Regulation of Cytochrome b5 Gene Transcription by Sp3, GATA-6, and Steroidogenic Factor 1 in Human Adrenal NCI-H295A Cells
Ningwu Huang1,
Andrea Dardis1 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: Prof. Walter L. Miller, 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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Sex steroid synthesis requires the 17,20 lyase activity of P450c17, which is enhanced by cytochrome b5, acting as an allosteric factor to promote association of P450c17 with its electron donor, P450 oxidoreductase. Cytochrome b5 is preferentially expressed in the fetal adrenal and postadrenarchal adrenal zona reticularis; the basis of this tissue-specific, developmentally regulated transcription of the b5 gene is unknown. We found b5 expression in all cell lines tested, including human adrenal NCI-H295A cells, where its mRNA is reduced by cAMP and phorbol ester. Multiple sites, between 83 and 122 bp upstream from the first ATG, initiate transcription. Deletional mutagenesis localized all detectable promoter activity within 327/+15, and deoxyribonuclease I footprinting identified protein binding at 72/107 and 157/197. DNA segments 65/40, 114/70 and 270/245 fused to TK32/Luc yielded significant activity, and mutations in their Sp sites abolished that activity; electrophoretic mobility shift assay (EMSA) showed that Sp3, but not Sp1, binds to these Sp sites. Nuclear factor 1 (NF-1) and GATA-6, but not GATA-4 bind to the NF-1 and GATA sites in 157/197. In Drosophila S2 cells, Sp3 increased 327/Luc activity 58-fold, but Sp1 and NF-1 isoforms were inactive. Mutating the three Sp sites ablated activity without or with cotransfection of Sp1/Sp3. In NCI-H295A cells, mutating the three Sp sites reduced activity to 39%; mutating the Sp, GATA, and NF-1 sites abolished activity. In JEG-3 cells, GATA-4 was inactive, GATA-6 augmented 327/Luc activity to 231% over the control, and steroidogenic factor 1 augmented activity to 655% over the control; these activities required the Sp and NF-1 sites. Transcription of cytochrome b5 shares many features with the regulation of P450c17, whose activity it enhances.
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INTRODUCTION
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CYTOCHROME P450c17 catalyzes steroid 17
-hydroxylase and 17,20 lyase activity (for review see Ref. 1). In the human adrenal, P450c17 serves as the qualitative regulator of steroid production (2). The zona glomerulosa lacks P450c17 and consequently produces mineralocorticoids, principally aldosterone. The zona fasciculata possesses abundant 17
-hydroxylase activity of P450c17 and produces glucocorticoids, whereas the zona reticularis possesses both 17
-hydroxylase and 17,20 lyase activities of P450c17, and produces precursors of sex steroids. Even though these two reactions are catalyzed on a single active site using the same chemistry (3), they are regulated independently. Increasing the ratio of 17,20 lyase activity to 17
-hydroxylase activity promotes adrenal secretion of dehydroepiandrosterone (DHEA). Therefore, the regulation of the 17,20 lyase activity is crucial to understanding the developmental regulation of DHEA in adrenarche and to the pathogenesis of the polycystic ovary syndrome (4).
Several factors posttranslationally regulate the 17,20 lyase activity of P450c17, including serine phosphorylation of P450c17 itself (5, 6) and increasing electron donation from reduced nicotinamide adenine dinucleotide phosphate (NADPH) through P450 oxidoreductase (POR) (7, 8, 9). Cytochrome b5 also enhances the 17,20 lyase activity (10, 11, 12, 13, 14, 15, 16), acting as an allosteric factor to promote the interaction of P450c17 and POR (15, 16). Because cytochrome b5 increases 17,20 lyase activity without affecting 17
-hydroxylation (10, 12, 15), changes in the level of expression of cytochrome b5 could be important in regulating adrenal DHEA production. Immunocytochemical studies indicate that cytochrome b5 is preferentially expressed in the primate zona reticularis (17, 18) and that its expression increases in children older than 5 yr old, roughly correlating with increases in circulating levels of DHEA sulfate during adrenarche (19). Similarly, cytochrome b5 expression is also high in the fetal zone of the fetal adrenal, which produces abundant DHEA, but is not detected in the definitive zone until the second half of gestation (20).
The human cytochrome b5 gene (CYB5) is located on chromosome 18q23, is about 39 kb long, consists of 6 exons (21, 22), and is expressed in two different isoforms generated by alternative splicing from the same precursor mRNA. Exons 14 encode the 98-amino-acid (AA) soluble form found in reticulocytes, and exons 13, 5, and 6 encode the 134-AA membrane-bound found in liver and other tissues (22). A third isoform, the 146-AA outer mitochondrial membrane cytochrome b5 (OMb) (23), which is encoded by a different gene (CYB5-M) located on chromosome 16q22.1, is less abundant than the microsomal cytochrome b5 in adrenal NCI-H295A cells (24). Because cytochrome b5 promotes the association of microsomal P450c17 with microsomal POR, we have investigated the transcription of microsomal cytochrome b5. Preliminary studies in nonsteroidogenic human liver HepG2 and leukemia K562 cells suggested that the 5' flanking DNA sequence of the TATA-less cytochrome b5 gene contains regulatory regions (25). However, little is known about the transcriptional regulation of the cytochrome b5 gene or about which factors might promote its selective expression in the adrenal zona reticularis. Therefore, we studied the transcriptional regulation of cytochrome b5 gene in human adrenal NCI-295A cells, a zonally undifferentiated model of the human fetal adrenal (26).
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RESULTS
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Expression of Human Cytochrome b5
Cytochrome b5 is expressed in three forms: a microsomal membrane-bound and a soluble form, both encoded by a single gene, and OMb5, encoded by a different gene (21, 22, 23). To identify the forms of cytochrome b5 mRNA expressed in transformed human cell lines, we performed RT-PCR using oligonucleotides that would amplify both the microsomal membrane-bound and the soluble form, permitting their discrimination on a gel (Fig. 1
, upper panel), or OMb5 (Fig. 1
, lower panel). RNA from leukocytes produced both the 434-bp product corresponding to the 98-AA soluble form of cytochrome b5 and the 410-bp product corresponding to the 134-AA membrane-bound form of cytochtome b5, and a 510-bp glyceraldehyde-3-phosphate dehydrogenase (GAPDH) PCR product, used as an internal control. RNA from seven cell lines: human adrenal NCI-H295A, cervical carcinoma HeLa, placental choriocarcinoma JEG-3, hepatocarcinoma HepG2, skin fibrocarcinoma HT-1080, human embryonic kidney (HEK) 293, and embryonic lung fibroblast L132 cells, produced only the 410-bp cytochrome b5 product and the 510-bp GAPDH internal control, but not the 434-bp product (Fig. 1
, upper panel). Sequencing of the 410-bp PCR products from all seven cell lines confirmed that each PCR product lacked exon 4 and were identical with the membrane-bound, hepatic form of cytochrome b5 cDNA. RNA from leukocytes and the seven cell lines all produced the 441-bp product corresponding to the 146-AA OMb5 (Fig. 1
, lower panel), but this form was less abundant in NCI-H295A cells, as reported previously (24).

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Fig. 1. Expression of Cytochrome b5 mRNA
Upper panel, Microsomal cytochrome b5. The PCRs contained two pairs of primers: one to amplify a 501-bp product from GAPDH cDNA as an internal control and one to amplify mRNA encoding both the 434-bp product corresponding to the soluble form of cytochrome b5 and the 410-bp product corresponding to the membrane-bound form of cytochrome b5. Lower panel, OMb5. The PCRs contained primers for GAPDH (501 bp) and for OMb5 (441 bp). The RNA sources are indicated on the top of the figure.
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Cytochrome b5 Transcription Start Site
Cytochrome b5 gene transcription in human liver HepG2 cells and leukemia K562 cells is initiated from multiple sites located 100110 bp upstream from the first ATG codon (25), but its transcriptional start site in human adrenal NCI-H295A cells is unknown. To locate this transcriptional start site, we used ribonuclease (RNase) protection assays. RNA from NCI-H295A cells and HeLa cells yielded a single sharp band with a control probe comprising a fragment of GAPDH cDNA (bases 664804), consistent with GAPDH mRNA having only one transcript and confirming that the nuclease digestion conditions were effective. By contrast, the probe comprising the 5' flanking region of cytochrome b5 generated multiple bands of 116155 bp, indicating that cytochrome b5 mRNA arises from multiple transcriptional start sites lying 83122 bp upstream from the ATG translational start codon (Fig. 2
). The pattern of band sizes and intensities did not differ significantly between the NCI-H295A cells and HeLa cells, indicating a lack of cell-specific use of transcriptional start sites. This result is generally consistent with the findings in HepG2 and K562 cells (25). The transcriptional start site nearest to the initial ATG codon (83 bases 5' from the ATG) was designated as base +1, and all the promoter/reporter constructs were built to include this base and all the alternative upstream start sites.

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Fig. 2. Identification of the Human Cytochrome b5 Gene Transcription Start Site by RNase Protection Assay
RNA probes for an interior fragment of GAPDH (left lanes) or for the 5' flanking region of cytochrome b5 (right lanes) were hybridized with yeast tRNA, with total RNA from HeLa cells, or with total RNA from NCI-H295A cells as indicated, and digested with RNase A and RNase T1. Size markers (10-bp ladder) are shown at the left. GAPDH mRNA produced a single fragment of 144 bp. Cytochrome b5 mRNA produced multiple fragments, ranging from 116155 bp. Because the probe contains 33 nucleotides of protein-coding region, this indicates that cytochrome b5 has multiple transcriptional start sites that lie between 83 and 122 bp upstream from the ATG translational start codon. The same array of start sites is used both in NCI-H295A cells and in HeLa cells.
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Regulation of Cytochrome b5 mRNA in Human Adrenal NCI-H295A Cells
We considered four factors that might influence b5 mRNA expression in NCI-H295A cells: 8Br-cAMP, a generic activator of the protein kinase A pathway; phorbol 12-myristate 13-acetate (PMA), a generic activator of the protein kinase C pathway; CRH, a hypothalamic factor reported to stimulate adrenal C19 steroid secretion directly (27), and insulin, which exerts multiple effects on steroidogenic cells. Phosphorimage analysis of Northern blots, adjusted for the content of ß-actin mRNA as an internal control, showed that 24 h treatment with 1 mM 8Br-cAMP decreased the abundance of cytochrome b5 mRNA to 38% of control (P = 3 x 106), 1 µM PMA decreased it slightly to 80% of control (P = 3.2 x 104), and neither 10100 nM CRH nor 10100 nM insulin had a detectable effect (Fig. 3A
).

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Fig. 3. Hormonal Regulation of Cytochrome b5 mRNA
A, Northern blot of mRNAs for cytochrome b5 and GAPDH in NCI-H295A cells treated with 8 Br-cAMP, PMA, CRH, insulin, or vehicle for 24 h. The abundance of each mRNA was quantitated by phosphorimaging and normalized with ß-actin and shown as a percentage of the value in untreated cells. Data are means ± SD of four independent experiments. B, Northern blot of mRNAs for cytochrome b5 and ß-actin in NCI-H295A, HepG2, HEK293, and JEG-3 cells treated with or without 1 mM 8 Br-cAMP. C, The inhibitory effect of cAMP on cytochrome b5 is at the level of trancription. The 327/Luc and 1300/Luc promoter/reporter constructs were transfected into NCI-H295A and treated with 1 mM 8 Br-cAMP for 24 h (open bars) or vehicle (closed bars), showing that cAMP inhibits both constructs equally. Luciferase activities were shown as a percentage of the value in untreated cells. Data are mean ± SEM of three independent experiments, each performed in triplicate.
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To determine whether the suppression of cytochrome b5 mRNA by 8Br-cAMP is specific to adrenal NCI-H295A cells, we treated liver HepG2, kidney HEK293, and placental JEG-3 cells with 1 mM 8Br-cAMP for 24 h and assayed the mRNAs for cytochrome b5 and ß-actin by Northern blot. Phosphorimage analysis showed that cAMP decreased the abundance of cytochrome b5 mRNA in steroidogenic NCI-H295A and JEG-3 cells but did not change the abundance of cytochrome b5 mRNA in nonsteroidogenic HepG2 or HEK293 cells (Fig. 3B
).
To determine whether the effect of 8Br-cAMP was at the level of cytochrome b5 gene transcription, we built constructs containing either 327 or 1300 bp of cytochrome b5 DNA upstream from the transcriptional start site and examined their expression in transiently transfected NCI-H295A cells. Treatment with 1 mM 8Br-cAMP reduced luciferase activity produced by both constructs to about 40% of control (Fig. 3C
), the same level of inhibition seen in the RNA analysis. Thus, cAMP decreases cytochrome b5 mRNA by directly inhibiting the transcription of the cytochrome b5 gene via elements within 327 bp from the transcriptional start site.
Identification of the Proximal Promoter
To locate transcriptionally active segments of the cytochrome b5 gene, we built a series of constructs containing 79 to 1300 bp of 5' flanking DNA fused to the luciferase reporter and transfected them into NCI-H295A cells. The shortest construct, 79/Luc, had minimal activity, only 2.4-fold over the vector control (Fig. 4A
). Constructs containing either 157 or 185 bp of the 5' flanking DNA had substantially greater activity, 15-fold greater than the vector control, and longer constructs containing from 225-1300 bp of 5' flanking DNA had about 40-fold more activity than the vector control. These constructs showed similar activity in HeLa cells (Fig. 4B
). One-way ANOVA showed no difference among the constructs containing 225, 327, 730, 965, and 1300 bp (P = 0.4). Thus, deletional mutagenesis analysis identified strong basal elements lying between 79 and 157, and between 185 and 225.

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Fig. 4. Deletional Mutagenesis
Deletional mutants containing up to 1300 bp of the 5' flanking region of the human cytochrome b5 gene were transiently transfected into NCI-H295A cells (panel A) or HeLa cells (panel B). In both panels, data are shown as mean ± SEM of three independent experiments, each performed in triplicate.
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Identification of Specific Functional Elements in the Proximal Promoter
To locate the specific functional elements in the proximal promoter, we performed deoxyribonuclease (DNase) I footprinting assays of the region extending from 327 bp to +15 bp. Nuclear proteins from NCI-H295A cells protected two regions on each strand. Footprint 1 (F1) encompassed bases 72/107, and footprint 2 (F2) encompassed bases 157/197 (Fig. 5
). The location of F1 correlates well with the deletional mutagenesis results showing that important elements lie between 79 and 157, and the location of F2 partially overlaps with the region between 185 and 225 that showed activity in the deletional mutagenesis assay, but includes all of the DNA between 157 and 185, for which the deletional mutagenesis showed no activity. This suggests that one or more proteins bind between 157 and 197, but that their transcriptional activity requires interaction with other proteins that bind upstream, between 185 and 225.

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Fig. 5. DNase I Footprinting
A PCR-amplified 342-bp double strand probe (bases 327 to +15) 32P-end labeled on the sense or antisense strand was incubated with NCI-H295A nuclear extract (N.E.) and digested with DNase I. Left, Footprint of DNA labeled on the sense strand. Two regions corresponding to sequences 72 to 107 (F1) and 157 to 197 (F2) are protected. Right, Footprint of DNA labeled on the antisense strand. The same regions (F1 and F2) are protected.
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To assess the role of the DNA elements comprising F1 and F2, we assessed their ability to stimulate transcription from a heterologous promoter. A series of 45-bp oligonucleotides extending from +26 to 334, including one comprising F1 (114/70) and one comprising F2 (201/157), were cloned into the TK32/Luc minimal promoter/reporter system and transiently transfected into NCI-H295A cells (Fig. 6A
). The oligonucleotide containing F1 increased activity more than 9-fold compared with the vector control, whereas oligonucleotide containing F2 increased activity only 3.7-fold over the vector control. Two other regions, 65/20 and 289/245 also increased activity substantially (Fig. 6A
) even though these DNA segments were not identified by either the deletional mutagenesis experiment (Fig. 4A
) or the DNase I footprinting (Fig. 5
). To define the regions responsible for the activity conferred by 65/20, 114/70 and 289/245, we synthesized overlapping 24-bp oligonucleotides for each and assessed their activities in TK32/Luc. The activity of 65/20 was found in 65/40; the activity of 114/70 was found in 114/90; and the activity of 289/245 was found in 270/245 (Fig. 6B
). Analysis of these regions using TESS (http://www.cbil.upenn.edu/tess/index.html), a web tool for predicting transcription factor binding sites in DNA sequences, predicted Sp1/Sp3 sites in all three regions. The putative Sp site (CCGCCC) was mutagenized by replacing it with TTAGTA in 65/40-TK32/Luc and 270/245-TK32/Luc, and the putative Sp site (GGCGG) was mutagenized to TTACC in 114/90-TK32/Luc. All three mutants abolished all activity, indicating that these three Sp sites are essential for activity (Fig. 6B
).

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Fig. 6. Activity of Promoter Segments Fused to the Heterologous TK32/Luc Promoter/Reporter System
A, Activity of eight 45-bp oligonucleotides spanning from 327 bp to +26 bp of the cytochrome b5 gene. DNA segments 65/20, 114/70, and 289/245 had 9-fold more activity than the TK32/Luc vector, whereas the other DNA segments increased activity less than 4-fold compared with TK32/Luc. B, The active 65/20, 114/70 and 289/245 regions identified in panel A were recreated as two overlapping 24-bp oligonucleotides and analyzed in TK32/Luc. Sp mt (mutant) indicates that the putative Sp-factor binding site in the oligonucleotides is mutated. Activity was found at 65/40, 114/70 and 270/245, which contain Sp-factor binding sites. Mutation of 5 or 6 bp in the putative Sp-factor binding sites in each region abolished activity. Data are mean ± SEM of three independent experiments, each performed in triplicate.
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Characterization of DNA/Protein Interaction
To test the abilities of the three DNA segments identified in Fig. 6
to bind to Sp proteins, we used 32P-labeled double-stranded oligonucleotides 65/40 (probe I), 114/90 (probe II), and 270/245 (probe III), and their mutants (Table 1
) to perform electrophoretic shift mobility assays (EMSAs). Incubating probe I with NCI-H295A nuclear extracts formed several protein/DNA complexes (Fig. 7A
). Incubation with a 100-fold molar excess of unlabeled probe displaced the labeled probe from the protein/DNA complex, whereas incubation with a 100-fold excess of unlabeled probe, containing mutations in the putative Sp site did not displace the labeled probe from the protein/DNA complex. Incubation with a 100-fold excess of an oligonucleotide containing a Sp1/Sp3 consensus sequence also displaced the labeled probe. Finally, the complexes formed by the wild-type probe I were partially supershifted and partially inhibited by antibody against Sp3, but not by antibody against Sp1 (Fig. 7A
). Thus, Sp3 is a key factor in promoting cytochrome b5 gene transcription by binding to a consensus Sp site at 65/40. Similar results were seen with probes II and III (Fig. 7
, B and C). These results strongly suggest that Sp3 interacts with DNA in all the three regions.

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Fig. 7. Gel Mobility Shift Assays
Nuclear proteins from NCI-H295A cells were incubated with 32P-labeled probe I (bases 65/40) (panel A), 32P-labeled probe II (bases 114/70) (panel B), 32P-labeled probe III (bases 270/245) (panel C). Protein/DNA complexes were competed with 100-fold molar excess of the wild type oligonucleotides, and with Sp1/Sp3 consensus oligonucleotides, but not with oligonucleotides containing mutations at the putative Sp sites of probes I, II, or III. Polyclonal antibodies against Sp1 and Sp3 were used to supershift protein/DNA complexes. Protein/DNA complexes formed with probes I, II, and III were partially supershifted and partially inhibited by anti-Sp3, but not by anti-Sp1. mt, Mutant; wt, wild type; cons., consensus; n.s., nonspecific.
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Sp3 Stimulates the Human Cytochrome b5 Promoter in Drosophila S2 Cells
To confirm that regions I, II, and III act by binding Sp1 or Sp3, we assessed the activities of these sites in Drosophila S2 cells, which lack endogenously produced Sp1 and Sp3 (28, 29). We inserted the same mutations used above in each Sp site in regions I, II, and III in the 327/Luc construct, and transfected wild-type and mutant constructs into S2 cells with or without cotransfection of vectors expressing Sp1 or Sp3. The 327/Luc basal promoter had minimal activity when cotransfected with the empty pPacO expression vector or with the pPac vector expressing Sp1, but its activity increased 58-fold when cotransfected with the pPac vector expressing Sp3 (Fig. 8
). Surprisingly, cotransfection of both the Sp1 and Sp3 expression vectors increased the activity of 327/Luc only 22-fold compared with vector control, suggesting an inhibitory effect of Sp1, possibly by competing for Sp3 sites. Furthermore, the mutant 327/Luc construct in which the three Sp sites were mutated was inactive in Drosophila S2 cells, and cotransfection with Sp1, Sp3, or both Sp1 and Sp3 did not increase its activity (Fig. 8
). These data indicate the Sp sites in regions I, II, and III are the only functional Sp sites, and that Sp3 binds to all three regions, even though computational analysis with TESS suggested that the 327/+15 region contained additional Sp binding sites. Because we previously found that nuclear factor 1C (NF-1C) isoforms play a key role in regulating human P450c17 gene transcription in NCI-H295A cells (30, 31), we also sought to assess the potential role of NF-1C factors in the regulation of the human cytochrome b5 promoter in Drosophila S2 cells, which lack endogenously produced NF-1C factors. Cotransfection of the 327/Luc construct and with each of three vectors expressing the NF-1C isoforms termed CCAAT transcription factor (CTF)-1, -2, -5 did not elicit any more activity than cotransfection with an empty vector (Fig. 8
). Thus, NF-1C factors alone are insufficient to drive expression of the cytochrome b5 promoter in S2 cells.

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Fig. 8. Sp3 Transactivates Human Cytochrome b5 Promoter/Reporter Constructs in Drosophila S2 Cells
Wild-type 327/Luc (closed bars) and the mutant 327/Luc in which the three Sp sites were mutated (open bars) were cotransfected into S2 cells with empty pPacO vector, or with vectors expressing Sp1, Sp3, or both Sp1 and Sp3, or with vectors expressing the NF-1C factors CTF-1, -2, and -5. Data are mean ± SEM of three independent experiments, each performed in triplicate.
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Sp1/3, NF-1, and GATA Binding Sites Mediate Cytochrome b5 Promoter Activity in NCI-H295A Cells
Although the data in the Drosophila S2 cells demonstrated a key role for the three Sp sites, computational analysis with TESS indicated that footprint F2 also contains a putative NF-1 binding site at 170/176 and a putative GATA binding site at 194/199. This array of transcription factor binding sites is reminiscent of the gene for P450c17, which requires the action of Sp1, Sp3, NF-1C, and GATA factors for expression in NCI-H295A cells (30, 31). To investigate whether NF-1 and GATA factors bind to the putative NF-1 binding site at 170/176 and the putative GATA binding site at 194/199, we performed EMSAs with 32P-labeled oligonucleotides 182/159 (probe IV), and 209/186 (probe V) (Table 1
). Both of these probes formed protein/DNA complexes with NCI-H295A nuclear proteins (Fig. 9
). Incubation with a 100-fold molar excess of unlabeled probe displaced the labeled probe from the protein/DNA complex, whereas incubation with a 100-fold excess of unlabeled probe, containing mutations in the putative NF-1 site (Fig. 9A
), or in the putative GATA site (Fig. 9B
), did not displace the labeled probe from the protein/DNA complex. Incubation with a 100-fold excess of an oligonucleotide containing consensus sequences for NF-1 (Fig. 9A
) and GATA (Fig. 9B
) also displaced the labeled probe from the protein/DNA complex. Finally, the formation of a complex by wild-type probe IV was inhibited by antibody against NF-1 (Fig. 9A
), and the formation of a complex by wild-type probe V was inhibited by antibody against GATA-6, but not GATA-4 (Fig. 9B
). Thus, the cytochrome b5 promoter contains DNA binding sites for NF-1 and GATA-6.

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Fig. 9. Gel Mobility Shift Assays
Nuclear proteins from NCI-H295A cells were incubated with 32P-labeled probe IV (bases 182/159) (panel A) and 32P-labeled probe V (bases 209/186) (panel B). Protein/DNA complexes were competed with 100-fold molar excess of the wild-type oligonucleotides, and with consensus oligonucleotides for NF-1 (in panel A) and GATA (in panel B), but not with oligonucleotides containing mutations with oligonucleotides containing mutations at the putative NF-1 site in probe IV, or at the putative GATA site in probe V. Polyclonal antibody against NF-1 competed for formation of the complex with probe IV (panel A). Polyclonal antibody against GATA-6, but not GATA-4 competed for formation of the complexes with probe V (panel B). wt, Wild type; mt, mutant; cons., consensus; n.s., nonspecific.
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To investigate the potential functional roles of these sites, we built a series of 327/Luc vectors in which we mutated the three Sp sites, the NF-1 site and the GATA-6 site, solely and in combination, and assessed the activity of each in NCI-H295A cells (Fig. 10
). The wild-type 327/Luc, which elicited 31-fold more luciferase activity than did the pGL3-basic vector without the 327/+15 fragment of the cytochrome b5 promoter, is defined as 100%. Mutation of the three Sp sites reduced activity dramatically, to 39 ± 7.3% of the wild-type 327/Luc construct, but did not eliminate activity. This 39% of residual activity in the absence of the three Sp sites indicated that other factors are also important. Mutation of both the GATA-6 and the NF-1 sites reduced activity slightly, to 86 ± 14.4% of the wild-type 327/Luc construct, suggesting that the effects of the two sites and the three Sp sites might be additive. Mutation of the GATA-6 site in addition to the three Sp sites increased activity significantly (P = 0.003), from 39 ± 7.3% to 96 ± 16.3% of the wild-type construct. A similar positive effect of mutating the GATA-6 site is seen when it alone is mutated, increasing activity significantly to 175 ± 37.8% of the wild-type construct (P = 0.01). A similar positive effect of mutating a GATA site is seen in the closely related regulation of the human gene for P450c17 in NCI-H295A cells. In that case, GATA acts by binding to Sp factors rather than to a GATA-binding site in the DNA; hence, mutation of the DNA increases the availability of GATA, which then increases activity by binding to Sp factors (31). Mutation of the NF-1 site alone had no effect (118 ± 33.2%), and mutation of the NF-1 site in combination with the three Sp sites yielded 39 ± 7.4% of the activity of the wild-type construct, which was the same as the activity when the three Sp sites are mutated and the NF-1 site remained intact. These data might suggest that the NF-1 site plays no role. However, mutation of the three Sp sites, the NF-1 site and the GATA site, eliminated all detectable activity, indicating that these five sites are the overwhelmingly most important, and possibly sole regulatory sites acting in human adrenal NCI-H295A cells.

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Fig. 10. Interplay among Sp, NF-1, and GATA Factors in Expression of the b5 Gene in Human Adrenal NCI-H295A Cells
Cells were transfected with wild-type or mutants of 327/Luc. The activity of wild-type 327/Luc is defined as 100%, and the activities of the other constructs, including mutation of all three Sp sites (closed ovals), mutation of GATA (closed triangle) and NF-1 (closed diamond), are shown as percentage of the value in the wild-type 327/Luc construct. Data are means ± SEM of three independent experiments, each performed in triplicate.
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Steroidogenic Factor 1 (SF1) and GATA-6, But Not GATA-4, Augment Cytochrome b5 Transcription in JEG-3 Cells
In addition to Sp1, Sp3, NF-1C, GATA-4, and GATA-6, the expression of P450c17 in NCI-H295A cells requires SF1 (30). Because cytochrome b5 is widely expressed in many tissues that do not express SF1, and because there is no apparent SF1 binding site in cytochrome b5 promoter region, it was clear that SF1 was not required for basal cytochrome b5 gene transcription, but we considered whether SF1 might contribute to the high levels of cytochrome b5 transcription seen in the adrenal. To clarify the potential roles of GATA-4, GATA-6 and SF1 in cytochrome b5 expression, we used human placental JEG-3 cells, which lack endogenous GATA-4, GATA-6, and SF1 (31), and which are unable to support expression of P450c17 promoter constructs (32), which require these factors (31). Transfection of JEG-3 cells with an SF1 expression vector had little effect on endogenously produced cytochrome b5 mRNA, transfection of a GATA-6 expression vector increased endogenously produced cytochrome b5 mRNA by about 115% and cotransfection of both SF1 and GATA-6 expression vectors increased endogenously produced cytochrome b5 mRNA by about 157% (Fig. 11A
). To explore the potential roles of SF1 and GATA factors on cytochrome b5 gene transcription in greater detail, we cotransfected JEG-3 cells with expression vectors for GATA-4, GATA-6, or SF1 along with vector control, the wild-type 327/Luc, and each of the seven 327/Luc mutant constructs used in Fig. 10
, and measured luciferase activity (Fig. 11B
). As seen in Fig. 10
in NCI-H295A cells, the wild-type 327/Luc construct elicited 34-fold more luciferase activity than the pGL3-basic vector without the 327/Luc fragment; this level of activity is defined as 100%. This was not increased further by coexpression of GATA-4 (116 ± 71.2%). However, coexpression of GATA-6 increased expression to 231 ± 95.1% over the vector control, and coexpression of SF1 increased activity to 655 ± 154.9% over the vector control (Table 2
). As there is no apparent SF1 binding site in the cytochrome b5 promoter region, this stimulatory effect of SF1 on b5 promoter activity suggests that SF1 acts through interaction with other factors, as has been reported for interactions between SF1 and Sp1 (33, 34), or GATA factors (35, 36, 37, 38). Mutation of the three Sp sites decreased activity by about half in each of the cotransfections, consistent with the previously shown data indicating that these three sites play a major quantitative role in cytochrome b5 expression. However, unlike the situation in NCI-H295A cells (Fig. 10
), mutation of the GATA-6 and NF-1 sites reduced luciferase activity dramatically, mutation of the NF-1 site alone also reduced activity, and mutation of the three Sp sites and the NF-1 site together eliminated almost all activity. Thus, the activity of NF-1 factors appears to be more important in placental than in adrenal expression of cytochrome b5. However, the role of the GATA site is similar in both cell lines because mutation of the GATA site alone increases basal activity, and mutation of the three Sp sites plus the GATA site increases activity in comparison to mutation of the three Sp sites alone. Thus GATA factors, principally GATA-6, increase cytochrome b5 gene transcription in JEG-3 cells, but not by action on the GATA site in the cytochrome b5 promoter. This action is very similar to the role of GATA factors in P450c17 transcription where GATA acts by protein/protein interaction with Sp factors to promote transcription (31). Thus, transcription of the cytochrome b5 gene in steroidogenic cell lines uses a similar transcriptional strategy to that employed by P450c17, with important differences in the specific Sp factors and GATA factors involved.

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Fig. 11. Interplay among Sp, NF-1, and GATA Factors in Human Placental JEG-3 Cells
A, Northern blot. JEG-3 cells were transfected with vectors expressing SF1 or GATA-6 (indicated by + signs), and the blot was probed for the mRNAs indicated at the left. B, Activity of cytochrome b5 promoter. The 327/Luc construct and the construct with mutations at all three Sp sites or NF-1 site or GATA site were cotransfected into JEG-3 cells with empty pCDNA3 vector (open bars), or with pCDNA3 vectors expressing GATA-4 (hatched bar), or GATA6 (closed bar), or SF1 (cross-hatched bars). Data are shown in Table 2 as mean ± SEM of three independent experiments, each performed in triplicate.
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DISCUSSION
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Cytochrome b5 is a small hemoprotein that participates in electron transfer in multiple biochemical reactions. Its principal physiological role is in the NADPH-dependent reduction of methemoglobin to hemoglobin in erythrocytes (39, 40), but it also participates in sterol metabolism (41), fatty acid desaturation (42), and cytochrome P450-catalyzed reactions (43). Its role in P450-mediated reactions has been controversial. Although cytochrome b5 acts as an electron transfer factor in other reactions and can receive electrons from P450 oxidoreductase (44), the redox potential of cytochrome b5 is unfavorable for donation of a second electron to a cytochrome P450 (45). In certain P450-mediated reactions (46, 47), including the 17,20 lyase activity of P450c17 (15), the action of cytochrome b5 can be reproduced with apo-b5 (cytochrome b5 devoid of its heme group), indicating that in these situations cytochrome b5 acts as an allosteric factor and not as an electron donor. Consistent with the essential role of cytochrome b5 in the reduction of methemoglobin, hereditary methemoglobinemia is generally caused by disorders in cytochrome b5 reductase (48). Only one patient has been described with a genetic disorder of cytochrome b5 (48); that patient was a male pseudohermaphrodite initially thought to have 17
-hydroxylase deficiency until a splicing mutation was found in the cytochrome b5 gene (49). Thus, the role of cytochrome b5 in androgen biosynthesis is established by studies both in vivo and in vitro.
Cytochrome b5 is preferentially expressed in the fetal adrenal (20) and in the zona reticularis, but not in the zona fasciculata of the adult adrenal (19), but the basis of this zona-specific expression is not clear. In the absence of cellular models of each zone, we used human adrenocortical NCI-H295A cells, which are an excellent model of the human fetal adrenal (26). NCI-H295A cells respond to cAMP with increases in the transcription of genes and accumulation of mRNAs for P450scc and P450c17 (26) and increased secretion of cortisol and DHEA (50), just like primary cultures of human fetal adrenal cells (51). Furthermore, NCI-H295A cells make abundant IGF-II mRNA (26), which is abundantly expressed in the fetal but not the adult adrenal (52, 53). Finally, NCI-H295A cells have relatively low expression of 3ßHSDII (54) and produce more
5 than
4 steroid (55), similar to the fetal adrenal and the adult zona reticularis. Thus, it is likely that the behavior of the cytochrome b5 gene in NCI-H295A cells closely models its behavior in the human fetal adrenal and in the human zona reticularis.
Consistent with its multiple roles and broadly based expression, the gene for cytochrome b5 lacks a TATA box in its proximal promoter, a feature typical of housekeeping genes, which often lack factors causing tissue-specific or hormonally regulated expression. However, because the zone-specific pattern of adrenal expression of cytochrome b5 is similar to that of P450c17, and because both cytochrome b5 and P450c17 increase in the zona reticularis at the time of adrenarche, we sought to determine whether their transcription was regulated similarly. We previously showed that adrenal P450c17 transcription requires NF-1C, Sp1/Sp3, SF1, GATA-4, and GATA-6 in NCI-H295A cells (30, 31). Similarly, we found that adrenal expression of the cytochrome b5 gene is also regulated by Sp3, SF1, and GATA-6, but not by GATA-4, and requires the DNA binding site for NF-1C.
SF1 is required for adrenal and gonadal (but not placental) steroidogenic gene expression and is essential for adrenal development (56, 57, 58, 59, 60). SF1 regulates steroidogenic gene expression by interacting with Sp1 (33, 34) and GATA factors (35, 36, 37, 38), which are coexpressed with SF1 in steroidogenic tissues. Therefore, it is not surprising that SF1 stimulates cytochrome b5 327/Luc activity, even though this DNA lacks an apparent SF1 binding site. It is likely that SF1 stimulates cytochrome b5 expression by interaction with Sp3 or GATA-6 because Sp3 and GATA-6 bind to the cytochrome b5 promoter and stimulate cytochrome b5 gene expression.
GATA factors can be divided into two groups based on similarities in their primary amino acid sequences and spatiotemporal expression patterns. GATA-1, -2, and -3 are expressed mainly in hematopoietic cells, where they influence differentiation (61), and GATA-4, -5, and -6 are expressed mainly in heart, gut, gonads and adrenal (62, 63). The human fetal adrenal expresses both GATA-4 and GATA-6, whereas the adult adrenal expresses GATA-6 and very little GATA-4 in both the zona fasciculata and reticularis (63). By contrast, human adrenal malignancies usually express vastly more GATA-4 than GATA-6 (64), but even though NCI-H295A cells derive from an adrenocortical carcinoma (55), they remain differentiated and express both GATA-4 and GATA-6 (31). GATA-4, -5, and -6 are important in programming cell-specific expression and mediating cellular specificity by physical interactions with other factors, such as Sp1 (31, 65) and SF1 (35, 36, 37, 38). GATA-4 partners with SF1 in regulating the transcription of the genes for Müllerian inhibitory substance, steroidogenic acute regulatory protein, inhibin and P450aro (35, 37), whereas GATA-6 partners with SF1 in regulating steroid sulfotransferase 2A1 gene transcription (66). Furthermore, GATA-4 (38) and SF1 (67) mediate the cAMP-responsive induction of genes for many steroidogenic enzymes, which lack classic cAMP response elements that bind CREB. By contrast to the genes for P450scc or P450c17, whose transcription is induced by cAMP in NCI-H295A cells (26), cAMP decreased the abundance of mRNA for b5. This may reflect the inability of b5 gene to respond to GATA-4 in NCI-H295A cells.
In addition to the activation of the genes for both b5 and P450c17 by GATA factors, members of the NF-1 family of transcription factors also regulate expression of both of these genes in NCI-H295A cells. There are four genes for NF-1 generally termed NF-1-A, -B, -C, and -X, each of which can undergo alternative splicing, yielding multiple mRNAs and proteins (68). Splice variants of NF-1C can bind to CCAAT sequences found in many promoters and are termed CTFs. CTF-2 and -5 are the principal forms of NF-1C found in NCI-H295A cells (30, 31). These factors appear to interact with Sp1, and possibly SF1, but not Sp3 in regulating expression of P450c17 in NCI-H295A cells. NF-1C similarly plays a crucial role in expression of b5 in NCI-H295A cells, but its precise mode of action is not yet known. Thus, the regulation of adrenal expression of b5 and P450c17 are very similar, yet distinct.
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MATERIALS AND METHODS
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Cell Culture, Transfection, and Luciferase Assay
Human HeLa cervical carcinoma cells and HEK293 cells were cultured in DMEM/Hams 21 medium (DME-H21) supplemented with 10% fetal bovine serum and penicillin/ streptomycin. NCI-H295A cells (69), an adherent subline of human adrenocortical NCI-H295 cells (26, 55) were cultured in RPMI 1640 medium supplemented with 2% fetal bovine serum, 5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml selenite, and penicillin, as described (26). Human placental JEG-3 cells (70) were cultured in DME-H21 supplemented with 5% fetal bovine serum, 5% horse serum, and 50 µg/ml gentamycin. Human liver HepG2 cells were cultured in DMEM with Eagles BSS medium supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 1% sodium pyruvate, and 50 µg/ml gentamycin. All cell types were plated on 12-well tissue culture plates 24 h before transfection with calcium phosphate precipitates (1.5 µg plasmid DNA/well) for 20 h, except for JEG-3 cells, which was transfected for 6 h. During calcium phosphate precipitation, cells were incubated in their growth medium, except for NCI-H295A cells, which were incubated in DMEM-H16 supplemented with 10% fetal bovine serum (55). After aspirating the calcium phosphate-DNA precipitates, the cells were washed and incubated with fresh growth medium for 24 h before assaying luciferase activity. Twenty hours after adding calcium phosphate precipitates, the culture medium was changed with or without 1 mM 8Br-cAMP, and the cells were incubated for another 24 h. Luciferase activity was measured and normalized by Renilla luciferase activity expressed by the cotransfected plasmid DNA pRL-CMV (Promega, Madison, WI) as described (71). NCI-H295A cells were treated with 1 µM 8Br-cAMP, 1 µM PMA, 10 nM or 100 nM CRH in growth medium; or treated with 10 nM or 100 nM insulin in RPMI 1640 medium without fetal bovine serum.
Drosophila S2 Schneider cells were cultured at 25 C in Shields and Sang M3 insect medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum. The cells were plated on 12-well tissue culture plates the day before transfection with 150 ng plasmid DNA per well using Effectene for 48 h (QIAGEN, Santa Clarita, CA) according to the manufacturers protocol. Luciferase assays, normalized by Bradford protein (Bio-Rad Laboratories, Richmond, CA), were performed 48 h after transfection.
RT-PCR
Total RNA was isolated from NCI-H295A cells, HeLa cells, JEG-3 cells, HepG2 cells, HEK293 cells, HT1080 cells, and L132 cells using Tri Reagent (Molecular Research Center, Cincinnati, OH), and from leukocytes using the SV total RNA isolation system (Promega, Madison, WI). First-strand cDNA was synthesized from 2 µg of total RNA from each cell type using random primers (200 ng) and Superscript III reverse transcriptase (Invitrogen Life Technologies, Rockville, MD). A 410-bp fragment, corresponding to the 134-AA membrane-bound form; or a 434-bp fragment, corresponding to the 98-AA soluble form of human cytochrome b5 cDNA and a 501-bp fragment of human GAPDH cDNA were coamplified by PCR using 2 µl of the first strand cDNA product and primer pairs b5-s/b5-as and GAPDH-s-1/GAPDH-as-1 (Table 1
). Similarly, a 441-bp fragment for the 146-AA OMb5 cDNA and a 501-bp fragment for GAPDH were coamplified by PCR with the primer pairs OMb5-s/OMb5-as and GAPDH-s-1/GAPDH-as-1 (Table 1
). PCR products were separated by electrophoresis on 2% agarose gel.
Northern Blot
Total RNA (10 µg) was separated by electrophoresis on 1% agarose formaldehyde gel, and blotted to nylon membrane (Hybond NX, Amersham Biosciences, Piscataway, NJ) in 10x SSC overnight [1x SSC is 150 mM NaCl, 15 mM sodium citrate (pH 7.0)]. A 410-bp cDNA fragment of human cytochrome b5 cDNA (coding nucleotides 1429) and a 511-bp PCR product of ß-actin cDNA (coding nucleotides 781-1310) were labeled with [
-32P]deoxy-CTP and random primers, and used as probes. The RNA blot was first incubated for 15 min at 65 C with 3x SET, 1% sodium dodecyl sulfate (SDS), [1x SET is 150 mM NaCl, 30 mM Tris-HCl (pH 8.0), 2 mM EDTA (pH 8.0)], 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 in addition of 250 µg/ml yeast transfer ribonucleic acids (Sigma). The blot was hybridized with the same prehybridization buffer in 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, then with 0.3x SSC, 0.1% SDS for 1 h at 65 C and analyzed by phosphorimaging.
RNase Protection Assay
To prepare a 32P-labeled, single-strand RNA probe for the 5' flanking region of human cytochrome b5, bases 327 to +33 (where the transcriptional start site nearest to ATG start codon is base +1) was generated by PCR of genomic DNA using primers 327 and +33 (Table 1
), which placed SacII and HindIII sites at the 5' and 3' ends. This fragment was cloned into the SacII and HindIII sites of pBluescript II sk+, then linearized with EagI. Transcription in vitro with T7 RNA polymerase generated a 215 base 32P-labeled RNA probe, which included 160 bp of the 5' flanking region of the cytochrome b5 gene. Similarly, a fragment of human GAPDH cDNA 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, subcloned into the SacII/XbaI sites of pBluescript II sk+, and linearized with SacI so that transcription with T7 RNA polymerase generated a 234-base 32P-labeled RNA probe that included 144 bp from GAPDH.
Total RNA (20 µg) from HeLa and NCI-H295A cells was hybridized with the 32P-labeled RNA probe at 42 C for 20 h. The hybridized products were then treated with RNase A and RNase T1 (Ambion, Austin, TX) for 30 min at 37 C to degrade unhybridized probe before electrophoresis on 6% polyacrylamide gel containing 7 M urea; the gel was then dried and analyzed by phosphorimaging.
Promoter/Reporter Constructions
A fragment of the 5' flanking region of cytochrome b5 gene extending from 1300 bp to +15 bp (where the transcription start site nearest to the ATG start codon is designated as +1) was generated by PCR using the primers 1300 and 3R (Table 1
), which placed KpnI and BglII sites at the 5' and 3'ends, permitting cloning into KpnI/BglII digested pGL3-basic, upstream from the firefly luciferase reporter gene (Promega, Madison, WI). Deletion mutants comprising 965/+15, 730/+15, 327/+15, 225/+15, 185/+15, 157/+15 and 79/+15 were generated by PCR using the primer pairs 965/3R, 730/3R, 327/3R, 225/3R, 185/3R, 157/3R and 79/3R (Table 1
), which placed KpnI or NheI and BglII sites at the 5' and 3'end, permitting cloning into KpnI/BglII or NheI/BglII digested pGL3-basic.
Oligonucleotides were synthesized corresponding to the DNA sequences between 19/+26, 65/20, 114/70, 159/115, 201/157, 244/200, 289/245 and 334/290, thus spanning the entire 327/+26 promoter region. Each oligonucleotide included a SacI site at the 5' end and a BamHI site at the 3'end, permitting subcloning into SacI/BamHI digested TK32/Luc, which has the minimal promoter from the herpes simplex virus thymidine kinase upstream from the firefly luciferase gene (72).
PCR-based site-directed mutagenesis (73) was used to alter CCGCCC to TTAGTA in regions I (65/40) and III (270/245) and GGCGG to TTACC in region II (114/90) using the oligonucleotides listed in Table 1
. The same method was used to alter the NF-1 binding site from GCCAAT to ATGCGT and alter the GATA binding site from AGATTG to ATCCTG (Table 1
). PCR was performed using 100 ng wild-type plasmid DNA as template in reactions containing 500 mM deoxynucleotide triphosphates, 2 U Pfu polymerase, 100 ng each sense and antisense oligonucleotides, which contain the mutated nucleotides in the center (Table 1
). The PCR program was 95C for 30 sec followed by 17 cycles of 95 C for 30 sec, 55 C for 1 min and 65 C for 13 min (2.5 min/kb DNA). The PCR products were digested with 20 U of DpnI for 2 h at 37 C, and then transformed propagated in Escherichia coli. The mutant constructs were verified by sequencing.
DNase I Footprinting
Oligonucleotides 327 (sense strand) and 3R (antisense strand) were end-labeled using [
-32P]ATP and T4 polynucleotide kinase (New England Biolabs, Beverly, MA), then labeled/unlabeled pairs were used for PCR, thus producing double strand DNAs labeled either on the sense or antisense strand. The 32P-labeled DNA (70,000 cpm) was incubated with various concentrations of nuclear extracts from NCI-H295A cells in 50 µl of binding buffer containing 50 mM NaCl, 0.5 mM EDTA, 20 mM HEPES buffer (pH 7.9), 10% glycerol, 0.5 mM dithiothreitol, and 1 µg poly(deoxyinosine-deoxycytosine) at 25 C for 15 min. After adding 2.5 mM CaCl2/10 mM MgCl2, DNase I (0.1 U) was added, the samples were incubated for 50 sec at 25 C, the DNA was extracted with phenol/chloroform, then precipitated with 2 vol 100% ethanol and washed with 1 ml 75% ethanol, and dissolved in 10 µl of H2O. The DNA fragments were separated by electrophoresis on 6% polyacrylamide gel containing 7 M urea, the gel was then dried and analyzed by phosphorimaging.
EMSAs
Nuclear protein extracts were prepared from NCI-H295A cells as described (74), and gel shift assays were performed as described (60). Wild-type oligonucleotides I (65/40), II (114/90), III (270/245), IV (182/159), and V (209/186) (Table 1
) were end-labeled with [
-32P] ATP and T4 polynucleotide kinase, then purified by Sephadex G50 spin columns (Amersham Biosciences). EMSA for Sp and GATA factors, nuclear protein extracts (8 µg) were first preincubated with 1 µg poly(deoxyinosine/deoxycytosine) as a nonspecific competitor, with or without unlabeled competitor oligonucleotides in a 20 µl volume in 20 mM HEPES (pH 7.9), 20 mM KCl, 20 mM NaCl, 1.5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 10% glycerol, 0.1 mg/ml BSA at 25 C for 15 min. After adding 40,000 cpm of 32P probe, the mixture was incubated at 25 C for 15 min. EMSA for NF-1 factors, 1.5 µg of nuclear protein extracts and 500 cpm of 32P probe was used. For supershift assays, rabbit polyclonal antibodies against human Sp1, Sp3, GATA-4, GATA-6 (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and NF-1 (Bethyl, Inc., Montgomery, TX) were added to the mixture 15 min after adding the 32P probe and incubated at 25 C for an additional 15 min. The reactions were separated on 6% native polyacrylamide gel (4% for NF-1 complex) in 50 mM Tris base, 38 mM glycine, and 2 mM EDTA (pH 8.0), and 0.35 µl of ß-mercaptoethanol, and the gels were dried and then analyzed by phosphorimaging.
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FOOTNOTES
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This work was supported by National Institutes of Health (NIH) Grant KO1 DK02939 (to H.N.) and NIH Grants DK37922 and HD41958 (to W.L.M.).
First Published Online April 14, 2005
1 N.H. and A.D. contributed equally to this work. 
Abbreviations: AA, Amino acids; CTF, CCAAT transcription factor; DHEA, dehydroepiandrosterone; DNase, deoxyribonuclease; EMSA, electrophoretic mobility shift assay; F1/F2, footprint 1 or 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HEK, human embryonic kidney; OMb, outer mitochondrial membrane cytochrome b5; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NF-1, nuclear factor 1; PMA, phorbol 12-myristate 13-acetate; POR, P450 oxidoreductase; RNase, ribonuclease; SDS, sodium dodecyl sulfate; SF1, steroidogenic factor 1.
Received for publication October 13, 2004.
Accepted for publication April 7, 2005.
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