Multiple Orphan Nuclear Receptors Converge to Regulate Rat P450c17 Gene Transcription: Novel Mechanisms for Orphan Nuclear Receptor Action

Peilin Zhang and Synthia H. Mellon

Department of Obstetrics, Gynecology, and Reproductive Sciences (P.Z.)and The Metabolic Research Unit (S.H.M.), University of California, San Francisco, California 94143-0556


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The orphan nuclear receptor steroidogenic factor-1 (SF-1) plays a key role in regulating the expression of the rat P450c17 gene in testicular Leydig and in adrenocortical cells. Other DNA sequences, not bound by SF-1, are also involved in transcriptional regulation of the rat P450c17 gene in both cell types. The region from -447/-399 or from -447/-419 increased both basal and cAMP-induced transcription, and the region from -418/-399 increased basal transcription to a greater extent than the intact -447/-399 DNA. The -447/-399 DNA sequence contains three imperfect copies of the orphan nuclear receptor-binding motif, AGGTCA, and at least three known orphan nuclear receptors, chicken ovalbumin upstream promoter transcription factor (COUP-TF), SF-1, and an early response gene induced by nerve growth factor (NGFI-B), bind to -447/-399 DNA. The AGGTCA triad is bound by one set of nuclear proteins when these three elements are colinear and is bound by a different set of proteins when these elements are separated. When the elements are separated, COUP-TF no longer binds, and the region -418/-399 is bound by a protein that greatly stimulates basal transcription. The region -447/-419 is bound by two different proteins that mediate both basal and cAMP-stimulated transcription. We call the protein binding to -418/-399 steroidogenic factor inducer of transcription-1 (StF-IT-1), and one of the proteins binding to -447/-419, StF-IT-2. SF-1 binds to a second AGGTCA element in the -447/-419 region. StF-IT-1 and StF-IT-2 are both found in Leydig and adrenal cells, and transcriptional regulation is similar in both cell types. SF-1 and NGF-IB may increase transcription by displacing COUP-TF (a transcriptional repressor) because these proteins share DNA-binding domains. However, neither SF-1 nor NGF-IB alone, binding as monomers, increases transcription. Rather, these proteins must interact with another DNA-binding protein, e.g. StF-IT-2, to increase transcription. StF-IT-2 also requires interaction with SF-1 (or NGF-IB) bound to DNA and cannot increase transcription by itself. This mechanism of action is different from the mechanism by which SF-1 regulates transcription from the -84/-55 region of the rat P450c17 gene. Thus, we have defined a novel mechanism of action for orphan nuclear receptors that bind to DNA as monomers.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroidogenesis is initiated by conversion of cholesterol to pregnenolone by the mitochondrial cholesterol side chain cleavage enzyme, P450scc. The cell type-specific synthesis of various steroids in the gonads and adrenals is determined by cell-specific expression of a variety of additional steroidogenic enzymes. The conversion of pregnenolone and progesterone to their 17{alpha}-hydroxylated products and then to dehydroepiandrosterone and androstenedione, respectively, is catalyzed by a single protein, P450c17, encoded by a single gene (1, 2) Thus, P450c17 mediates two enzymatic reactions, 17{alpha}-hydroxylation and cleavage of the C17-C20 carbon bonds (lyase reaction) (3, 4, 5). The expression of the P450c17 gene in steroidogenic tissues is species specific: it is expressed in the rodent gonad (6, 7, 8) and placenta (9, 10), but not adrenal (11), and is expressed in the human adrenal and gonad, but not placenta (11, 12, 13).

P450c17 gene expression is regulated in a tissue-specific and species-specific fashion by trophic hormones via cAMP as a second messenger. Bovine, but not human or rodent, adrenals lack P450c17 in the absence of tropic stimulation (14, 15). Human adrenals (11, 12, 13) and human and rodent gonads (6, 7, 8, 12) contain P450c17 mRNA in the absence of tropic hormones or cAMP and hence exhibit basal transcription (7, 8, 13, 16, 17). Thus, the P450c17 gene is regulated by different mechanisms in various species and in various tissues. Although the P450c17 gene is not expressed in the rodent adrenal in vivo (11), the human or rodent P450c17 promoter is readily expressible when transfected into rodent adrenocortical cells, suggesting that the transcription factors necessary for its expression exist in both rodent adrenal and Leydig cells (2, 18, 19). One such factor is steroidogenic factor-1 (SF-1), which regulates the expression of the rat P450c17 gene in both adrenal and Leydig cells (2, 18). This regulatory diversity of P450c17 in various species and tissues may reflect the involvement of different nuclear transcriptional factors. Therefore, we sought to determine whether nuclear transcription factors other than SF-1 bind to the rat P450c17 promoter and are involved in regulation of rat P450c17 in mouse Leydig MA-10 cells. We now identify a segment of the 5'-flanking region of the rat P450c17 gene that is involved in both basal and cAMP-mediated transcriptional regulation, identify the multiple different transcription factors that bind to this region, and characterize two previously undescribed transcription factors.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of cis-Active DNA Sequences in the Rat P450c17 Gene
Previous studies showed that a DNA element located at -84/-55 of the rat P450c17 gene mediated both basal and cAMP-inducible transcription (2, 18). The nuclear protein bound to this element was identified as the orphan nuclear receptor, SF-1 (20). Our transfection studies (2) suggested that the region between -476/-267 was also important for basal and cAMP-mediated transcription of this gene in both mouse Leydig MA-10 and adrenal Y-1 cells. Therefore we studied the sites and activities of protein-DNA interactions in the -476/-267 region.

DNase I footprinting of bases -476/-267 using cell extracts from MA-10 cells identified a broad region from about -399 to -447 containing both DNase I-sensitive bands and newly created DNase I-hypersensitive bands, suggesting the binding of several nuclear proteins (Fig. 1Go). The DNA sequence of the footprinted region is shown beside the footprint. This footprinted region contains multiple imperfect copies of the common DNA estrogen receptor half-site motif, AGGTCA, which can be bound by a number of steroid/retinoid intracellular receptors.



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Figure 1. DNase Footprint of the -447/-399 Region of the Rat P450c17 Gene

P450c17 DNA from -476 to -267 was incubated with nothing (probe) or with nuclear extracts from MA-10 cells (MA-10) and digested with DNase I. One large region of DNA was bound by protein(s). The sequence to which they bind are indicated beside the autoradiogram. Lane A + G contains A + G chemical sequencing reactions.

 
Gel mobility shift assays using bases -447/-399 and MA-10 cell extracts identified three major protein-DNA complexes, called I, II, and III (Fig. 2AGo). Complex II may contain two different protein-DNA complexes, but these complexes are not well resolved by gel electrophoresis. All three complexes could be competed by adding 500-fold molar excess of unlabeled -447/-399 oligonucleotide (Fig. 2AGo, lane 3). The DNA segment from -84 to -55 binds SF-1 (18). When the DNA corresponding to -84/-55 was used as competitor, complex I could be competed completely (lane 4), and complexes II and III were also competed, but to a lesser extent. This suggests that SF-1 may be involved in complex I and that SF-1 binding may affect binding of other proteins in complexes II and III. SF-1 binds to the estrogen receptor element half site, AGGTCA, but there is also a strong preference for the sequence TCA at the 5'-end. Although the -447/-399 DNA contains three AGGTCA-like motifs, there is no consensus SF-1 site (TCAAGGTCA) in the -447/-399 DNA, suggesting that SF-1 may recognize a modified estrogen response element (ERE) half-site and may thus recognize different DNA sequences in different contexts.



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Figure 2. Gel Shift Analysis of Nuclear Extracts from MA-10 Cells Binding to a -447/-399 Oligonucleotide

A, Autoradiograph of an incubation of labeled oligonucleotide containing a sequence from -447 to -399 bp of the rat P450c17 gene, displayed on a 5% nondenaturing acrylamide gel (lane 1). Three protein-DNA interactions, labeled I, II, and III, are observed when incubated with MA-10 cell extract (lane 2). Each complex can be competed by incubation with 500 x excess unlabeled oligonucleotide (lane 3); competition by an oligonucleotide from -84/-55 of the rat P450c17 gene, to which SF-1 binds (2, 18), is in lane 4. B, Autoradiograph of an incubation of labeled oligonucleotide containing a sequence from -447/-399 bp of the rat P450c17 gene, displayed on a 5% nondenaturing acrylamide gel (lanes 1–3 and lanes 10–12). Competition of complex formation by 500-fold concentration of mutant oligonucleotides, lanes 4, 5, 6, 9, and 13. Competition of complex formation by 500-fold concentration of wild type oligonucleotides from -432/-399 (lane 7) and from -418/-399 (lane 8) of the rat P450c17 gene.

 
Because protein binding in complex I appears to affect the protein binding in complexes II and III, we used various oligonucleotides (Table 1Go) to study these protein-DNA interactions in greater detail. The -447/-399 DNA contains three variations of the AGGTCA motif, listed in Table 1Go as sites 1, 2, or 3, and hereafter referred to as sites 1, 2, or 3. We created mutant oligonucleotides in which we deleted a single ERE half-site at site 1 or 3 or mutated three bases within the ERE half-sites (changing AGG to TTT) and used these as competitors for the wild type -447/-399 sequence in gel shift assays (Fig. 2BGo).


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Table 1. Oligonucleotides Used in the Studies

 
Oligonucleotides containing a mutation at site 1 (-447/-399{Delta}1, lane 13) were able to compete with the wild type -447/-399 oligonucleotide for formation of all three complexes. Similarly, oligonucleotides containing a deletion of site 1 and mutation of either site 2 (Mut 2, lane 4) or site 3 (Mut 3, lane 5) or a deletion of site 3 (WT -432/-399, lane 7) were also able to compete with the wild type -447/-399 oligonucleotide for the formation of all three complexes. However, when sites 2 and 3 were mutated simultaneously (Mut 2/3, lane 6), the resulting oligonucleotide could no longer compete with the wild type -447/-399 oligonucleotide. We observed competition with the mutant oligonucleotides when only one ERE half-site at either site 2 or 3 remained intact. Oligonucleotides containing a single ERE half-site at site 1 (WT -418/-399, lane 8) or a mutant ERE at site 1 (Mut 1, lane 9) were unable to compete with the wild type -447/-399 DNA for protein binding in any of the complexes, indicating that protein(s) binding to the site 2 or 3, but not at site 1 alone, affect protein binding at all three ERE half-sites. Because mutations at ERE half-sites affected protein binding, the data further indicate that the formation of complexes I, II, and III all require protein binding to intact AGGTCA motifs.

-447/-399 DNA Contains Novel Basal- and cAMP-responsive Transcriptional Elements
We determined whether the -447/-399 region of the P450c17 gene was transcriptionally active by ligating the -447/-399 oligonucleotide into a luciferase expression vector containing the herpes simplex virus thymidine kinase (TK) minimal promoter (TK32LUC) and transfecting the resulting construct into mouse Leydig MA-10 and adrenocortical Y-1 cells. As shown in Fig. 3AGo, addition of the -447/-399 sequences to the TK32LUC vector increased basal luciferase activity 15-fold in MA-10 cells. Stimulation with cAMP for 6 h further increased luciferase activity approximately 12-fold. Thus, DNA sequences between -447/-399 mediate both the basal and cAMP-regulated transcription of the rat P450c17 gene. Qualitatively similar results were obtained in Y-1 cells (Fig. 3BGo), but the magnitude of the responses was less than in MA-10 cells.



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Figure 3. Luciferase Assays of P450c17-TK32LUC Constructs

Luciferase reporter gene constructs containing the -447/-399 rP450c17 oligonucleotide (A and B) or mutants of this oligonucleotide (C) were ligated to 32 bp DNA from the TK promoter (-447/-399 TKLUC). These constructs, or the TK minimal promoter alone (TKLUC), were transfected into cultured mouse Leydig MA-10 cells (A and C) and cultured mouse adrenocortical Y-1 cells (B). Other wild type and mutant oligonucleotides from -447/-419 or -418/-399 were similarly cloned into the TK32LUC reporter plasmid and used to transfect A-10 (A) or Y-1 (B) cells. Cells were not treated (open bars) or were treated with 1 mM 8-Br-cAMP for 6 h (black bars) before luciferase activity was measured. Transfections were performed in triplicate for each construct. This figure is one representative of three separate transfection experiments with each cell type. Error bars represent ±SD.

 
We removed site 1 by deleting sequences from -418 to -399 and ligated the remaining -447/-419 oligonucleotide, containing sites 2 and 3 to the TK32LUC vector. This construct, -447/-419TK32LUC, was transfected into MA-10 and Y-1 cells to determine its basal and cAMP-induced transcriptional activity. In MA-10 cells, basal luciferase activity of the -447/-419TK32LUC plasmid was less than that of the intact -447/-399TK32LUC plasmid, but was still 6-fold greater than the vector alone. This truncated plasmid still showed a 7-fold increased response to stimulation with cAMP. By contrast, this construct showed only a 2-fold response to cAMP in Y-1 cells. We mutated sites 2 and 3 individually (mutants 2 and 3) (Table 1Go) or together (mutant 4), ligated each to the TK32LUC plasmid, and transfected these plasmids into Y-1 and MA-10 cells. All three of these mutants completely lost both basal and cAMP-induced transcription. Mutant 4 demonstrated some cAMP stimulation in MA-10 cells, although neither mutant 2- or 3-TK32LUC showed activity. Similar results were obtained when the constructs were transfected into Y-1 cells (Fig. 3BGo), suggesting that the-447/-399 sequence is functional in both Leydig and adrenocortical cells, and that the same transcription factors may be functioning in both types of steroidogenic cells.

We also analyzed the activity of the -418/-399 DNA, after ligation to the TK32LUC plasmid, in transfected MA-10 and Y-1 cells (Fig. 3Go, A and B). Basal luciferase activity of the -418/-399TK32LUC plasmid in MA-10 cells was 4 times greater than the activity of the intact -447/-399TK32LUC and was 45 times greater than the vector alone. However, cAMP had a minimal effect on -418/-399TK32LUC activity. When this construct was transfected into Y-1 cells, the results were similar, but again, the magnitude of the responses was less than in MA-10 cells (Fig. 3BGo). When site 1 in -418/-399TK32LUC was mutated to TTTAGA, (called Mut 1 -418/-399 TK32LUC), it had no basal or cAMP-induced luciferase activity in MA-10 or Y-1 cells. These data indicate that an intact AGGTCA-like motif is required for transcriptional activation, and that the sequence between -418 and -399 contains a strong basal transcription activator whose activity is attenuated by sequences between -447 and -419.

Transcriptional activities were also assessed in the intact -447/-399 DNA, but in which site 1 was individually mutated (called -447/-399{Delta}1). This oligonucleotide was ligated to TK32LUC and transfected into MA-10 cells (Fig. 3CGo). The data demonstrate that basal activity of the -447/-399{Delta}1TK32LUC plasmid was slightly greater than the intact -447/-399TK32LUC plasmid. The -447/-399{Delta}1TK32LUC plasmid showed a 12-fold response to cAMP stimulation, similar to the response seen with the -447/-419TK32LUC construct (Fig. 3AGo).

Finally, we analyzed the activity of the -432/-399 DNA after ligation to the TK32LUC plasmid (Fig. 3Go, A and B). This DNA contains sites 1 and 2, but lacks site 3. This plasmid had neither basal nor cAMP-stimulated activity in either MA-10 or Y-1 cells. These data, together with the data from the other constructs, indicate that transcription from -432/-399 DNA is repressed. Thus, the basal transcription and cAMP induction from -447/-399 P450c17 DNA is due to the combination of activating and repressing interactions, and this transcriptional activation requires intact AGGTCA-like motifs.

Chicken Ovalbumin Upstream Promoter-Transcription Factor (COUP-TF) Binds to the -447/-399 Region of the Rat P450c17 Gene
Our functional data (Fig. 3Go) suggest that the intact -447/-399 sequence has less transcriptional activity than the truncated -418/-399 sequence, and that the -432/-399 sequence had no activity. This suggests that a repressor may be involved in attenuating transcriptional activity in the intact fragment. COUP-TF is a factor that can bind to AGGTCA-like sequences and repress transcription (21, 22) and the DNA sequence between -447/-399 contains a potential COUP-TF binding site. To determine whether COUP-TF is involved in rat P450c17 gene transcription, we performed gel mobility shift assays with MA-10 cell extracts and several oligonucleotides encompassing the -447/-399 region, in the absence and presence of a COUP-TF antibody. As shown previously in Fig. 2AGo, the entire -447/-399 element formed three protein-DNA complexes with MA-10 cell extracts (Fig. 4Go, lane 2). Addition of COUP-TF antibody decreased the formation of complexes II and III, increased the amount of complex I, and generated an additional band (lane 4), possibly a supershift of either complex II or III. As the antibody binds to both COUP-TF I and COUP-TF II, the data suggest that one or both of these forms of COUP-TF binds to the 5'-flanking DNA of the rat P450c17 gene. COUP-TF not only bound to the -447/-399 DNA, but also bound to -432/-399 DNA (Fig. 4BGo). This binding is indicated by a supershift of the protein-DNA complex by antibody to COUP-TF. These data suggest that COUP-TF binding to the -447/-399 DNA occurs at sites 1 and 2. The data are consistent with a lack of transcriptional activation (i.e. repression) seen with the -432/-399TK32LUC construct (Fig. 3Go).



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Figure 4. COUP-TF Antibody Supershift Assay

Autoradiographs of incubations of labeled oligonucleotide containing a sequence from -447/-399 bp of the rP450c17 gene (A) or -432/-399 bp of the rP450c17 gene (B) with MA-10 cell nuclear extract, displayed on a 5% nondenaturing acrylamide gel. A, Three complexes, I, II, and III, are identified (lanes 2) and are competed with 500-fold molar excess of unlabeled oligonucleotide (lane 3). In lane 4, the probe was incubated with MA-10 cell extract and COUP-TF antibody, resulting in a decrease in the intensity of complexes II and III and the formation of an additional, super-shifted complex that we call IIIa. B, One complex is formed with the -432/-399 DNA. Incubation with a COUP-TF antibody results in the formation of an additional, supershifted complex.

 
Identification of Additional Protein-DNA Complexes within the -447/-399 Region of the Rat P450c17 Gene
Transfection data using smaller DNA fragments from within the -447/-399 region suggested that each AGGTCA-like motif may function independently when isolated from the other motifs, but that these motifs may function cooperatively, and differently, when arranged in a group of three motifs. Therefore, we determined whether additional nuclear proteins bound to sites 1, 2, and 3 when these regions were isolated from each other. We used -447/-419 (containing sites 2 and 3) or -418/-399 oligonucleotide (containing site 1) as probes in gel shift experiments (Fig. 5AGo). We detected two protein-DNA complexes, called complexes IV and V, when -447/-419 DNA was used as probe (lane 2), and we detected one protein-DNA complex, called complex VI, when -418/-399 DNA was used as probe (lane 9). Complexes IV and V could be competed using 500-fold excess of unlabeled -447/-419 oligonucleotide (lane 3) but could not be competed using an unlabeled -418/-399 oligonucleotide (lane 4). Complexes IV and V could also be competed with -432/-399 DNA (lane 5), even though this DNA lacks site 3. Complex VI could be competed with 500-fold excess unlabeled -418/-399 oligonucleotide (lane 10), but it could also be competed using unlabeled -432/-399 (lane 12) and -447/-419 (lane 11) oligonucleotides. These data indicate that the proteins in complexes IV and V were different from the protein in complex VI. The data also suggest that the protein in complex VI had a permissive DNA sequence requirement, as its binding could be competed with an unrelated, but similar, DNA sequence found in the -447/-419 oligonucleotide.



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Figure 5. Analyses of Protein-DNA Interactions with -447/-419 and with -418/-399 rP450c17 DNA

A, Gel shift assay of MA-10 cell nuclear extract to rP450c17 oligonucleotide probes -447/-419 (lanes 1–7) and -418/-399 (lanes 8–14) Incubation of the -447/-419 probe with MA-10 cell nuclear extracts yields two protein-DNA complexes, IV and V, indicated on the left side of the autoradiogram. Competition for these complexes was examined using 500-fold unlabeled wild type oligonucleotides -447/-419 (lane 3), -418/-399 (lane 4), -432/-399 (lane 5), or by mutant -447/-419 oligonucleotides mutant 2 (lane 6) and mutant 3 (lane 7). Incubation of the -418/-399 probe with MA-10 cell nuclear extracts yields one protein-DNA complex, VI (lane 9), indicated on the right side of the autoradiogram. Competition for this complex was by unlabeled wild type oligonucleotides -418/-399 (lane 10), -447/-419 (lane 11), -432/-399 (lane 12), -447/-399 (lane 13) or by mutant -418/-399 oligonucleotide mutant 1 (lane 14). B, Direct binding of MA-10 cell nuclear proteins to mutant oligonucleotides. Wild type oligonucleotide -447/-419 was used as probe in lanes 1–4; -447/-419 mutant 2 oligonucleotide was used as probe in lanes 5–7; -447/-419 mutant 3 oligonucleotide was used as probe in lanes 8–10. Two protein-DNA complexes are formed with wild type -447/-419 oligonucleotide (complexes IV and V; lanes 1–2) and is competed by 500-fold molar excess unlabeled oligonucleotide (lane 3); complex V is formed with mutant 2 oligonucleotide probe (lane 6) and competed with 500-fold excess unlabeled mutant 2 probe (lane 7); complex IV is formed with mutant 3 oligonucleotide probe (lane 9) and competed with 500-fold excess unlabeled mutant 3 probe (lane 10). Complex IV formation is competed by -84/-55 rP450c17 oligonucleotide probe (lane 4). C, Rat recombinant SF-1 binding to -447/-399 oligonucleotide. Autoradiogram of a gel shift assay using -447/-399 oligonucleotide as probe (lane 1) with rat recombinant SF-1 prepared from bacteria (lane 2). SF-1-DNA complex is competed by 500-fold molar excess -447/-399 oligonucleotide (lane 3) and by -447/-419 mutant 3 oligonucleotide (lane 5) but not by -447/-419 mutant 2 oligonucleotide (lane 4). D, Protein-DNA interactions within the -447/-399 region of the rat P450c17 gene. In vivo, this region of the rat P450c17 gene is bound by at least three factors, COUP-TF, SF-1, and an unidentified nuclear protein. All these proteins appear to bind to AGGTCA-like sequences. COUP-TF binds at site 1, and either at site 2 and/or 3, and therefore needs an intact DNA sequence. There are also additional nuclear factors in MA-10 and Y-1 cells that will bind to this region. They are not apparent on gel shift assays, either due to the relative abundance of the nuclear proteins, or the binding affinities of these proteins, relative to the affinity of COUP-TF. By separating the AGGTCA-like elements in vitro, thereby displacing COUP-TF binding, we identified two additional nuclear proteins that bind to this DNA. Since these proteins increase transcription (either basal transcription or both basal and cAMP-induced transcription), we call them steroidogenic factor inducer of transcription, StF-IT-1 and -2.

 
We used various mutant oligonucleotides as competitors of the wild type probes to determine which bases were required to form protein-DNA complexes. Mutant 2 competed with the -447/-419 probe for formation of complex V (lane 6), but also competed slightly for complex IV. Mutant 3 competed mainly for formation of complex IV (lane 7), but also slightly competed for complex V. These data indicate that the protein in complex IV binds to site 2 (Table 1Go) and the protein in complex V binds to site 3. Mutant 1 did not compete with the -418/-399 probe for formation of complex VI (lane 14), indicating that this complex probably also requires an ERE half-site.

To identify specific bases required for formation of complex IV, V, and VI, we used mutant oligonucleotides (Table 1Go) as probes in gel shift experiments (Fig. 5BGo). Mut 2 -447/-419 oligonucleotide generated a single protein-DNA interaction, corresponding to complex V (lane 6). Mut 3 -447/-419 oligonucleotide formed a single protein-DNA interaction corresponding to complex IV (lane 9). Mut 1 -418/-399 did not generate complex VI (not shown).

Our data indicated that SF-1 bound to the intact -447/-399 DNA (Fig. 2AGo). We determined whether SF-1 also bound to -447/-419 or to -418/-399 and whether it was the protein in either complex IV, V, or VI. We used oligonucleotide -84/-55, to which SF-1 binds in another region of the rat P450c17 gene (2, 18) (see Fig. 2AGo), as a competitor of the -447/-419 DNA (Fig. 5BGo) or of the -418/-399 DNA (not shown). The -84/-55 oligonucleotide did not compete with -418/-399 DNA for formation of complex VI (not shown) but did compete with -447/-419 for formation of complex IV, but not complex V (lane 4). Thus, complex IV is due to an interaction of SF-1 with site 2. These data were confirmed by displacement of complex IV with an antibody against mouse SF-1 (not shown) and by using recombinant rat SF-1 and -447/-399 as probe (Fig. 5CGo). SF-1 bound to this DNA, and binding was not competed by Mut 2 -447/-419, but was competed with Mut 3 -447/-419. Thus, SF-1 binds to site 2 but not to site 3.

We also determined whether any of the proteins in complexes IV, V, or VI was COUP-TF by adding antibody to COUP-TF in the binding reaction. Although COUP-TF bound to the intact -447/-399 oligonucleotide, it did not bind to either the -447/-419 or the -418/-399 oligonucleotides because formation of complexes IV, V, and VI was not supershifted by antibody to COUP-TF (not shown). Likewise, the data in Fig. 4Go demonstrated that COUP-TF binds to -447/-399 DNA and to -432/-399 DNA. These findings suggest that COUP-TF binds simultaneously to two sequences: one is within the -447/-419 region, most likely site 2, and the second is in the -418/-399 region, and binding requires these two regions to be colinear. These results further indicate that COUP-TP does not bind to sites 2 and 3 in the -447/-419 region alone. COUP-TF binding to sites 1 and 2 likely represses the activating function of the protein that binds to site 1 alone. Thus, removing COUP-TF binding, by separating sites 1 and 2, results in increased transcription (Fig. 3Go).

Thus it appears that there are two different sets of proteins that bind to the -447/-399 region of the rat P450c17 gene. One set of proteins (SF-1, COUP-TF, and an additional unidentified protein) binds to this region when it is intact, and an additional two proteins bind when the region is cut into two pieces. As these proteins affect basal and/or cAMP transcription, we call these two novel proteins Steroidogenic Factor Inducer of Transcription-1 and -2, or StF-IT-1 (which forms complex VI) and StF-IT-2 (which forms complex V). The data further indicate that in vivo, binding of COUP-TF to the intact DNA and formation of a ternary protein-DNA complex consisting of complexes I, II, and III would preclude binding of StF-IT-1 or StF-IT-2 (Fig. 5EGo). Protein binding and transcriptional activation data are summarized in Table 2Go.


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Table 2. Summary of Protein-DNA Complexes and Associated Transcriptional Activities

 
Cell-Specific Expression of StF-IT-1 and StF-IT-2
To determine the tissue distribution of expression of StF-IT-1 and StF-IT-2, we performed gel shift experiments using nuclear extracts from mouse adrenal Y-1, mouse adrenal AN4Rpp7 (23), human placental JEG-3, rat glial C6, monkey COS-1, and human HeLa cells, and from mouse testis, adrenal, and liver, using either the -447/-419 or the -418/-399 oligonucleotides as probe (Fig. 6Go). One protein-DNA complex is formed with extracts from MA-10, Y-1, AN4Rpp7, and rat glioma C6 cells, and from mouse testis and adrenals when the -418/-399 DNA is used as probe. HeLa cell extracts also formed a single complex of slightly lesser mobility than the complex from the other cells. Thus, StF-IT-1 is expressed in Leydig, adrenal, and glial cells and may also be expressed in nonsteroidogenic HeLa cells. The slight difference in mobility with HeLa cell extract may represent human/mouse differences in the molecular weight of StF-IT-1.



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Figure 6. Gel Shift Assay of Nuclear Extracts from Various Cell Lines

A, Tissue specificity of StF-IT-1. A -418/-399 oligonucleotide probe was incubated with nuclear extracts from mouse MA-10, Y-1, AN4Rpp7 (23), rat C6 glioma, and human HeLa cells, and from mouse adrenal and testis. Lanes 2–5 and 7–8 show one protein-DNA complex, and lane 6 shows a protein-DNA complex of different mobility. B, Tissue specificity of StF-IT-2. A -447/-419 oligonucleotide probe was incubated with nuclear extracts from mouse MA-10, Y-1, rat C6, monkey COS-7, human HeLa, and JEG-3 cells and from rat liver, mouse adrenal, and mouse testis. Two protein-DNA complexes IV and V are found with extracts from MA-10 and Y-1 cells (lanes 2 and 4) and from mouse adrenal and testis (lanes 9 and 10), and one protein-DNA complex migrating faster than complex V is found with extracts from COS-7 and JEG-3 cells (lanes 5 and 6). No complexes are formed with extracts from HeLa or C6 cells or from extracts of rat liver (lanes 3, 7, and 8).

 
When the -447/-419 oligonucleotide was used as probe, two protein-DNA complexes were formed only with extracts from Leydig and adrenal cells or from testis and adrenals (Fig. 6BGo). Extracts from COS-1 and JEG-3 cells gave one major protein-DNA complex that migrated faster than complex V, and extracts from C6, mouse liver, or HeLa cells showed a diffuse protein-DNA complex or no complex at all. Thus, StF-IT-2 (complex V) is expressed in steroidogenic cells and may be expressed in COS-1 cells, but not in HeLa cells or rat liver. Again, differences in the mobilities of the protein-DNA complex in COS-1 or JEG-3 cells vs. the rodent cell lines may reflect species differences in the molecular weight of StF-IT-2.

NGF-IB Increases Transcription from -447/-419 Rat P450c17 DNA
The DNA sequence between -447/-419 contains the sequences 5'-CAAAGGTTA-3' (site 2) and 5'-ATAAGGTCA-3' (site 3) on the noncoding strand of DNA, which are variant sites for the nuclear receptor NGF-IB, whose consensus binding site is 5'-AAAAGGTCA-3' (24, 25). NGF-IB, a member of the immediate early response gene family, is involved in the transcriptional regulation of the related steroidogenic enzyme, P450c21, in adrenal Y-1 cells (26). Therefore, we determined whether NGF-IB was involved in the regulation of the rat P450c17 gene in mouse Leydig MA-10 cells. Our bacterially expressed rat NGF-IB binds to -447/-419 DNA (Fig. 7AGo, lane 2). This binding is not competed by Mut 2 -447/-419 (lane 4) but is competed by Mut 3 -447/-419 (lane 5), thus indicating that NGF-IB binds to site 2. NGF-IB binding is also competed by WT -447/-399, or by WT -432/-399 oligonucleotides, consistent with binding at site 2.



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Figure 7. Luciferase Assays of Reporter Gene Constructs

Cotransfection of various wild type and mutant rP450c17-TKLUC reporter gene constructs, or the TK minimal promoter alone (TKLUC), together with a plasmid expressing NGF-IB, into cultured mouse Leydig MA-10 cells (A and C) and cultured mouse adrenocortical Y-1 cells (B). Luciferase activity from reporter constructs alone is shown in open bars, and from cells cotransfected with an NGF-IB expression vector is shown in hatched bars. Transfections were performed in triplicate for each construct. This figure is one representative of three separate transfection experiments with each cell type. Error bars represent ±SD.

 
To determine whether NGF-IB participates in P450c17 gene regulation, we cotransfected a vector expressing NGF-IB cDNA (26) and either the WT -447/-419 TK32LUC, mutants 2 or 3 -447/-419 TK32LUC into MA-10 and Y-1 cells (Fig. 7BGo and C), or -447/-399{Delta}1- and 447/-399{Delta}2-TK32LUC into MA-10 cells (Fig. 7DGo). MA-10 and Y-1 cells yielded qualitatively similar results, but stimulation of transcription by NGF-IB in MA-10 cells was much greater than in Y-1 cells. In MA-10 cells, NGF-IB caused a 10-fold increase in the activity of -447/-399-TK32LUC, a 7-fold increase with -447/-419-TK32LUC, and a negligible increase with -418/-399-TK32LUC. When site 1 is mutated in the -447/-399 DNA (Fig. 7DGo), NGF-IB elicits a stimulation similar to its effect on wild type -447/-399 DNA. However, when site 2 is mutated, NGF-IB has no effect on transcription. These data are consistent with NGF-IB binding to site 2 to increase transcription directly or they could indicate that NGF-IB binding prevents repression by COUP-TF. Since both the -447/-399 and -447/-419-TK32LUC plasmids contain these ERE half-sites but are stimulated differently by NGF-IB in Y-1 cells, it is also possible that NGF-IB may interact with a protein (e.g. StF-IT-1) that binds to sequences between -418/-399.

When site 2 is mutated (mutant 2 -447/-419-TK32LUC), NGF-IB elicits a 3-fold stimulation in luciferase activity in MA-10 cells, but not in Y-1 cells, even though the gel shift data (Fig. 7AGo) indicated that NGF-IB did not bind to this DNA. When site 3 is mutated (mutant 3 -447/-419 TK32LUC), NGF-IB elicits a 3-fold stimulation in luciferase activity in MA-10 cells, compared with a 7- to 10-fold stimulation with the wild type. This stimulation of mutant-TK-LUC constructs was not seen in Y-1 cells, as neither mutant 2- nor 3-TK32LUC could be stimulated by cotransfection with NGF-IB. These data suggest that NGF-IB binds to site 2, that by itself, NGF-IB does not activate transcription, and that the action of NGF-IB may require interaction with another protein that binds to DNA at site 3 (i.e. StF-IT-2). The slight stimulation of Mut3-TK-LUC in MA-10 cells indicates that NGF-IB binding to DNA in those cells may result in some transcriptional activation by itself; however, it is puzzling that Mut2-447/-419-TK32LUC, a plasmid to which NGF-IB does not bind, is activated similarly.

Our gel shift data (Fig. 5Go) indicated that SF-1 bound to the same site (site 2) as NGF-IB in MA-10 and in Y-1 cells. Like NGF-IB, the functional data (Fig. 3Go, mutant 3-447/-419-TK32LUC construct) also indicated that although SF-1 binds to mutant 3, this binding does not activate transcription. However, when proteins bind both to sites 2 and 3 (i.e. SF-1 and StF-IT-2; Fig. 5Go, A and B), transcription is activated (Fig. 3Go; -447/-419 TKLUC construct). Thus, it appears that SF-1, like NGF-IB, must interact with StF-IT-2 and, together, this protein-protein-DNA complex induces transcription.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Multiple Nuclear Proteins Regulate Steroidogenic P450 Genes
The transcriptional regulation of the genes encoding the steroidogenic enzymes is complex, involving tissue-specific, gene-specific, and species-specific factors. One such factor, SF-1/Ad4BP, is a member of the orphan nuclear receptor family and binds as a monomer to sequences having the core consensus sequence AGGTCA. An SF-1-binding site has been found within 100 bp of the cap site of all the steroidogenic P450 enzyme genes and regulates these genes in various steroidogenic tissues (1, 2, 18, 20, 27, 28, 29, 30, 31, 32, 33, 34, 35). However, SF-1 is not absolutely required for steroidogenic gene regulation. P450scc gene transcription is regulated in C6 glioma cells (32) and in JEG-3 cells (36, 37), and rat P450c17 is regulated in rat Rcho-1 (38) and in human JEG-3 placental cells (preliminary data) even though C6, Rcho-1, and JEG-3 cells do not express SF-1.

Multiple Members of the Orphan Nuclear Receptor Gene Family Regulate P450c17 Gene Transcription
A second factor that is involved in steroidogenic P450 gene regulation is NGF-IB, which is also a member of the orphan nuclear receptor family. NGF-IB binds as a monomer to the same AGGTCA core sequence as SF-1, but its DNA sequence requirements 5' to this core region are distinct from those required by SF-1. NGF-IB regulates the transcription of the mouse P450c21 gene in adrenal Y-1 cells (26, 39) and of the rat P450c17 gene in Leydig MA-10 cells (Fig. 7Go). Ablation of the NGF-IB gene in transgenic animals has no effect on adrenal or gonadal function (40). Thus, NGF-IB expression is not uniquely crucial to rodent adrenal or gonadal development or steroidogenesis; it is possible that other proteins can compensate for the lost NGF-IB function in these knockout mice. This hypothesis is consistent with our demonstration that multiple factors, in addition to NGF-IB, regulate expression of P450c17 in both adrenocortical and Leydig cells.

A third factor that regulates P450c17 gene transcription is COUP-TF, a ubiquitous transcription factor that binds as a dimer to two core AGGTCA sequences. These sequences are usually found as direct repeats, spaced 0–12 bp apart, or can be palindromic repeats (21, 22). COUP-TF binding to the rat P450c17 gene is unusual because the spacing is 13 bp (if COUP-TF binds to sites 1 and 2),or 28 bp (if COUP-TF binds to sites 1 and 3). Although COUP-TF binding usually decreases transcription, there are additional elements in the COUP-TF binding site of the rat P450c17 gene that bind other factors that activate transcription. Nevertheless, disruption of the COUP-TF binding sequence increases transcription, suggesting that COUP-TF acts as a negative transcriptional modulator.

A fourth transcriptional factor that may regulate steroidogenic P450 genes is the homeobox protein Pbx (41). Pbx1a and Pbx1b bind to the -243/-225 region of the bovine P450c17 gene and enhance cAMP-mediated transcription. Although sequences similar to the Pbx-binding site have not been found in the rat P450c17 gene, it is not known whether Pbx also plays a role in the transcriptional regulation of this gene in the rat.

Identification of Novel Nuclear Proteins that Regulate Rat P450c17 Gene Transcription in Both Leydig and Adrenocortical Cells
We have also identified two proteins that bind to ERE half-sites in different regions of the rat P450c17 gene to activate transcription. We have named these proteins StF-IT-1 and StF-IT-2. These proteins appear to be novel as they have not been characterized as binding to and regulating the transcription of other steroidogenic P450 genes. Both of these factors are found in mouse testis, Leydig MA-10, adrenals, adrenocortical Y-1 and AN4Rpp7 cells, indicating that they are not expressed in one steroidogenic cell type only. StF-IT-1 binds to DNA and increases basal transcription. StF-IT-2 binding alone has no effect on P450c17 transcription but, in concert with other proteins such as SF-1 or NGF-IB, StF-IT-2 also induces transcription. Once bound to DNA, the interaction of these two proteins increases both basal and cAMP-induced transcription. Thus, the rat P450c17 gene is regulated by a number of factors that appear to bind to AGGTCA-like sequences, suggesting that they are bound by multiple orphan nuclear receptors.

Mechanism of Action of SF-1 and NGF-IB
The mechanism of SF-1 action is not well understood. As more genes that are bound by SF-1 are identified, there is increasing evidence that SF-1 functions by multiple mechanisms. These mechanisms may be tissue-specific or may depend upon the DNA sequence in the target gene. SF-1 can 1) bind DNA without altering transcription; 2) bind and activate basal transcription; and 3) bind and mediate a cAMP response. In the -84/-55 region of the rat P450c17 gene, SF-1 binding elicits both increased basal and cAMP-stimulated transcription (2, 18), whereas binding to-447/-419 (Fig. 5Go) has no direct effect on transcription (Fig. 3Go). In the rat P450scc gene, SF-1 binding can elicit both basal (32) and cAMP-stimulated transcription (27, 32). Similarly, SF-1 induces both basal and cAMP-induced transcription of the human P450arom gene (30).

We now show that bases -447 to -418 of the rat P450c17 gene participate in yet another mechanism of SF-1 action; although SF-1 binding alone has no effect on either basal or cAMP-stimulated transcription, it appears to interact with the novel DNA-binding protein, StF-IT-2. This SF-1/StF-IT-2 interaction, but neither protein by itself, increases transcription. Equivalent results were also seen for NGF-IB, which also required binding of StF-IT-2 to DNA for transcriptional activation. The nature of these interactions is unknown but requires that both SF-1 (or NGF-IB) and StF-IT-2 interact with the DNA. This may be similar to the synergism seen between the estrogen receptor and SF-1 in activating transcription of the salmon gonadotropin IIß subunit gene (42), a gene closely related to mammalian LHß. In both our case and in the case of estrogen receptor/SF-1 interaction, the two proteins may interact physically to synergize increased transcription. The role of StF-IT-2 in interacting with SF-1 or NGF-IB is different from the role of the retinoid X receptor in interacting with NGF-IB, as in the latter case, the retinoid X receptor need not interact with the DNA directly (43).

Displacement of COUP-TF as a Mechanism for Transcriptional Activation
Our experiments also show that both SF-1 and NGF-IB may displace COUP-TF binding from DNA, and that this displacement of the repressive action of COUP-TF may increase transcription. By this mechanism, both NGF-IB and SF-1 by themselves would not be activators of transcription but would activate transcription by removing a protein with repressor function, thereby allowing other activating proteins (e.g. StF-IT-1 and StF-IT-2) to bind to DNA.

Transcriptional Regulation by cAMP
The -447/-399 region of the rat P450c17 gene contains a cAMP-responsive element that is distinct from all consensus CRE and CRS sequences previously described in other steroidogenic genes (1, 2, 18, 19, 28, 30, 31, 33, 34, 37, 41, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56). This element may be related to SF-1 binding, as we have previously shown that SF-1 can mediate cAMP-induced transcriptional activation from another region of the rat P450c17 gene (18).

SF-1 may play a role in cAMP regulation of other steroidogenic genes. Others have shown that SF-1 can mediate cAMP-stimulated transcription of the P450aro and P450scc genes (27, 30, 32). Our previous data demonstrated that SF-1 could be phosphorylated by protein kinase A (18), suggesting that this might be a mechanism by which SF-1 mediates cAMP effects. Since the -447/-399 region of the rat P450c17 gene is also bound by SF-1 and is also regulated by cAMP, the actions of SF-1 may be similarly modified by protein kinase A at this region. However, when SF-1 binds to the -447/-419 region of the rat P450c17 gene, it does not appear to mediate cAMP stimulation. It is thus intriguing that a single protein has the ability to function by several different mechanisms. The different elements of the rat P450c17 gene provide an outstanding template for further studies of these mechanisms of orphan nuclear receptor action.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Preparation of Nuclear Extracts
Nuclear protein extracts from mouse Y-1 adrenal and MA-10 Leydig cells and from mouse tissues were prepared as described (2, 57). Protein concentrations were determined by the BCA protein assay system (Pierce Chemical Co., Rockford, IL).

Gel Shift Assays
Gel shift assays were performed as described previously (2). Oligonucleotides corresponding to -447/-399, -418/-399, -447/-419 of the rat P450c17 gene (2), and mutants of these oligonucleotides were used. These oligonucleotides are shown in Table 1Go. Bases that are underlined in the mutants are different from those in the wild type sequences. Oligonucleotide probes were end labeled using [{gamma}-32P]ATP and T4 polynucleotide kinase and mixed with the nuclear proteins in the presence of 100 µg/ml polydeoxyinosinic-deoxycytidylic acid, 50 µg/ml salmon sperm DNA, 5 mM dithiothreitol, and 1 mg/ml BSA, and incubated at room temperature for 40 min. One quarter of the total reaction was loaded onto a 6% nondenaturing polyacrylamide gel, using 0.5 x Tris-borate-EDTA as a running buffer, to separate the free labeled probe from probe bound by nuclear protein. The dried gel was then exposed to x-ray film.

DNase I Footprinting Assay
DNase I footprinting assays were performed as described (2, 58). An oligonucleotide corresponding to bases -447/-399 of the rat P450c17 gene was cloned into the BamHI site of pUC19. The recombinant plasmid was first digested with EcoRI and labeled by Klenow fragment of DNA polymerase I and [{alpha}-32P]dATP. The labeled plasmid was then digested with HindIII and purified on a 6% nondenaturing polyacrylamide gel. The probe was mixed with 25 µg nuclear proteins from Y-1 and MA-10 cells in buffer containing 10 mM Tris-Cl, pH 7.9, 5 mM MgCl2, 1 mM CaCl2, 2 mM dithiothreitol, 100 mM KCl, and 2 mg/ml poly dI/dC. Samples were kept on ice for 30 min, prewarmed to 26 C for 1 min before DNase I was added (0.02 U/reaction), and then incubated at 26 C for 90 sec. Reactions were stopped by digestion with proteinase K (20 mg/ml) in 0.1% SDS, extracted once with phenol/chloroform, precipitated with ethanol, and separated on 8% polyacrylamide denaturing gels. The protected regions were detected by autoradiography.

Construction of the Rat P450c17 Oligonucleotide-TK-LUC Expression Plasmids
Rat P450c17 oligonucleotides were cloned into a luciferase expression vector with a minimal promoter from TK gene of herpes simplex virus (TK32LUC) as described (2). The chimeric constructs were confirmed by DNA sequencing to determine the copy number and sequences. Plasmids containing a single copy of the wild type and mutant oligonucleotides cloned in the 5'->3' direction were used for transfection experiments.

Transfection of Y-1 and MA-10 Cells
Mouse adrenocortical Y-1 (59) and mouse Leydig MA-10 cells (60) were grown as described (2). Plasmid DNAs were transfected into Y-1 and MA-10 cells by calcium phosphate precipitation. When vectors expressing NGF-IB were cotransfected with reporter luciferase constructs, the molar ratio of DNA for these two plasmids was 1:1. DNA concentrations were equalized in all samples by addition of the cloning vector pKS. DNA precipitates were kept on the surface of the cells for 12 h before being replaced by fresh medium. 8-Bromo-cAMP (1 mM) was added for an additional 6 h before cells were harvested. Luciferase assays and data analysis were as described elsewhere (61), using a Monolight 1500 Luminometer (Analytical Luminescence Laboratory, San Diego, CA) using D-Luciferin (Sigma, St. Louis, MO) as substrate for the light reaction.

Preparation of Rat Recombinant SF-1 and Rat Recombinant NGF-IB
A rat SF-1 cDNA fragment (28), kindly provided by B. A. White (University of Connecticut, Farmington, CT) was cloned into the prokaryotic expression vector pET (Novagen, Madison, WI). Comparison of the rat SF-1 sequence to the full-length mouse SF-1 sequence (62) suggests that the rat cDNA fragment encodes amino acids 20–293 and lacks part of the ligand-binding domain of SF-1. SF-1 was overexpressed in bacteria strain BL21 and purified as inclusion bodies as described (63). Renatured SF-1 protein was used in gel shift assays.

A rat NGF-IB PvuII fragment encoding 477 amino acids was kindly provided by J. D. Milbrandt (Washington University, St. Louis, MO) and was similarly cloned into the prokaryotic expression vector pET. NGF-IB was overexpressed in bacteria and purified as inclusion bodies. Renatured NGF-IB was used in gel shift assays.


    ACKNOWLEDGMENTS
 
We thank Dr. Ming-Jer Tsai (Baylor College of Medicine, Houston, TX) for the anti-COUP antibody, and Dr. Jeffrey D. Milbrandt (Washington University, St. Louis, MO) for the NGFI-B expression vector.


    FOOTNOTES
 
Address requests for reprints to: Synthia H. Mellon Ph.D., Department of Obstetrics/Gynecology, University of California, San Francisco, Box 0556, San Francisco, California 94143-0556.

This work was supported by NIH Grants HD-27970 (to S.H.M.) and HD-11979 (to the Reproductive Endocrinology Center, UCSF) and a grant from the Academic Senate, University of California, San Francisco (to S.H.M.). P.Z. was supported in part by a grant from the Rockefeller Foundation (to the Reproductive Endocrinology Center, UCSF).

Received for publication July 17, 1996. Revision received February 11, 1997. Accepted for publication March 11, 1997.


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 INTRODUCTION
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 DISCUSSION
 MATERIALS AND METHODS
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