LBP Proteins Modulate SF1-Independent Expression of P450scc in Human Placental JEG-3 Cells
Ningwu Huang and
Walter L. Miller
Department of Pediatrics and The Metabolic Research Unit, University of California, San Francisco, San Francisco, California 94143-0978
Address all correspondence and requests for reprints to: Professor Walter L. Miller, M.D., Department of Pediatrics, University of California, San Francisco, San Francisco, California 94143-0978. E-mail: wlmlab{at}itsa.ucsf.edu.
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ABSTRACT
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The cholesterol side-chain cleavage enzyme, P450scc, initiates biosynthesis of all steroid hormones. Adrenal and gonadal P450scc expression requires steroidogenic factor-1 (SF1), but P450scc expression in human placental JEG-3 cells utilizes an SF1-independent element at 155/131 that is inactive in adrenals and gonads. We previously cloned two transcription factors, long terminal repeat binding protein (LBP)-1b and LBP-9, from JEG-3 cells. In transient transfection assays, LBP-1b activated the 155/131 element whereas LBP-9 suppressed its LBP-1b-stimulated expression. To assess the roles of these factors on the intact P450scc gene, we stably expressed LBP-1b or LBP-9 in JEG-3 cells. All cell lines stably expressing a fusion protein of LBP-1b and enhanced green fluorescent protein increased P450scc expression, but cell lines stably expressing LBP-9 fused to enhanced green fluorescent protein either increased or decreased P450scc expression. 8-Br-cAMP induced endogenous LBP-9, but not LBP-1b expression. Glutathione-S-transferase pull-down assays showed that LBP-1b and LBP-9 can dimerize with themselves and with each other; LBP-1b residues 300540 and LBP-9 residues 300479 were required for dimer formation. Glutathione-S-transferase pull-down assays, bandshifts, and transient transfection assays showed that TReP-132 (another factor that can bind to 155/131) does not interact with either LBP-1b or LBP-9, or influence their ability to induce or suppress transcription from the 155/131 element. Gal4 transactivation assays showed that transcriptional repression activity by LBP-9 requires residues 100200. RNAi interference of either LBP-1b or LBP-9 mRNAs decreased P450scc expression. LBP-1b is an important SF1-independent transcriptional activator stimulating P450scc expression in human placental JEG-3 cells, whereas LBP-9 modulates the action of LBP-1b, exerting both positive and negative effects.
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INTRODUCTION
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STEROID HORMONES, WHICH regulate a wide variety of physiological functions, are synthesized from cholesterol in the adrenals, gonads, and placenta (1). Conversion of cholesterol to pregnenolone in mitochondria is catalyzed by cytochrome P450scc, the cholesterol side-chain cleavage enzyme, and is the first, rate-limiting, and hormonally regulated enzymatic step in the synthesis of all steroid hormones (1). Human P450scc is encoded by a single gene, formally termed CYP11A (2), that is located on chromosome 15q23-q24 (3) and is expressed in the adrenals, gonads, placenta, and brain (4, 5, 6).
The orphan nuclear receptor steroidogenic factor-1 (SF1), also known as Ad4-BP, is essential for the expression of all the steroidogenic genes in the adrenals and gonads and also for adrenal and gonadal development (7, 8). However, although SF1 is expressed in many tissues, its expression is undetectable in some tissues that contain P450scc, including the rodent brain and embryonic gut, and the human placenta (8, 9, 10, 11). There appear to be substantial differences in the identity and location of the cis-acting elements responsible for the transcription of the P450scc genes from different species (12), but all contain sites that bind SF1. However, transcription of the P450scc gene in the human placenta (13, 14, 15) and mouse brain (16, 17) does not require these sites. Transcription of the P450scc gene in human placenta requires a cis-acting element between nucleotides 155 and 131 (155/131) (13, 14), which is not required for P450scc expression in the adrenals and gonads and is independent of SF1. Using a yeast one-hybrid approach, we previously cloned two transcription factors, termed LBP-1b and LBP-9 (officially termed TFCP2A and TFCP2L1), from human placental JEG-3 cells, that appear to modulate P450scc reporter activity through the 155/131 element (15). A zinc-finger, 132-kDa transcriptional regulating protein (TReP-132) isolated from human placental JEG-3 cells also interacts with the 155/131 element and stimulates P450scc reporter activity in JEG-3 cells (18) and interacts with SF1 to promote P450scc reporter activity in human adrenal NCI-H295 cells (19); hence its role in SF1-independent placental transcription of P450scc is unclear.
The LBP-1 family of mammalian transcription factors was initially described in HeLa cells where they stimulate transcription from the major late promoter of simian virus 40 in vitro; the name LBP refers to long terminal repeat binding protein (20). There are two related human LBP-1 genes, each of which encodes two alternatively spliced transcripts; LBP-1a and LBP-1b arise from one gene (TFCP2A), and LBP-1c and LBP-1d arise from a second gene (TFCP2C) (20). LBP-1c is identical to the
-globin transcription factor CP2 (21), also referred to as LSF (22, 23), and plays an essential role in the transcription of bone morphogenic protein-4 (24). LBP-1d is also known as LSF-ID (23). LBP proteins are related to the Drosophila transcription factor Elf-1/NTF-1 (25, 26), as they share sequence similarity at the region required for DNA binding (21). These proteins bind DNA as homodimers (23, 27), homotetramers (28, 29), or heteromultimers (30). Although most members of the LBP family of proteins act as transcription activators (15, 20, 31, 32), LBP-9 (15) and the closely related mouse protein termed CRTR-1 (CP2-Related Transcriptional Repressor-1) (Tfcp2l1) (33), which shares 88% identity to LBP-9 and 60% identity to LBP-1b, appear to act as transcriptional repressors.
The human placenta produces large amounts of progesterone, which are required for suppressing uterine contractility so that pregnancy may proceed (34). During human pregnancy, progesterone is initially provided by the mothers ovarian corpus luteum, but after about 810 wk virtually all progesterone is produced by the placenta, a fetal tissue (34). Therefore, without placentally produced progesterone, spontaneous abortion will ensue, and interfering with the action of progesterone (e.g. by administering RU-486) will terminate pregnancy. As human placental P450scc expression and progesterone synthesis are mandatory for successful pregnancy, it is important to understand the SF1-independnet mechanisms of regulating human placental P450scc transcription. We previously found that LBP-1b stimulated transcription of the 155/131 element of the P450scc gene fused to the minimal promoter/reporter construct TK32/LUC (32 bp of the thymidine kinase promoter fused to the luciferase gene), and that LBP-9 suppressed this LBP-1b-induced transcription (15). However, the potential roles of these factors on the intact P450scc promoter in placental cells have not been established. We now show that LBP-1b promotes P450scc gene expression in human placental JEG-3 cells and provide evidence that LBP-9 modulates this activity.
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RESULTS
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Characterization of Human Placental JEG-3 Cells Stably Expressing LBP-1b-Enhance Green Fluorescent Protein (EGFP) and LBP-9-EGFP
We previously cloned LBP-1b and LBP-9 from JEG-3 cells by a yeast one-hybrid screening approach, based on their ability to interact with 155/131 element from the human P450scc gene (15). Transient cotransfection of an LBP-1b expression vector and 155/131 fused to TK32/LUC activated LUC expression; transient cotransfection of an LBP-9 expression vector and 155/131 TK32/LUC had no effect on basal LUC expression, but transient cotransfection of the LBP-1b and LBP-9 vectors showed that LBP-9 suppressed LUC induction by LBP-1b in a dose-dependent fashion (15). Because these initial experiments used transient transfection, it was not possible to determine whether LBP-1b and LBP-9 interacted or competed with each other, or whether they could regulate the intact P450scc promoter in addition to regulating the 155/131 TK32/LUC construct. To investigate this, we first used retrovirally mediated gene transfer into JEG-3 cells to create cell lines that stably express LBP-1b or LBP-9, each fused to EGFP. Control cell lines were also created expressing EGFP alone. We selected cells expressing EGFP fusion proteins first by a fluorescence-activated cell sorter, then by growing in G418 for 3 wk, and finally grew clones from individual cells.
Twenty clones expressing LBP-1b-EGFP, 20 clones expressing LBP-9-EGFP, and 10 control clones expressing EGFP alone were randomly selected for further study. The mRNAs for LBP-1b, LBP-9, P450scc, and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) from each cell line were assayed simultaneously by performing a single ribonuclease (RNase) protection assay using probes for all four mRNAs that produced fragments of readily distinguishable lengths, so that all four mRNAs could be seen on a single gel (Fig. 1
). The levels of mRNAs for LBP-1b, LBP-9, and P450scc were normalized to GAPDH in the 10 cell lines expressing EGFP alone, providing an indication of the basal variability of these mRNAs in JEG-3 cells. The level of LBP-1b mRNA, which was about 0.5% of the level of GAPDH mRNA, was arbitrarily set equal to 1 (range, 0.71.2-fold; mean, 1.0 ± 0.17-fold) and was used for comparison to the levels of LBP-9 and P450scc mRNAs, as the probes for LBP-1b, LBP-9, and P450scc had the same specific activity (Table 1
). The levels of P450scc mRNA were similar (range, 0.60.9-fold; mean, 0.8 ± 0.11-fold), and neither LBP-1b nor P450scc mRNA varied substantially among the various cell lines. By contrast, LBP-9 mRNA was substantially more abundant and varied substantially more among these 10 control cell lines (range, 1.93.3-fold; mean, 2.5 ± 0.55-fold).

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Fig. 1. LBP-1b and LBP-9 Regulate P450scc Expression When Stably Overexpressed in JEG-3 Cells
RNA from the stably transfected JEG-3 cell lines indicated was analyzed by RNase protection assay using combined probes for LBP-1b, LBP-9, P450scc, and GAPDH. The lane designated tRNA is a negative control (20 µg yeast tRNA hybridized with probes). Overexpression of LBP-1b-EGFP in clones 16 and 19 increased P450scc expression, and overexpression of LBP-9-EGFP in clone 4 increased P450scc expression, but overexpression of LBP-9-EGFP in clone 18 decreased P450scc expression.
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In all 20 cell lines stably expressing LBP-1b-EGFP, the amount of P450scc mRNA was significantly (P < 0.005) increased (range, 1.34.5-fold; mean, 2.5 ± 0.83-fold) (Table 2
) compared with the mean value for P450scc mRNA in the 10 control cell lines. The expression of P450scc mRNA was the highest in clone 19 (shown in Fig. 1
compared with control line 6). Although these experiments cannot exclude an indirect action of LBP-1b to stimulate P450scc gene transcription, the results with these stably transfected lines are consistent with our initial report that LBP-1b stimulates P450scc reporter activity through the 155/131 element in human placental JEG-3 cells (15). Furthermore, overexpression of LBP-1b had no impact on the expression of LBP-9. Thus, expression of LBP-1b-EGFP increased expression of P450scc in all 20 stably transfected cell lines, but the levels of LBP-1b expression did not correlate directly with the levels of P450scc or LBP-9 expression.
Transient transfection data indicated that LBP-9 suppressed LBP-1b-induced transcriptional activity of the 155/131 element from P450scc (15); hence we examined the abundance of P450scc, LBP-1b, and LBP-9 mRNA in 20 randomly chosen JEG-3 cell lines stably expressing LBP-9-EGFP. RNase protection assays showed that 19 of the 20 cell lines overexpressed LBP-9 as designed (range, 0.95.9-fold; mean, 2.7 ± 1.45-fold) (Table 3
) compared with the mean value for LBP-9 in the 10 control cell lines. This overexpression of LBP-9 did not affect the expression of LBP-1b (range, 0.71.6-fold; mean, 0.9 ± 0.23-fold) (Table 3
). P450scc expression was highly variable; overall, expression was increased 1.5-fold (P = 0.0014), with six cell lines (2, 5, 7, 9, 18 and 20) having decreased expression (range, 0.5 -0.9-fold compared with control), most cell lines having slightly increased expression (range, 1.21.7-fold) and three cell lines (1, 4, and 6) having substantially increased P450scc expression (range, 2.43.8-fold) (Table 3
) compared with the mean value for P450scc in the 10 control cell lines. Thus, overexpressing LBP-9 in stably transfected JEG-3 cells had a minor effect on P450scc expression.
To investigate further the possible impact of LBP-1b and LBP-9 on the expression of P450scc, we transiently transfected LBP-1b-EGFP clones 16 and 19, which express high levels of P450scc mRNA (Table 2
), with an expression vector for LBP-9. Similarly, we transiently transfected LBP-9-EGFP clones 4 and 18, which, respectively, had high and low levels of P450scc mRNA (Table 3
), with an expression vector for LBP-1b. RNase protection assays showed the mRNA abundance of LBP-1b and LBP-9 increased when an expression vector for LBP-1b or LBP-9 was transiently transfected into the cells, indicating good transfection efficiency. However, the RNase protection data revealed no effect on the abundance of P450scc mRNA in either experiment (data not shown). Therefore, we sought other means to explore the potential interactions between LBP-1b and LBP-9, and their potential impact on P450scc expression in JEG-3 cells.
Regulation of Endogenous LBP-1b and LBP-9 by cAMP
In JEG-3 cells, both the accumulation of P450scc mRNA (35, 36) and the transcription of the P450scc gene (13, 36) are induced by cAMP. As LBP-1b and LBP-9 influence P450scc expression when stably expressed in JEG-3 cells, we sought to determine whether LBP-1b or LBP-9 is regulated by cAMP. Selected, stably transfected JEG-3 cell lines overexpressing LBP-1b-EGFP (clones 16 and 19), LBP-9-EGFP (clones 4 and 18), and EGFP alone (clones 6 and 8) were treated with 1 mM 8-Br-cAMP for 24 h. Northern blotting showed that the abundance of the endogenously expressed 4.42-kb LBP-1b mRNA was not regulated by cAMP in any of the cell lines (Fig. 2A
). The expression of the 3.76-kb mRNA encoding by the stably transfected LBP-1b-EGFP construct in LBP-1b cell lines 16 and 19 was induced, consistent with the known cAMP responsiveness of the cytomegalovirus (CMV) promoter driving expression of this construct (Fig. 2A
). However, the endogenously encoded 4.9-kb LBP-9 mRNA was not detected in any cell line, but the 3.38-kb mRNA encoded by the stably transfected LBP-9-EGFP construct in LBP-9 cell lines 4 and 18 was also induced by cAMP (Fig. 2A
). Although Northern blotting was not sufficiently sensitive to detect the endogenous LBP-9 mRNA, RNase protection assays showed that cAMP increased the abundance of endogenously expressed LBP-9 mRNA (Fig. 2B
). P450scc responded dramatically in all cell lines, consistent with the known action of cAMP to induce P450scc in JEG-3 cells (13, 35, 36), and did not vary with the presence or absence of the stably transfected LBP transcripts (Fig. 2A
). Thus, the LBP proteins endogenously produced in these cells are sufficient to induce any LBP-responsive effect on P450scc.

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Fig. 2. Effects of 8-Br-cAMP
A, Northern blotting. The indicated lines of stably transfected JEG-3 cells expressing LBP-1b-EGFP, LBP-9-EGFP, or EGFP alone were treated with or without 1 mM 8-Br-cAMP for 24 h. A single Northern blot containing 10 µg total RNA from each cell line was probed sequentially for LBP-1b, LBP-9, P450scc, and GAPDH. Endogenously produced P450scc mRNA (1.87 kb) is detected in all cell lines and responds strongly to cAMP. The endogenously produced 4.42-kb LBP-1b mRNA is expressed in all cell lines and does not respond to cAMP. The 3.76-kb LBP-1b-EGFP mRNA is found only in the cell lines stably transfected with vector for LBP-1b-EGFP and is responsive to cAMP. The endogenously produced LBP-9 mRNA is expressed at a level below the limit of detection by Northern blotting; only the 3.38-kb LBP-9-EGFP mRNA from the stably transfected cells is detected and is responsive to cAMP. B, RNase protection assay of RNA from JEG-3 cells treated with or without cAMP using combined probes for LBP-1b, LBP-9, P450scc, and GAPDH. The endogenously produced P450scc and LBP-9 mRNAs respond to cAMP, but the endogenously produced LBP-1b mRNA does not respond to cAMP.
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LBP Proteins Can Form Homodimers and Heterodimers
Both LBP-1b and LBP-9 bind to the same 155/131 region of the P450scc promoter (15), suggesting that these LBP proteins might form homodimers or heterodimers. Moreover CP2, a member of the LBP protein family, can form homodimers (23, 27) or heterodimers with other proteins (30). To determine whether LBP-1b and LBP-9 can interact, we prepared glutathione-S-transferase fusion proteins with LBP-1b (GST-LBP-1b) and LBP-9 (GST-LBP-9). GST-LBP-1b, GST-LBP-9, or GST alone was bound to glutathione-coupled Sepharose 4B and incubated with 35S-labeled LBP-1b or LBP-9 protein, after which the bound, labeled LBP proteins were boiled off and analyzed by sodium dodecyl sulfate (SDS) gel electrophoresis (Fig. 3A
). LBP-1b interacted with itself and with LBP-9, but not with GST protein. Similarly, GST-LBP-9 also interacted with itself and with 35S-labeled LBP-1b protein. Thus both LBP-1b and LBP-9 can form homodimers or they can heterodimerize with one another. To identify the regions of LBP-1b responsible for this protein-protein interaction, we built vectors expressing a series of LBP-1b deletion mutants, prepared the 35S-labeled proteins by in vitro transcription/translation, and incubated them with GST-LBP-1b, GST-LBP-9, or GST alone. Pull-down assays showed that LBP-1b residues 300540 were required for the interaction with LBP-9 and with itself (Fig. 3B
). Similarly, to identify the regions of LBP-9 that are responsible for homodimerization and heterodimerization, we built a series of vectors expressing LBP-9 deletion mutants and prepared the 35S-labeled proteins and incubated them with GST-LBP-1b, GST-LBP-9, or GST alone. The GST pull-down results showed that LBP-9 residues 300479 were required for the interaction with LBP-1b and with itself (Fig. 3C
).

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Fig. 3. LBP-1b and LBP-9 Form Homodimers and Heterodimers
A, GST pull-down experiments with full-length LBP-1b and LBP-9. GST-LBP-1b, GST-LBP-9, or GST alone (indicated above the lanes) was immobilized on a matrix, incubated with 35S-labeled LBP-1b or LBP-9 (indicated by the + signs), eluted, and analyzed by 10% SDS-PAGE. The "input" lanes for LBP-1b and LBP-9 contain one tenth the amount of 35S-labeled LBP proteins used in the incubation. Both the GST-linked LBP-1b and LBP-9 could pull down labeled LBP-1b (56 kDa) or LBP-9 (48 kDa), but GST alone pulled down neither. B, GST pull-down assays with segments of LBP-1b. Segments of LBP-1b (indicated at the top of the figure) labeled with [35S]methionine by in vitro transcription/translation were incubated with GST-LBP-1b, GST-LBP-9, or GST alone (indicated by the + signs). GST-LBP-1b and -9 are both able to pull down the 48-kDa 102540 segment, the 38-kDa 200540 segment of LBP-1b, and somewhat more weakly, the 26-kDa 300540 segment. Thus residues 300540 of LBP-1b are required for the interaction with itself and with LBP-9. C, GST pull-down assays with segments of LBP-9. Segments of LBP-9 (indicated at the top of the figure) were labeled with [35S]methionine by in vitro transcription/translation, and incubated with GST-LBP-1b, GST-LBP-9, or GST alone (indicated by the + signs). GST-LBP-1b and -9 are both able to pull down the 42-kDa 100479 segment, the 31-kDa 200479 segment, and the 20-kDa 300479 segment. Thus, LBP-9 residues 300479 are required for the interaction with itself and with LBP-1b.
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In addition to LBP-1b and LBP-9 binding to 155/131, the zinc-finger protein TReP-132 also binds to this element and stimulates P450scc transcription in JEG-3 cells (18); hence we sought to determine whether either LBP-1b or LBP-9 interacts with TReP-132. Therefore, we prepared 35S-labeled TReP-132 and 35S-labeled LBP-1b proteins and performed GST pull-down experiments with GST-LBP-1b and GST-LBP-9 fusion proteins bound to glutathione-coupled Sepharose 4B. 35S-labeled TReP-132 did not bind to the GST-LBP-1b or GST-LBP-9 fusion protein, whereas 35S-labeled LBP-1b bound to either GST-LBP-1b or GST-LBP-9, as before (Fig. 4A
). Similarly, EMSAs showed that LBP-1b, LBP-9, and TReP-132 bound to 32P-labled 155/131, but no combination of any of these three proteins yielded a supershift pattern (Fig. 4B
). The generation of a supershift pattern requires high binding affinity between the two proteins, but functional interactions may occur at low affinities. To examine the possibility that TReP-132 might functionally interact with LBP proteins, we transiently cotransfected JEG-3 cells with vectors expressing LBP-1b, LBP-9, and TReP-132 and assessed their ability to transactivate the 155/131 sequence fused to TK32/LUC, a minimal promoter-reporter system. As reported previously with a single copy of 155/131 fused to TK32/LUC (15), LBP-1b alone induced 3x(155/131) TK32/LUC activity significantly (P = 0.014), LBP-9 alone suppressed 3x(155/131) TK32/LUC activity significantly (P = 0.005), and LBP-9 abolished the induction by LBP-1b (P = 0.02) (Fig. 4C
). TReP-132 alone did not change the activity of 3x(155/131) TK32/LUC significantly (P = 0.38), TReP-132 did not alter the level of transcription elicited by LBP-1b (P = 0.65), and TReP-132 did not relieve the suppression effect by LBP-9 (P = 0.18). Thus, whereas TReP-132 binds to the same DNA sequence in the P450scc promoter that can also bind LBP-1b and/or LBP-9, TReP-132 does not appear to interact with the LBP proteins or exert a significant effect on the isolated 155/131 element.

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Fig. 4. TReP-132 Does Not Interact with LBP-1b, or LBP-9
A, GST pull-down assay. TReP-132 (left) and LBP-1b (right) were labeled with [35S] methionine by in vitro transcription/translation and incubated with GST-LBP-1b, GST-LBP-9, or GST alone (indicated by the + signs). None of the GST fusion proteins was able to pull down 35S-TReP-132, whereas both the GST-LBP-1b and GST-LBP-9 could pull down 35S-LBP-1b. B, EMSA. Double-stranded, 32P-labeled DNA probe corresponding to bases 155/131 of the human P450scc promoter was incubated with LBP-1b, LBP-9, or TReP-132 or a combination of these proteins produced by in vitro transcription/translation without or with 100-fold excess unlabeled competitor. C, Functional assay. A vector containing three copies of the human P450scc promoter sequence 155/131, fused to TK32/LUC, was cotransfected into JEG-3 cells with expression vectors for LBP-1b, LBP-9, and/or TReP-132 as shown. Luciferase data are mean ± SEM for three independent experiments, each performed in triplicate.
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The Transcriptional Repression Domain of LBP-9 Lies in Residues 100200
LBP-9 was identified as a factor that suppressed the activity of the 155/131 TK32/LUC construct stimulated by LBP-1b in transient transfection assays (15), and CRTR-1, the mouse homolog of LBP-9, also exerts transcriptional repression, acting through a domain located at its N terminus (33). However, as overproduction of LBP-9-EGFP in stably transfected JEG-3 cell lines had little impact on P450scc expression (Table 3
), we used GAL4 transactivation to assess the potential impact of LBP-9 on P450scc transcription. We cotransfected JEG-3 cells with a vector expressing full-length LBP-9 fused to the DNA binding domain (DBD) of the yeast transcription factor Gal4 (37) and the pTK-MH100x4-Luc reporter plasmid containing the luciferase reporter gene regulated by the TK promoter and four upstream copies of the Gal4 binding site (37). Compared with basal luciferase activity produced by the empty pCMX-GAL4 vector (expressing only GAL4-DBD), cotransfection of full-length pGAL4-LBP-9 and pTK-MH100x4-Luc decreased luciferase activity by 68% (Fig. 5
). To locate the domain of LBP-9 responsible for this repression, we built constructs expressing various segments of LBP-9 fused to GAL4-DBD and cotransfected JEG-3 cells with these GAL4-DBD-LBP-9 fusions and pTK-MH100x4-Luc. GAL4-DBD fusions retaining LBP-9 residues 100200 decreased luciferase activity by 58%, but constructs lacking this region had no effect (Fig. 5
). There was no significant difference in the level of repression obtained from full-length (1479) LBP-9 and the construct containing only residues 100200 of LBP-9 (P > 0.5). Thus LBP-9 can act as a transcriptional repressor, and residues 100200 of LBP-9 are both necessary and sufficient for this activity.

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Fig. 5. LBP-9 Represses Transcription
JEG-3 cells were cotransfected with vectors expressing fusion proteins of the GAL4-DBD fused to the segments of LBP-9 indicated and the reporter plasmid pTK-MH100x4-Luc. The ability of each construct to repress transcription of the reporter plasmid was assessed by luciferase assays normalized against expression of Renilla luciferase expressed from cotransfected plasmid pRL-TK. Results are expressed as a percentage of the luciferase activity obtained from the pCMX-GAL4 vector expressing only the GAL4-DBD. The data are the means of three independent transfections, each performed in triplicate (±SEM). The repression activities of full-length LBP-9 (1479), and its shorter segments comprising residues 47479 and 100200, are not significantly different (P > 0.5), but all suppress more than the 1106 or 200479 segments. Thus the trans-repression activity by LBP-9 requires residues 100200.
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Knockdown of Either LBP-1b or LBP-9 by Small Interfering RNA (siRNA) Decreased P450scc Expression
To determine whether reducing the expression of LBP-1b and LBP-9 mRNA would affect P450scc expression, we used siRNA to knock down the expression of LBP-1b and LBP-9 in placental JEG-3 cells. We built vectors expressing 19-base siRNA directed against P450scc, LBP-1b, and LBP-9, transiently transfected them into JEG-3 cells, and isolated RNA 72 h later for analysis by RNase protection assays. To test the effectiveness of the siRNA procedure in JEG-3 cells, we first knocked down P450scc, reducing P450scc mRNA to 45% of the level in control cells transfected with empty vector; this reduction in P450scc mRNA did not change the abundance of either LBP-1b or LBP-9 mRNA (Fig. 6
). Knockdown of LBP-1b reduced the abundance of LBP-1b mRNA to 38% of control, and decreased P450scc mRNA to 63% of control (P = 0.00035), but did not affect the abundance of LBP-9 mRNA (Fig. 6
), consistent with our other data that LBP-1b is a transcriptional activator. Knockdown of LBP-9 reduced the abundance of LBP-9 mRNA to 63% of control (P = 0.0012) and decreased P450scc mRNA to 42% of control (P = 0.04), but did not affect the abundance of LBP-1b mRNA. The decreased P450scc expression in the LBP-9 knockdown suggests that LBP-9 also acts as a transcriptional activator stimulating P450scc transcription in JEG-3 cells, similarly to its action in the cell lines stably transfected with LBP-9 (Table 3
).

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Fig. 6. Knockdown of LBP-1b, LBP-9, and P450scc by siRNA
A, RNase protection assay. JEG-3 cells were transfected with empty vector or vectors expressing siRNA directed against LBP-1b, LBP-9, or P450scc, and total RNA isolated 72 h after transfection was assayed for mRNA abundance for P450scc, LBP-1b, LBP-9, and GAPDH by RNase protection. B, Quantitation of mRNA abundance. The abundance of mRNAs for P450scc (black bars), LBP-1b (hatched bars), and LBP-9 (open bars) are shown relative to the abundance of GAPDH mRNA. Data, shown as the percentage of each mRNA found in vector-transfected JEG-3 cells, are from three independent transfection experiments (±SEM). Knockdown of P450scc reduced P450scc mRNA to 45% of control, but did not change the abundance of LBP-1b and LBP-9 mRNA significantly. Knockdown of LBP-1b reduced the abundance of LBP-1b mRNA to 38% of control. Knockdown of LBP-1b did not affect the abundance of LBP-9 mRNA, but decreased P450scc mRNA to 63% of control. Knockdown of LBP-9 reduced the abundance of LBP-9 mRNA to 63% of control. Knockdown of LBP-9 did not affect the abundance of LBP-1b mRNA, but decreased P450scc mRNA to 42% of control.
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DISCUSSION
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Transcription factors of the LBP family activate various promoters in vivo and in vitro (15, 20, 21, 22, 29, 31, 32, 38), but only LBP-1b and LBP-9 appear to be involved in steroidogenesis (15). These two factors have different patterns of expression in cultured cell lines: LBP-1b is very widely expressed, whereas LBP-9 is apparently expressed only in JEG-3 cells (15). Transient transfection studies suggested that LBP-1b stimulates P450scc reporter activity through the 155/131 element, and LBP-9 suppresses P450scc reporter activity stimulated by LBP-1b (15). TReP-132 also binds to 155/131, but appears to be a weak regulator. We found no activity of TReP-132 on the effects of LBP proteins. Also, whereas we found effects of LBP proteins on a reporter containing one copy of 155/131 (15), multiple copies of 155/131 were fused to a reporter to detect activity of TReP-132 (18), and equal activity is found in NCI-H295 cells (19) where the 155/131 element is not essential (13, 14). Thus, the effects of LBP proteins predominate over any effects of TReP-132 in human placental JEG-3 cells.
The roles of LBP-1b and LBP-9 in regulating the intact P450scc promoter have been unclear. Using two independent approaches, we have now established that LBP-1b induces P450scc gene expression in JEG-3 cells. First, stably overexpressing LBP-1b in JEG-3 cells increased P450scc gene transcription. Second, knocking down the abundance of LBP-1b mRNA by siRNA decreased P450scc gene transcription. Thus LBP-1b is a transcriptional activator that is essential for the SF1-independent transcription of the P450scc gene in human placental JEG-3 cells. Other approaches, such as mouse knockouts, are not applicable. The mouse P450scc promoter lacks an element similar to the 155/131 sequence, and the mouse placenta produces little progesterone during pregnancy. In human pregnancy, the production of progesterone shifts from the maternal corpus luteum to the placental syncytiotrophoblast cells (a fetal tissue) at 810 wk gestation and thereafter requires very high levels of placental P450scc transcription, whereas in the mouse the maternal corpus luteum supplies progesterone throughout pregnancy.
The physiological role of LBP-9 is less clear. Our initial studies using transiently transfected JEG-3 cell showed no direct role of LBP-9 on the 155/131 segment of the P450scc gene fused to a minimal promoter and the luciferase reporter (TK32/LUC), but cotransfection experiments showed a clear dose-response inhibition by LBP-9 on LBP-1b-stimulated expression of this 155/131 TK32/LUC construct (15). The view that LBP-9 suppresses LBP-1b-induced transcription was strengthened by the demonstration that CRTR-1, the mouse homolog of LBP-9, also acts as a transcriptional suppressor (33). However, overexpression of LBP-9 by stable transfection of JEG-3 cells yielded variable results, with different cell lines showing increased, decreased, or unchanged P450scc expression. As the inductive and suppressive effect on P450scc expression by LBP-9 did not correlate with the amount of LBP-9 mRNA expressed, these differences are probably unrelated to the copy number or site of integration of the LBP-9 expression cassette into the JEG-3 genomic DNA. The net positive effect on P450scc expression by LBP-9 overexpression seen in the stable cell lines is consistent with the suppression of P450scc mRNA after LBP-9 knockdown. By contrast to these effects on the intact P450scc promoter, LBP-9 suppressed transcription when fused to GAL4-DBD, in a fashion similar to the LBP-9-mediated suppression of LBP-1b-induced expression of the 155/131 segment of the P450scc promoter fused to TK32/LUC. Thus LBP-9 acts as a suppressor in in vitro systems, but it can elicit either mild inductive or suppressive effects on the intact P450scc gene. We found that the endogenous levels of LBP-9 mRNA in JEG-3 cells are both higher than those of LBP-1b and are more variable. Thus LBP-9 appears to acts as modulator of the action of LBP-1b, which is the key regulator of placental P450scc gene transcription. The nature of these complex interactions remains uncertain but may involve protein-protein interactions with LBP-1b, shown by our GST pull-down experiments, and/or interactions with other factors.
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MATERIALS AND METHODS
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Tissue Culture and Transient Transfection
JEG-3 cells, a steroidogenic human placental cell line derived from choriocarcinoma (39), were obtained from the American Type Culture Collection (Manassas, VA) and grown at 37 C and 5% CO2 in DMEM/Hams 21 medium (DME-H21) with 50 µg/ml gentamycin, 5% fetal bovine serum, and 5% horse serum. JEG-3 cells were grown to 5080% confluence on 12-well tissue culture plates 24 h before transfection. For transient transfectios with TK32/LUC, 0.7 µg of expression vectors for TReP-132, LBP-1b, or LBP-9 were cotransfected with 0.1 µg of 3x(155/131) TK32/LUC into JEG-3 cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) at a ratio of 1 µg DNA to 1 µl of Lipofectamine 2000. After transfection for 20 h, the cells were washed and incubated in fresh growth medium for an additional 24 h. For transient transfections with GAL4-LBP-9, 1.0 µg of the promoter-reporter plasmid pTK-MH100x4-Luc and 0.5 µg of pGAL4-LBP-9 plasmid DNA were cotransfected into each well of JEG-3 cells by calcium phosphate precipitation for 6 h. After the calcium phosphate-DNA precipitates were aspirated, the cells were washed with 1 ml PBS and incubated for an additional 36 h in JEG-3 growth medium. Firefly luciferase activity was measured with Dual-Luciferase system (Promega Corp., Madison, WI) and normalized by Renilla luciferase activity expressed by the cotransfected plasmid DNA pRL-CMV (Promega Corp.).
Retrovirus-Mediated Gene Transfer
Full-length open reading frames for LBP-1b and LBP-9 were amplified from our previously described cDNAs (15). The primer pairs 1s-12/1as-7 and 9s-9/9as-8 (Table 4
) placed XhoI and SalI sites on the 5'- and 3'-ends, permitting cloning into XhoI/SalI-cleaved retroviral vector pLEGFP-N1 (CLONTECH, Palo Alto, CA) to create the plasmids pLEGFP-N1-LBP-1b and pLEGFP-N1-LBP-9, respectively. These plasmids, under the control of the human CMV promoter, permitted the expression of the LBP proteins fused to green fluorescent protein. The neomycin resistance (Neor) gene in pLEGFP-N1 permitted the selection of stably integrated plasmid by growth in medium containing G418.
The retroviral packaging cell line phoenix-Ampho, a gift from Dr. Gary Nolan (Stanford University, Stanford, CA), was maintained as described elsewhere (40) (http://www.stanford.edu/group/nolan/pheonix_info.html). These cells express the Moloney murine leukemia virus gag and pol genes and Moloney murine leukemia virus amphotropic envelope gene (41). For retroviral gene transfer (40, 42), the pheonix-Ampho cells were transfected at 80% confluence with 10 µg of pLEGFP-N1-LBP-1b or pLEGFP-N1-LBP-9 by the calcium phosphate method. Replication-defective retrovirus was harvested from the cell supernatant 48 h after transfection and filtered under sterile conditions. JEG-3 cells were then infected by the retrovirus in the presence of 5 µg/ml polybrene (Sigma Chemical Co., St. Louis, MO) and then were sorted by a fluorescence-activated cell sorter 24 h after infection. Cells having green fluorescence were pooled and selected by growth in 500 µg/ml G418 for 3 wk, and 20 individual clones were selected expressing LBP-1b-EGFP and 20 expressing LBP-9-EGFP. Vector control cells were generated similarly using the empty vector pLEGFP-N1, and 10 individual clones expressing EGFP alone were selected.
RNase Protection Assay
32P-labeled single-stranded RNA probes for human P450scc, LBP-1b, LBP-9, and human GAPDH were generated as follows. Full-length (1.8 kb) human P450scc cDNA was excised from its pUC18 cloning vector with EcoRI (5), subcloned into the EcoRI site of pBluescript II sk+, and then linearized with PstI. In vitro transcription with T7 RNA polymerase generated a 252 base 32P-labeled RNA probe, which included 192 bp from P450scc. A fragment of LBP-1b cDNA extending from bases 793 to 1420 was excised with BamHI and EcoRI and subcloned into pBluescript II sk+ plasmid cleaved with BamHI and EcoRI. This construct was then linearized with HaeII so that transcription using T7 RNA polymerase generated a 322-base 32P-labeled RNA probe that included 266 bp from LBP-1b. A fragment of LBP-9 cDNA extended from bases 608838 was generated by PCR using primers 9s-2 and 9as-1 and subcloned into pCR2.1 by TA cloning (Invitrogen). This construct was linearized with BamHI so that transcription using T7 RNA polymerase generated a 335 base 32P-labeled RNA probe that included 230 bp from LBP-9. A fragment of human GAPDH cDNA extended from bases 664804 was generated by PCR using primers GAPDH-s-1 and GAPDH-as-1. The PCR product was digested with SacII and XbaI and then subcloned into pBluescript II sk+ at SacII and XbaI sites. This construct was linearized with SacI so that transcription with T7 RNA polymerase generated a 234-base 32P-labeled RNA probe that included 144 bp from GAPDH. To assay LBP-1b, LBP-9, P450scc, and GAPDH simultaneously, the probe for GAPDH was synthesized with a specific activity that was 200-fold lower than the rest of probes, as the abundance of GAPDH is higher than LBP-1b, LBP-9, and P450scc.
Total RNA from JEG-3 cell lines stably expressing LBP-1b-EGFP, LBP-9-EGFP, or EGFP alone was prepared using TRI Reagent (Molecular Research Center, Cincinnati, OH), and 20 µg were hybridized with the mixture of 32P-labeled RNA probes for LBP-1b, LBP-9, P450scc, and GAPDH at 42 C for 20 h. The hybridized products were treated with RNase A and RNase T1 (Ambion, Inc., Austin, TX) at 37 C for 30 min to degrade unhybridized probes before electrophoresis on 5% polyacrylamide gel containing 7 M urea; the gel was then dried and analyzed by phosphorimaging.
Northern Blot
Total RNA was isolated from cell lines, and 10 µg aliquots were separated by electrophoresis on 1% agarose formaldehyde gel and blotted to nylon membrane (Hybond NX, Amersham Biosciences, Piscataway, NJ) in 10x saline sodium citrate (SSC) overnight (1x SSC is 150 mM NaCl, 15 mM sodium citrate, pH 7.0). A 428-bp cDNA fragment of LBP-1b (bases 992-1420), a 485-bp cDNA fragment of LBP-9 (bases 133618), a 750-bp cDNA fragment of human P450scc (bases 747-1497), and a 501-bp PCR product of GAPDH cDNA (bases 566-1067) were labeled with [
32P]dCTP and random primers, and used as probes. The RNA blot was first incubated for 15 min at 65 C with 3x SET, 1% SDS, (1x SET is 150 mM NaCl; 30 mM Tris-HCl, pH 8.0; 2 mM EDTA, pH 8.0), and then prehybridized for 1 h at 65 C in hybridization buffer containing 3x SET, 1% SDS, 2 mg/ml ficoll (Sigma), 2 mg/ml polyvinylpyrrolidone (Sigma), and 2 mg/ml BSA (Sigma), followed by 2 h at 65 C with the same buffer with the addition of 250 µg/ml yeast transfer ribonucleic acids (Sigma). The blot was hybridized with the same prehybridization buffer with the addition of 20 mM sodium phosphate (pH 7.0), 10% dextran sulfate, and the 32P-labeled probe for 20 h at 65 C. The blot was then washed with 1x SSC, 0.1% SDS for 1 h at 65 C, and then with 0.3x SSC, 0.1% SDS for 1 h at 65 C and analyzed by phosphorimaging.
GST Pull-Down Assay
Full-length open reading frames for LBP-1b and LBP-9 were amplified by PCR using the primer pairs 1s-17/1as-5 and 9s-14/9as-6, which placed SmaI and XhoI sites at the 5'- and 3'-ends, permitting cloning into SmaI/XhoI digested pGEX-4T-1 (Amersham Biosciences) to produce the plasmids pGST-LBP-1b and pGST-LBP-9. This permitted the expression of the LBP proteins fused to GST. These plasmids were propagated in Escherichia coli BL21 (DE3) and induced with 0.5 mM isopropyl ß-D-thiogalactoside at 30 C for 4 h to produce the GST-fusion proteins. The bacteria were resuspended in 10 ml of 50 mM Tris-HCl (pH 8.0), 100 mM KCl, 0.1 mM dithiothreitol, 10 µM ZnCl2, 1 mM phenylmethylsulfonyl fluoride, proteinase inhibitors (1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 µg/ml pepstatin A), and then lysed with 50 µg/ml lysozyme for 30 min at 4 C, and then 2 min at 37 C, followed by three cycles of freeze/thaw, and then disrupted further by sonication for 4 x 30 sec at 4 C. After centrifugation at 12,000 x g for 30 min, the GST-fusion proteins were immobilized on glutathione Sepharose 4B for 1 h at 4 C in binding buffer (50 mM Tris-HCl, pH 8.0; 100 mM KCl; 0.3 mM dithiothreitol; 10% glycerol; 10 mM MgCl2; 0.1% Nonidet P-40; 1 mM phenylmethylsulfonyl fluoride; 1 µg/ml leupeptin; 1 µg/ml aprotinin; and 1 µg/ml pepstatin A). The beads were then washed three times with PBS and three times with 800 µl binding buffer, after which 5 µg of beads were incubated with 10 µl [35S] methionine-labeled LBP-1b, LBP-9, or TReP-132 proteins produced by in vitro transcription/translation in the same binding buffer (final volume, 200 µl) for 2 h at 4 C. The beads were then washed five times with 800 µl of binding buffer. Bound proteins were released from the Sepharose 4B beads by boiling in SDS sample buffer and were separated by 10% or 15% SDS-PAGE according to the size of the proteins.
In Vitro Transcription/Translation
Full-length open reading frames for LBP-1b and LBP-9 were amplified by PCR using the primer pairs 1s-12/1as-5 and 9s-9/9as-6. These primers placed XhoI sites at both ends, permitting cloning into XhoI-digested pcDNA3.1 (Invitrogen) to produce the plasmids pcDNA3.1-LBP-1b and pcDNA3.1-LBP-9. Deletion mutants of LBP-1b, comprising residues 102540, 200540, 300540, and 403540, were amplified by PCR with the primer pairs 1s-koz-102/1as-5, 1s-koz-200/1as-5, and 1s-koz-300/1as-5, and cloned into pcDNA3.1 using TOPO cloning (Invitrogen) to produce the plasmids pcDNA3.1-LBP-1b (102540), pcDNA3.1-LBP-1b (200540), and pcDNA3.1-LBP-1b (300540). Deletion mutants of LBP-9, comprising residues 100479, 200479, and 300479 were amplified by PCR with the primer pairs 9s-21/9as-6, 9s-22/9as-6, and 9s-23/9as-6, and cloned into pcDNA3.1 as above to produce the plasmids pcDNA3.1-LBP-9 (100479), pcDNA3.1-LBP-9 (200479), and pcDNA3.1-LBP-9 (300479). Full-length TReP-132 cDNA in pcDNA3 vector (18) was a generous gift from Dr. Dean W. Hum (Genfit, Lille, France).
LBP-1b, LBP-9, their various deletion mutants, and TReP-132 were synthesized by in vitro transcription/translation from pcDNA3.1-LBP-1b, pcDNA3.1-LBP-9, or TReP-132 pcDNA3 plasmids, encoding full-length or deletion mutants, using rabbit reticulocyte lysate (TNT system, Promega) in the presence of [35S]methionine, according to the manufacturers protocol. The radiolabeled proteins were separated by 10% or 15% SDS-PAGE, and the gels were dried and analyzed by phosphorimaging.
Plasmid Construction of GAL4-DBD Fusions
In-frame fusions between the yeast GAL4-DBD (amino acids 1147) and fragments of LBP-9 were generated in the plasmid pCMX-GAL4-DBD (kindly provided by Dr. Ronald M. Evans, Salk Institute, San Diego, CA) (37). PCR fragments encoding the full-length open reading frame of LBP-9 were generated with primer pairs of 9s-14/9as-6. These primers placed SmaI and XhoI at the 5'- and 3'-ends, permitting cloning into SmaI/SalI digested pCMX-GAL4-DBD to generate pGAL4-LBP-9 (1479). The LBP-9 fragments comprising residues 1106, 100200, and 200479 were amplified with the primer pairs 9s-14/9as-106, 9s-18/9as-200, and 9s-19/9as-6. These primers placed SmaI and XhoI at the 5'- and 3'-ends, permitting cloning into SmaI/SalI-digested pCMX-GAL4-DBD to generate pGAL4-LBP-9 (1106), pGAL4-LBP-9 (47479), pGAL4-LBP-9 (100200), and pGAL4-LBP-9 (200479).
EMSAs
Double-stranded DNA corresponding to bases 155/131 of the human P450scc promoter (15) was 5'-end labeled with [
-32P]ATP and T4 polynucleotide kinase, and then purified through a Sephadex G50 spin column (Amersham Pharmacia Biotech). A 1-µl aliquot of in vitro transcription/translation mix containing LBP-1b, LBP-9, or TReP-132 proteins was incubated at room temperature for 20 min with 40,000 cpm (
0.4 ng) of 32P-end-labeled double-stranded probe in a final volume of 20 µl in 10 mM Tris-HCl (pH 7.5), 100 mM KCl, 1 mM EDTA, 4% glycerol, 5 mM dithiothreitol, 0.1 mg/ml BSA with 1 µg poly(deoxyinosine-deoxycytosine) added as a nonspecific competitor. DNA-protein complexes were separated by electrophoresis in 4% native polyacrylamide gel in 50 mM Tris base, 38 mM glycine, 2 mM EDTA (pH 8.0), and 0.35 µl of ß-mercaptoethanol, and analyzed by phosphorimaging.
RNA Interference
Small interfering RNA (siRNA) was expressed using the vector pSUPER (OligoEngine, Seattle, WA) (43, 44). For interference of P450scc mRNA, an oligonucleotide was synthesized consisting of the 19 bp of P450scc cDNA from bases 171189 (where the A of the ATG start codon is base 1) (Table 4
), followed by the noncomplementary nine-base sequence TCTCTTGAA, and then followed by the reverse complement of P450scc bases 171189. BglII and HindIII sites were placed at each end, permitting cloning into the BglII/HindIII site of pSUPER, downstream from the H1-RNA promoter. Transcription of the resulting pSUPER-P450scc vector produces a 19-bp double-stranded P450scc siRNA linked by the 9-bp hairpin. The vectors pSUPER-LBP-1b and pSUPER-LBP-9 were constructed in the same fashion, using the 19-base sequences 715733 of LBP-1b and 12471265 of LBP-9 (Table 1B
). The pSUPER vectors were transfected into JEG-3 cells in 10-cm plates using Lipofectamine 2000 (Invitrogen), and RNA was isolated from the cells 72 h later for analysis by RNase protection assays for P450scc, LBP-1b, LBP-9, and GAPDH. Results of siRNA experiments represent the means of three independent transfections, RNA preparations, and assays.
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ACKNOWLEDGMENTS
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We thank Dr. Gary Nolan (Stanford University, Stanford, CA) for the retroviral packaging cell line phoenix-Ampho, Dr. Ronald M. Evans (Salk Institute, San Diego, CA) for the plasmids pCMX-GAL4-DBD and pTK-MH100x4-Luc, Dr. Dean W. Hum (Genfit, Inc., Lille, France) for the TReP-132 plasmid, and Ms. Izabella Damm for technical assistance.
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FOOTNOTES
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This work was supported by Mentored Investigator Grant KO1 DK02939 (to N.H.) and National Institutes of Health Grants DK37922 and HD41958 (to W.L.M.).
First Published Online October 7, 2004
Abbreviations: CMV, Cytomegalovirus; CRTR-1, CP2-related transcriptional repressor-1; DBD, DNA binding domain; EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione-S-transferase; LBP, long terminal repeat binding protein; RNase, ribonuclease; SDS, sodium dodecyl sulfate; SF1, steroidogenic factor 1; siRNA, small interfering RNA; SSC, saline sodium citrate; TK32/LUC, thymidine kinase 32/luciferase.
Received for publication March 2, 2004.
Accepted for publication October 1, 2004.
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