the TEF-5 expression vector in BeWo cells resulted in increased transcriptional activity. The most likely explanation for the different results obtained in the current study to that reported by Jiang et al. is that TEF-5 had a much higher affinity for the HSD-En element than the DF-4 of the hCS-B enhancer, suggesting that the TEF-5 binding site in HSD-En was already saturated with JEG-3 or BeWo endogenous TEF-5, thus the addition of more TEF-5 in the cotransfection experiment would not result in higher transcriptional activity. [The amount of TEF-5 expressed in JEG-3 or in BeWo cells is not significantly different (Jiang, S.-W., unpublished data).] The ineffectiveness of showing transactivation of the HSD3B1-53-bp enhancer in HEK293 cells can be explained by the absence in HEK293 cells of the protein(s) needed in addition to TEF-5 for mediating enhancer transcriptional activity.
The binding site comprising complex I of the HSD-En represents a binding site for GATA proteins. We demonstrated competition with a GATA oligonucleotide that binds all of the known GATA proteins. In EMSA studies using HSD-En as a probe and all of the GATA antisera available against human GATA proteins, we observed a weak supershift with the GATA-4 antiserum. This finding suggests that the nuclear protein in complex I represents GATA-4. GATA-4 was also shown to be the most abundant GATA protein in JEG-3 cells, and furthermore, GATA-4 protein is present in first trimester chorionic villi and first and second trimester cytotrophoblast cells. Mutations, specific in the GATA binding site, resulted in marked reduction in placental-specific transcriptional activity indicating that GATA-4 or a GATA-like protein is essential in addition to TEF-5 in determining placental-specific expression of 3ßHSD I. From our studies, we cannot exclude the possibility that the relatively weak supershift represents an as-yet-unidentified GATA protein that shows weak cross-reactivity with the GATA-4 antiserum. Wang and Melmed (15) identified a specific binding site for a GATA-like protein in the placental-specific enhancer of the human leukemia-inhibitory receptor. However, none of the GATA (1, 2, 3, 4) antisera resulted in a supershift of the GATA-like protein in an EMSA with the oligonucleotide representing the human leukemia-inhibitory receptor GATA binding site (15). It has been reported that GATA-2 and GATA-3 proteins are involved together with at least three other proteins in conferring placental-specific expression of the human chorionic gonadotropin-
subunit gene (16). As can be concluded from the current study and the reports by Wang and Melmed (17) and by Steger et al. (16), GATA factors act in conjunction with other transcription factors to confer placental-specific expression.
Although the tissue-specific expression of mouse 3ßHSD VI and human 3ßHSD I are very similar, it may not be too surprising that the placental-specific expression is regulated by different transcription factors in the two species. Placental progesterone production in humans, which requires 3ßHSD I, is absolutely essential for maintenance of pregnancy, whereas we have shown that mouse trophoblast cells during mid-pregnancy have the capacity for progesterone production (4). There is no evidence to date that trophoblast-generated progesterone is required for maintenance of mouse pregnancy. The difference in the transcription factors that determine the placental-specific expression does not appear to be the result of the absence of expression of the factors identified that mediate human or mouse trophoblast-specific expression. As discussed above, AP-2 and Dlx-3 are pre-sent in human placental JEG-3 cells as well as in mouse trophoblast cells. TEF-5 (11), GATA-2, and GATA-3 (18) are expressed in mouse giant trophoblast cells. Whether GATA-4 or other GATA-like proteins are expressed in mouse trophoblast cells is not currently known. A somewhat analogous finding to our study has been reported for the CYP19 aromatase gene. Kamat et al. (19) reported that a 501-bp of DNA flanking the 5' end of the human placental-specific CYP19 exon I.1 introduced as a fusion gene into transgenic mice resulted in labyrinthine trophoblast cell-specific expression, although endogenous aromatase is not expressed in the mouse placenta. Human and rodent trophoblast cells differ in the expression of other steroidogenic enzymes. Rodent trophoblast cells express CYP17 (cytochrome P450 17-hydroxylase/C17–20 lyase) (20, 21), which is not expressed in human placental cells. From these findings, it can be concluded that although the mouse and human placental cells express the same transcription factors, this property does not necessarily indicate that these transcription factors act in an identical manner in regulating the tissue-specific expression of steroidogenic enzymes of the different species.
Human placental-specific expression of CYP11A, P450scc, the other steroidogenic enzyme required for progesterone biosynthesis, is yet to be established. Huang and Miller (22) identified two transcription factors related to human immunodeficiency virus-inducible LBP proteins, LBP-1b and LBP-9, which were found to modulate expression of P450scc initiated by other placental-specific transcription factors that as yet have not been identified. Because the expression of P450scc and 3ßHSD I is essential for the biosynthesis of progesterone, one would assume that their expression should be coordinated. It will be of interest in future experiments to investigate whether TEF-5 and GATA-proteins also determine the human placental-specific expression of P450scc.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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. All of the primers had KpnI site added at the 5' end. To characterize the promoter region of the human HSD3B1, a series of promoter fragments that included the first exon and intron (Fig. 1) were subcloned into a promoterless luciferase reporter vector pA3LUC at the KpnI site (4). To make the heterologous constructs, the different fragments within the –2861 to –2395 enhancer region were subcloned into a heterologous thymidine kinase promoter driven luciferase reporter vector TK164LUC (4) at the KpnI site. Site-directed mutations were introduced into the potential TEF-5 and/or GATA-like binding sites using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) after the instructions given in the manual. PCR fidelity, orientations of inserts, introduced mutations were verified by restriction enzyme digestion and sequencing. Construction of the TEF-5 expression plasmid was as described previously (7).
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0.4 µg DNA) and pSV2ß-Gal (24) (0.1 µg DNA) were cotransfected in triplicate. All transfections were normalized with carrier DNA to equal 0.7 µg DNA/well. After transfection (42 h), cells were lysed using Reporter Lysis Buffer (Promega Corp., Madison, WI). The activities of luciferase and ß-galactosidase were measured according to the manufacturer’s description. The luciferase activity of each construct was normalized to the cotransfected ß-galactosidase activity. JEG-3 cells used for transfection of the TEF-5 expression plasmid (pDR2-TEF-5) (7) were plated in 10-cm dishes and grown to 50% confluency before transfection. Lipofectamine-mediated transfection was performed with 30 µg of TEF-5 expression plasmid or 30 µg of empty vector pDR2 (Stratagene) control DNA. After 28 h, cells were washed three times with cold PBS and collected for preparation of cell extracts.
Tissue and Cell Preparation of Nuclear Extracts
Preparation of crude nuclear extract from nontransfected JEG-3 and HEK293 cells were prepared as described (25). Human placentas were collected under a protocol approved by the Stanford University Panel on the Use of Human Subjects in Medical Research, after informed consent. Placentas were obtained at elective termination of genetically normal pregnancies. Chorionic villi and purified cytotrophoblasts cells from first and second trimester human placentae were prepared as previously described by Fisher et al. (26). Trophoblast cells, which included a mixed population of synciotrophoblasts and cytotrophoblasts, were obtained as described (27). Nuclear extracts from these placental trophoblast cells were isolated using a 1-h minipreparation technique as described by Deryckere and Gannon (28). Cell extracts of the TEF-5 and the pDR2 transfected JEG-3 cells were subjected to a micro-scale method for the isolation of the DNA-binding proteins (29).
In Vitro Translation
TEF-5 protein was generated in vitro by transcription-translation reaction with TNT Coupled Rabbit Reticulocyte Lysate (Promega) as described previously (7). Standard in vitro transcription-translation reaction was performed at 30 C for 2 h with 25 µl TNT, 2 µl reaction buffer, 1 µl T3 RNA polymerase, 1.5 µl ribonuclease inhibitor (RNasin, 40 U/µl; Roche Diagnostics Corp., Indianapolis, IN), 1 µl amino acid mixture, and 1 µg of pDR2-TEF-5 DNA.
EMSA
EMSAs were performed essentially as described previously (4, 7). The binding reaction contained the appropriate radiolabeled probes, 5–10 µg of crude nuclear extract, or 4–5 µg of TEF-5 transfected JEG-3 cell extract or in vitro generated TEF-5 protein as indicated in the legends. The sequences of the double-stranded oligonucleotides used for EMSAs are listed in Table 2. For competition assays, the indicated fold molar excess of unlabeled oligonucleotides was added to the binding reaction. For supershift assays, 2 µg of polyclonal antisera to GATA 1, 2, 3, 4, or 6 (Santa Cruz Biotechnology, Santa Cruz, CA) were added to the mixture before the addition of probe. The binding reactions were resolved on a 5% nondenaturing polyacrylamide gel.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Current address for F.J.: Women’s Hospital, Zhejiang University School of Medicine, Hangzhou, China 310006.
This work was supported by National Institute of Child Health and Human Development (NICHD)/National Institutes of Health (NIH) cooperative agreement (U54 HD 31398) as part of the Specialized Cooperative Centers Program in Reproductive Research (to A.H.P.), and NIH/HD 041577 (to S.-W.J.).
Abbreviations: AP-2, Activating protein-2; Dlx-3, the homeodomain protein, distaless-3; 3ßHSD, 3ß-hydroxysteroid dehydrogenase/isomerase; hCS, human chorionic somatomammotropin; HEK, human embryonic kidney; JEG-3, human choriocarcinoma cells; MA-10, mouse Leydig tumor cells; mHSD-En, mutated HSD-En; P450scc, cholesterol side-chain cleavage; TEF, transcription enhancer factor; TK, thymidine kinase.
Received for publication January 23, 2004. Accepted for publication April 27, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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5-4 isomerase gene family. J Endocrinol 150:S189–S207
5-4-isomerase in human adrenals and gonads. Mol Endocrinol 5:1147–1157[Abstract]
and the homeodomain protein distal-less 3 are required for placental-specific expression of the murine 3ß-hydroxysteroid dehydrogenase VI gene, Hsd3b6. J Biol Chem 277:7945–7954-subunit gene in the placenta and pituitary gland. Mol Cell Biol 14:5592–5602-hydroxylase gene expression in differentiating rat trophoblast cells. J Endocrinol 150:161–168[Abstract]
) and P450SCC gene expression and regulation in the rat placenta. Endocrinology 130:1309–1317[Abstract]
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 A. Kamat, M. E. Smith, J. M. Shelton, J. A. Richardson, and C. R. Mendelson Genomic Regions that Mediate Placental Cell-Specific and Developmental Regulation of Human Cyp19 (Aromatase) Gene Expression in Transgenic Mice Endocrinology, May 1, 2005; 146(5): 2481 - 2488. [Abstract] [Full Text] [PDF]
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A. H. Payne and D. B. Hales Overview of Steroidogenic Enzymes in the Pathway from Cholesterol to Active Steroid Hormones Endocr. Rev., December 1, 2004; 25(6): 947 - 970. [Abstract] [Full Text] [PDF]
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, Dlx-3 binding site;
, AP-2
, represents the 53-bp placental-specific enhancer element. Luciferase activity of each construct is expressed relative to the TK164LUC vector. Each value represents the mean ± SE of three separate transfections, each performed in triplicate.
, CTTAGGAAaGTActAATGT; 
, TEF-5-specific: CTTATctATGATAGAATGT; and
,
GATA-site-specific, CTTAGGAATGAgAtctATGT.