Characterization of the Regulatory Regions of the Human Aromatase (P450arom) Gene Involved in Placenta-Specific Expression

Amrita Kamat, Joseph L. Alcorn, Cheryl Kunczt and Carole R. Mendelson

Departments of Biochemistry (A.K., J.L.A., C.K., C.R.M.) and Obstetrics-Gynecology (C.R.M.), The Cecil H. and Ida Green Center for Reproductive Biology Sciences University of Texas Southwestern Medical Center at Dallas Dallas, Texas 75235


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Aromatase P450 (P450arom), a product of the CYP19 gene, catalyzes the conversion of C19-steroids to estrogens. Human P450arom is expressed in placental syncytiotrophoblast, ovarian granulosa cells, and adipose stromal cells by use of tissue-specific promoters that are located 5' of unique untranslated first exons. Mononuclear cytotrophoblasts isolated from midterm human placenta spontaneously fuse in culture to form multinucleated syncytiotrophoblast. These morphological changes are associated with a marked induction of P450arom gene expression. The majority of P450arom transcripts in placental syncytiotrophoblast contain sequences encoded by exon I.1, which lies more than 35 kb upstream of the translation initiation site in exon II. To functionally map genomic sequences required for placenta-specific P450arom expression, fusion genes containing various amounts of DNA flanking the 5'-end of placenta-specific exon I.1 linked to the human GH (hGH) gene, as reporter, were introduced into primary cultures of human trophoblast cells and other cell types. Since the trophoblast cells manifest high levels of aromatase P450 expression, we believe that this provides a physiologically relevant system for characterizing the regulatory regions of this gene. Expression of the fusion genes increased as a function of time in culture in concert with syncytiotrophoblast differentiation and induction of aromatase activity and of P450arom gene expression. P450arom-hGH fusion genes containing 923 and 501 bp of exon I.1 5'-flanking DNA were expressed at comparable levels; these levels were more than 3-fold greater than those of fusion genes containing 2400 bp of exon I.1 5'-flanking DNA, suggesting the presence of an upstream silencer element(s). Expression of these fusion genes was undetectable in cell lines that do not express aromatase or that express aromatase utilizing a nonplacental P450arom promoter. By contrast, P450arom I.1-hGH fusion genes containing 246, 201, or 125 bp of exon I.1 5'-flanking sequence were expressed both in trophoblast cells and in other cell lines. These findings demonstrate that 501 bp of exon I.1 5'-flanking DNA contain response elements required for trophoblast-specific expression of P450arom. These results also suggest the presence of regulatory elements between -501 bp and -246 bp of exon I.1 5'-flanking sequence that bind inhibitory transcription factors expressed in nontrophoblast cells. Deletion and site-directed mutagenesis experiments further suggest that cis-acting elements, including a GC box and two hexameric sequences present within 246 bp of sequence flanking the 5'-end of exon I.1, contribute to the high levels of P450arom promoter activity in primary cultures of placental cells. By competitive and supershift electrophoretic mobility shift assays, it was observed that the ubiquitously expressed transcription factor Sp1 comprises one of the proteins binding to the GC box in the 5'-flanking sequence of P450arom exon I.1.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Aromatase, an enzyme complex of the endoplasmic reticulum, catalyzes the conversion of C19-steroids to estrogens. Aromatase is comprised of two polypeptides, the ubiquitous flavoprotein, NADPH-cytochrome P450 reductase, and a unique form of cytochrome P450, P450arom (a product of the CYP19 gene), which is present exclusively in estrogen-producing cells (1, 2, 3). In humans, aromatase is expressed in various tissues including the syncytiotrophoblast layer of the placenta (4, 5), gonads (6), brain (7, 8), and adipose tissue (8, 9), as well as in different fetal tissues, including skin, intestine, and liver (10, 11, 12). The human placenta has a remarkable capacity to aromatize C19-steroids secreted by the fetal adrenals; after the ninth week of gestation, the placenta provides the primary source of circulating estrogens (13).

Human P450arom is encoded by a single-copy gene that spans more than 75 kb in the human genome (14, 15, 16). The protein-coding sequence is contained within nine exons (exons II–X); the translation initiation and termination codons are present in exons II and X, respectively. The 5'-untranslated regions of P450arom mRNA transcripts in human ovary, testis, adipose stromal cells, and placenta are encoded by alternative first exons that are spliced onto a common site that lies ~39 bp upstream of the start of translation in exon II (17, 18). The ovary-specific first exon lies proximal to exon II, whereas the major first exon encoding the 5'-untranslated region of P450arom in placenta (exon I.1) lies more than 35 kb upstream of exon II. The genomic clones containing exons I.1 and II do not overlap, so the exact distance between them is not known (16). The major human adipose-specific first exon (exon I.4) lies >=20 kb upstream of exon II, although alternative downstream first exons (exons I.3 and II) are used in human adipose stromal cells treated with cAMP and phorbol esters (19, 20).

In rodents, P450arom is not expressed in placenta; rather, P450arom expression is restricted to gonads and the brain. As is the case in humans, gonadal expression of P450arom appears to be under control of promoter II. Therefore, it appears that the ovary-specific promoter (PII) is the primordial promoter, whereas the placenta-specific promoter (PI.1) was recruited much later in phylogeny with the evolution of the primates and the hemochorial type of placenta expressing high levels of aromatase.

The rapid growth of human placenta during the first trimester of pregnancy is driven by the replication of mononuclear cytotrophoblast cells, which sit on a basement membrane. When these cells mature, they stop dividing and fuse to form the syncytiotrophoblast layer, which has numerous secretory and transport/processing functions (21). Syncytiotrophoblast differentiation entails the generation of a cascade of regulatory signals that result in expression of genes encoding different polypeptide hormones and steroid- metabolizing enzymes. However, the molecular events that promote and maintain syncytiotrophoblast differentiation and culminate in expression of various genes, including P450arom, are not clearly understood.

Cell transfection studies to functionally define the regulatory regions of the human P450arom gene required for trophoblast-specific expression have thus far been confined to use of choriocarcinoma cell lines (14, 22, 23, 24), which are undifferentiated cytotrophoblasts that express relatively low levels of aromatase activity (21). In the present investigation, we sought to develop a more relevant model system for defining critical regulatory regions of the human P450arom gene involved in placenta-specific expression by introduction of reporter gene constructs containing various amounts of DNA flanking the 5'-end of P450arom exon I.1 into human cytotrophoblasts in primary monolayer culture. These trophoblast cells, which were isolated from midterm human placenta, differentiate into syncytiotrophoblast, which manifests high levels of aromatase activity and P450arom gene expression. The results of these studies indicated the presence of putative enhancer elements between -501 bp and -42 bp that are necessary for trophoblast-specific expression of aromatase. Our findings also suggest the presence of negative regulatory elements between -501 and -246 bp that prevent P450arom expression in nonplacental cells.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Aromatase Expression Is Induced in Primary Cultures of Placental Trophoblasts from Midgestation Human Placenta in Association with Syncytiotrophoblast Differentiation
We have modified a primary cell culture system developed for term placenta by Kliman et al. (25) for isolation and culture of cytotrophoblast cells from midterm human placental tissue. We have found that the yield of cytotrophoblast cells per gram of tissue from midgestational placenta is at least 5-fold greater than that from term placenta. Upon isolation, the midgestation placental cytotrophoblasts were found to have low or undetectable levels of aromatase activity (Fig. 1AGo) and P450arom mRNA (Fig. 1BGo). Within 24 h after plating, aromatase activity and P450arom mRNA were detectable and continued to increase further between 24 and 72 h of incubation (Fig. 1Go). The two species of P450arom mRNA (3.4 kb and 2.9 kb, Fig. 1BGo) result from alternative use of different polyadenylation signals present in exon X of the P450arom gene (16). After 4 days in culture, aromatase activity was induced to levels as high as 8–12 pmol androgen metabolized to estrogen/mg of protein/min (Fig. 1AGo). As previously observed using cytotrophoblasts from term placenta (25, 26), as a function of time in culture, the human cytotrophoblast cells migrated toward each other and fused to form syncytiotrophoblast. Our findings, therefore, indicate that differentiation of placental cytotrophoblasts to syncytiotrophoblast is associated with a marked induction of aromatase activity and P450arom gene expression. In consideration of this finding, we reasoned that midgestational human placental cells in primary culture should provide a relevant model system for functional mapping of genomic elements that mediate tissue-specific expression of the P450arom gene in syncytiotrophoblast.



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Figure 1. Aromatase Activity (A) and Northern Blot Analysis of P450arom mRNA (B) in Human Trophoblasts before and after Differentiation to Syncytiotrophoblast in Culture

A, Freshly isolated cytotrophoblasts were suspended in DMEM containing 2% FBS and plated at a density of 2 x 106 cells per dish. The aromatase activity (picomoles androgen metabolized to estrogen per mg protein/min) was assayed over a 4-day period by the incorporation of tritium from [1ß-3H]androstenedione into water. The values are the mean ± SEM of data from triplicate dishes of cells. B, Total RNA (20 µg/lane) was obtained from freshly isolated cytotrophoblasts and syncytiotrophoblast after 24, 48, and 72 h in culture and analyzed for P450arom mRNA by Northern blotting using a full-length 32P-labeled human P450arom cDNA probe.

 
Cultures of replicating trophoblast cell lines derived from human choriocarcinomas are commonly used for study of placental function and trophoblast-specific gene expression (18, 22, 27, 28). The human choriocarcinoma cell line JEG-3, like normal trophoblasts, lacks 17{alpha}-hydroxylase/17,20 lyase activity but produces human CG, progesterone, and estradiol-17ß (21, 27). However, aromatase activity (Fig. 2AGo) of JEG-3 choriocarcinoma cells was found to be very low (~0.2 pmol of androgen metabolized to estrogen/mg protein/min) as compared with primary syncytiotrophoblast (~11 pmol of androgen metabolized to estrogen/mg protein/min); P450arom mRNA levels of the JEG-3 cells were essentially undetectable when compared with the trophoblast cells in culture (Fig. 2BGo).



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Figure 2. Aromatase Activity ( A) and Northern Blot Analysis of P450arom mRNA ( B) in Human Trophoblasts and JEG-3 Cells

A, Aromatase activity (picomoles androgen metabolized to estrogen per mg protein/min) in trophoblast cells in primary culture and in JEG-3 cells was assayed by analysis of the incorporation of tritium from [1ß-3H]androstenedione into water. Values shown are the mean ± SEM of aromatase activity of triplicate dishes of cells on day 4 of culture. B, Total RNA (20 µg) obtained from freshly isolated cytotrophoblasts, syncytiotrophoblast after 4 days in culture, and JEG-3 cells at 80–90% confluence was analyzed for P450arom mRNA by Northern blotting using a full-length 32P-labeled human P450arom cDNA as probe.

 
Expression of P450aromI.1-hGH Fusion Genes in Human Trophoblast Cells in Primary Monolayer Culture Increases in Association with Syncytiotrophoblast Differentiation
Sequence analysis of ~2.4 kb of 5'-flanking sequence of P450arom exon I.1 revealed the existence of a number of putative cis-acting elements including a purine-rich sequence or PU box (5'-GAGGAA-3') (29) at -164 bp, two hexameric sequences between -191 bp and -183 bp (5'-ATTCCAGAGGAGGTCATGC-3', shown in bold); the downstream sequence is similar to the binding site for an orphan member of the nuclear receptor superfamily, Ad4BP/SF1 (30, 31), a GC box consensus-binding site for Sp1 (5'-GGCGGG-3') (32) at -233 bp, a reverse Pit-1 site (5'-ATGAATAAA-3') (33) at -460 bp, a so-called trophoblast-specific element or TSE (5'-CCAATGGG-3') (34) at -825 bp and human aromatase cytochrome P450 gene transcriptional regulatory elements or hATRE-1 (5'-CTTTCTGGCCTAAGGGTTGGAGAGC-3') and hATRE-2 (5'-CTTTTATGTTGCCCAATACCTGCTCTGCCTCGAG-GGTCACTCTC-3') previously characterized by Toda and Shizuta (23) at -2238 and -2141 bp, respectively (Fig. 3Go). To functionally define the genomic regions involved in P450arom expression of promoter I.1 in primary trophoblast cultures, P450aromI.1-hGH fusion genes were constructed comprised either of 42, 125, 201, 246, 501, 923, or 2400 bp of DNA upstream and 103 bp of sequence downstream of the transcription initiation site of exon I.1, linked to the hGH structural gene, as reporter (Fig. 3Go). As a control, a P450aromII-hGH fusion gene containing 952 bp of 5'-flanking DNA and +29 bp of ovary-specific exon II linked to the hGH structural gene also was constructed. At this point, we found that the primary cultures of human placental cells were resistant to conventional methods of DNA transfection, such as calcium phosphate, diethylaminoethyl-dextran, lipofection, and electroporation (our unpublished observations). To circumvent this barrier, the fusion genes were incorporated into the genome of a replication-defective human adenovirus (35). The recombinant adenoviral particles were then used for transfer of the P450arom-hGH fusion genes into the placental cells in primary monolayer culture by infection. By this means, the fusion genes were transferred into the cells with high efficiency and without perturbation of cellular integrity. Fusion gene expression was analyzed by RIA of hGH secreted into the culture medium, which was collected at 24-h intervals.



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Figure 3. Schematic Diagram of Exon I.1 of the Human P450arom Gene and Its 5'-Flanking Region with Putative Regulatory Elements, and of P450aromI.1-hGH Fusion Genes

The positions of putative regulatory elements (indicated by labeled small boxes) within 2400 bp of DNA flanking the 5'-end of P450arom exon I.1 are shown in the top portion of the figure. These elements include hATRE-1 and hATRE-2, TSE, Pit-1, GC box, Hex sequences, and PU box (defined in text). Just upstream of exon I.1 at -27 bp from the transcription start site is the TATA box, ATAAA. The flanking DNA is indicated by the black lines, while the black boxes indicate exon I.1 sequences. The arrow indicates the transcription initiation site and the direction of transcription. The various P450aromI.1-hGH fusion gene constructs used in this study are shown in the lower part of the figure; the human P450arom DNA sequences are shown in black, and the hGH sequences are indicated by the white boxes. For cell transfection studies, the fusion genes were incorporated into the genome of a replication-defective human adenovirus (details of the procedure are described in Materials and Methods). The trophoblast cells in primary culture were incubated overnight with the appropriate recombinant adenoviruses. Media from infected cells were collected at 24-h intervals and assayed for hGH by RIA. Media were changed daily.

 
Equivalent numbers of cytotrophoblast cells (2 x 106 cells per dish) were infected with the same number of functional recombinant adenoviral particles in terms of plaque forming units (1 x 106 pfu); these were limiting with respect to the number of cells per dish (multiplicity of infection, ~0.5). Since titration of adenoviral particles in 293 cells provides a reliable index of the number of infectious recombinant viruses, the same number of trophoblast cells (1 x 106 cells) were infected in each experiment using each fusion gene construct. In this manner, highly reproducible results were obtained from one experiment to another, and the results of a number of independent experiments could be combined without normalization.

When placental cells were infected with recombinant adenoviruses containing P450aromI.1-hGH fusion genes carrying -923 bp or -501 bp of exon I.1 5'-flanking sequence, hGH production was essentially undetectable on the first day after infection. However, expression of these fusion genes increased as a function of time in culture in association with syncytiotrophoblast differentiation and the induction of P450arom expression and reached maximal levels after 4 days of incubation (Fig. 4Go). On the other hand, expression of the P450aromI.1-42-hGH fusion gene construct, which contains the minimal promoter region, remained essentially undetectable throughout the 5-day culture period (Fig. 4Go). A comparative tritiated water assay of aromatase activity indicated that infection of placental cells with recombinant adenoviruses did not affect the aromatase activity levels that increased as a function of time in culture and were similar to those of uninfected placental cells in culture (data not shown).



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Figure 4. Expression of P450aromI.1-hGH Fusion Genes in Human Trophoblasts as a Function of Time in Culture

Freshly isolated cytotrophoblasts in culture were infected with 1 x 106 recombinant adenoviruses con- taining P450aromI.1-923-hGH, P450aromI.1-501-hGH, or P450aromI.1-42-hGH fusion genes within an hour of plating. After an overnight incubation, the media were collected and replaced with DMEM containing 2% FBS. Media from the infected cells were collected over the next 4 days and replaced with fresh media containing 2% FBS. The collected media were assayed for hGH by RIA. Shown here are the levels of hGH secreted into the medium for each day of culture. Values are the mean ± SEM of data from three independent experiments, each conducted in triplicate.

 
To further delineate the genomic elements that mediate P450arom gene expression in placenta, trophoblast cells in primary culture were infected with recombinant adenoviruses containing P450aromI.1-hGH fusion genes containing either 2400, 923, 501, 246, 201, 125, or 42 bp of exon I.1 5'-flanking sequence. On the third day after infection, when most of the cells had fused to form syncytia, fusion genes containing 923 bp and 501 bp of P450arom exon I.1 5'-flanking sequence were found to be expressed at comparable levels; these levels of expression were ~2- to 3-fold higher than those of the P450aromI.1-2400-hGH fusion gene construct (Fig. 5Go). By contrast, P450arom-hGH fusion genes containing only 246 bp of exon I.1 5'-flanking DNA were expressed at levels ~3-fold greater than those of the -923 and -501 bp fusion gene constructs. These findings suggest the presence of upstream silencer elements between -2400 bp and -923 bp and also between -501 bp and -246 bp of exon I.1 5'-flanking sequence. Expression of a P450aromI.1-201-hGH fusion gene construct that does not contain the GC box (-233 bp) was ~50% lower than the -246 bp-containing fusion gene. Further deletion of exon I.1 5'-flanking sequence to 125 bp resulted in an 80% reduction of fusion gene expression when compared with the P450aromI.1-246-hGH fusion gene construct. The P450aromI.1-125-hGH fusion gene does not con-tain the Hex sequences between -191 bp and -183 bp or the PU box (-164 bp). In cells infected with recombinant adenoviruses containing P450aromI.1-42-hGH fusion gene construct or the fusion gene containing -952 bp of DNA flanking the 5'-end of ovarian-specific exon II, hGH production was essentially undetectable. Taken together, these results suggest that as little as -246 bp of exon I.1 5'-flanking sequence mediates maximal levels of P450arom gene expression in human syncytiotrophoblast in primary culture.



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Figure 5. Expression of P450aromI.1-hGH Fusion Genes Containing 42–2400 bp of P450aromI.1 5'-flanking DNA in Primary Cultures of Human Trophoblasts

Freshly isolated cytotrophoblasts in culture were infected with 1 x 106 recombinant adenoviral particles containing P450aromI.1-2400-hGH, P450aromI.1-923-hGH, P450aromI.1-501-hGH, P450aromI.1-246-hGH, P450aromI.1-201-hGH, P450aromI.1-125-hGH, P450aromI.1-42-hGH, and P450aromII-952-hGH fusion genes. Culture media were harvested and replaced with fresh medium every 24 h over a 5-day period. Shown here are the levels of hGH that accumulated in the culture medium between days 2 and 3 of culture. Values are the mean ± SEM of data from three independent experiments, each conducted in triplicate.

 
The 5'-Flanking Sequence of P450aromI.1 between -501 bp and -42 bp Is Sufficient for Placenta-Specific Expression
To delineate the genomic regions required for syncytiotrophoblast-specific expression of P450arom promoter I.1 activity, expression of these fusion gene constructs also was analyzed in JEG-3 cells and in a number of other cell lines including a human lung adenocarcinoma cell line (A549), a rat hepatoma cell line (H4IIE), the Madin-Darby canine kidney cell line, MDCK, and a rat Leydig tumor cell line (R2C). In JEG-3 cells, fusion gene expression was considerably lower than in the primary trophoblast cell cultures (Fig. 6Go). As shown in Fig. 2Go, JEG-3 cells express very low levels of aromatase activity and contain low levels of P450arom mRNA transcripts. Furthermore, in contrast to the pronounced differences in their relative levels of expression in primary cell cultures, expression in JEG-3 cells of the -246 bp and -923 bp-containing fusion gene constructs were comparable, whereas expression of P450aromI.1-501-hGH fusion gene construct was considerably lower than expression of the P450aromI.1-923-hGH fusion gene construct. The markedly reduced levels of aromatase activity, P450arom mRNA levels, and P450aromI.1-hGH fusion gene expression in JEG-3 cells, as compared with primary trophoblast cultures, indicate that this choriocarcinoma cell line may not provide a relevant system for functional mapping of cis-acting elements and trans-acting factors involved in syncytiotrophoblast-specific expression of the human P450arom gene.



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Figure 6. Expression of P450aromI.1-hGH Fusion Genes in Primary Human Trophoblast Cultures and in Various Cell Lines

The different cell types shown were infected with recombinant infectious viral particles containing P450aromI.1-2400-hGH, P450aromI.1-923-hGH, P450aromI.1-501-hGH, P450aromI.1-246-hGH, P450aromI.1-201-hGH, P450aromI.1-125-hGH, and P450aromI.1-42-hGH fusion genes. Shown are the levels of hGH that accumulated in the medium over a 24-h period between days 2 and 3 of culture. Values are the mean ± SEM of data from two to four independent experiments, each conducted in triplicate. UD, Undetectable.

 
To determine whether expression of P450aromI.1-hGH fusion genes is dependent upon trophoblast-specific factors, the levels of expression of the P450aromI.1-hGH fusion genes in trophoblast cells in primary culture also were compared with expression in a number of nonplacental cell lines, including R2C, H4IIE, A549, and MDCK (Fig. 6Go). The R2C Leydig cell line expresses relatively high levels of aromatase constitutively using the ovary-specific promoter, which lies just upstream of exon II (36, 37), whereas the other cell lines do not express aromatase (our unpublished observations). The different cell types also were infected with recombinant adenoviruses containing TK-hGH fusion genes (in which hGH was under the control of the thymidine kinase promoter) to monitor transfection efficiency, which was found to be equivalent in all cell types (data not shown). Although expression levels of fusion genes containing 2400 bp, 923 bp, 501 bp, and 42 bp of exon I.1 5'-flanking DNA were barely detectable in any of the above mentioned transfected cell lines, hGH production was detectable in these cell lines infected with adenoviruses carrying P450aromI.1-246-hGH, P450aromI.1-201-hGH, and P450aromI.1-125-hGH fusion gene constructs. In A549 cells transfected with adenoviruses carrying the P450aromI.1-246-hGH fusion gene construct, hGH production was as high as in the primary placental cell cultures transfected with the same fusion gene. On the other hand, as expected, the fusion gene containing 952 bp of DNA flanking the 5'-end of ovary-specific exon II was expressed only in the R2C Leydig cells (data not shown). Although the levels of hGH produced varied in the different cell lines, it is clear from these results that the region between -501 bp and -42 bp upstream of exon I.1 is sufficient for enhanced activity of P450arom promoter I.1 in primary human trophoblast cultures and that sequences between -501 and -246 bp mediate inhibition of expression of this promoter in nonplacental cell types.

GC Box and Hex Sequences Serve as Enhancers for P450arom Gene Expression in Primary Cultures of Syncytiotrophoblast
To characterize cis-acting elements present within the P450arom exon I.1 5'-flanking sequence required for enhanced expression in human placental cells, we analyzed the expression of a P450aromI.1-923-hGH fusion gene construct containing a mutation in a putative GC box at -233 bp (P450aromI.1-923Sp1 mut-hGH) and of a P450aromI.1-501-hGH fusion gene construct containing mutations in two hexameric sequences, present between -191 bp and -183 bp from the start site of transcription (P450aromI.1-501Hexmut-hGH). These constructs were incorporated into the genome of a replication-defective human adenovirus and introduced into midgestation human placental cells by infection as described above.

When placental cells were transfected with P450aromI.1-923Sp1 mut-hGH, there was a 50% reduction in hGH expression as compared with expression levels observed upon transfection of P450aromI.1-923-hGH fusion gene containing the wild-type sequence (Fig. 7Go). The levels of hGH production by trophoblast cells transfected with P450aromI.1-501Hexmut-hGH fusion genes, in which the hexameric sequences were mutated, were reduced by ~75% when compared with those of trophoblast cells transfected with P450aromI.1-501-hGH fusion genes containing the wild type sequences. These findings suggest that both the GC box and hexameric sequences serve as enhancers for P450arom gene expression in placental cells.



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Figure 7. Effect of Mutation of the GC Box and of the Hex Sequences on Expression of P450aromI.1-923-hGH and P450arom I.1-501-hGH Fusion Genes, Respectively, in Trophoblast Cells in Primary Culture

Freshly isolated cytotrophoblasts were infected with 1 x 106 recombinant viral particles containing P450aromI.1-923-hGH, P450aromI.1-923Sp1 mut-hGH, P450aromI.1-501-hGH, and P450aromI.1-501Hexmut fusion genes. Shown here are the levels of hGH that accumulated in the medium over a 24-h period between days 2 and 3 of culture. Values are the mean ± SEM of data from three independent experiments, each conducted in triplicate.

 
Sp1 Comprises One of the Proteins Binding to the GC Box in the 5'-Flanking Sequence of Exon I.1
To begin to characterize the proteins binding to the GC box at -233 bp upstream of the translation start site of exon I.1, competitive electrophoretic mobility assay (EMSA) was performed. A fragment spanning a region from -241 bp to -217 bp upstream of exon I.1 containing the GC box (GCaromI.1) was used as the radiolabeled probe and either a consensus GC sequence (GCcon) or an oligonucleotide in which the GC box of P450aromI.1 was mutated (GCaromI.1 mut) was used as a competitor (Fig. 8AGo). Syncytiotrophoblast nuclear proteins interacted with the radiolabeled GCaromI.1 fragment as three complexes of different mobilities (lane 2); the two slower mobility complexes were effectively competed by a 200-fold excess of nonradiolabeled GCaromI.1 oligonucleotide (self, lane 3) and by a 200-fold excess of GCcon (lane 4) but not by a 200-fold excess of GCaromI.1 mut oligonucleotide (lane 5). These findings indicate that factors that comprise the two slower mobility complexes are specific for the GC box of P450aromI.1. To determine whether Sp1 is a component of these complexes, antibody supershift EMSA was performed using a polyclonal antibody directed against Sp1 and either the radiolabeled GCaromI.1 or the radiolabeled GCcon sequences as probes (Fig. 8BGo). As can be seen, an identical gel shift pattern was obtained using syncytiotrophoblast nuclear proteins and either the GCaromI.1 or GCcon as radiolabeled probes (lanes 3 and 6, respectively). The addition of Sp1 antibody to the binding reaction containing the nuclear proteins and the radiolabeled GCaromI.1 probe supershifted the slowest mobility complex (lane 4). The antibody itself did not interact with the GCaromI.1 radiolabeled probe in the absence of nuclear proteins (lane 2). A similar supershifted complex was observed when the Sp1 antibody was included in the incubation with the nuclear proteins and the radiolabeled GCcon probe (lane 7). The results of the competitive EMSA (Fig. 8AGo) and the Sp1 antibody supershift EMSA (Fig. 8BGo), together with the finding that an identical gel shift pattern is obtained using GCaromI.1 or GCcon as probes (Fig. 8BGo), suggest that Sp1 comprises one of the proteins binding to the GC box in the 5'-flanking sequence of exon I.1.



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Figure 8. Competitive EMSA (A) and Supershift EMSA (B) Using Syncytiotrophoblast Nuclear Protein and Either Radiolabeled GCaromI.1 or GCcon as Probes

A, Syncytiotrophoblast nuclear proteins were incubated with radiolabeled GCaromI.1 probe in the absence ([-], lane 2) or in the presence of a 200-fold excess of nonradiolabeled GCaromI.1 oligonucleotide (self, lane 3), a 200-fold excess of nonradiolabeled oligonucleotide containing a consensus GC box sequence (GCcon, lane 4), or a 200-fold excess of nonradiolabeled oligonucleotide containing a mutated GC box in GCaromI.1 (GCaromI.1 mut, lane 5). No protein was incubated with the probe in the first lane (free probe, lane 1). B, Syncytiotrophoblast nuclear proteins (3 µg) were incubated for 1 h at 4 C in the absence (Syn, lanes 3 and 6) or presence of antisera (Ab) to Sp1 (lanes 4 and 7), and poly(dI-dC)-poly(dI-dC), as nonspecific competitor. The nuclear protein mix was then incubated either with 32P-labeled GCaromI.1 (lanes 1–4) or a GC box consensus (GCcon, lanes 5–7) probe for an additional hour at 4 C. The radiolabeled GCaromI.1 probe was incubated with the Sp1 antibody in the absence of nuclear proteins in lane 2. The DNA-protein complexes were resolved on a 5% polyacrylamide gel and visualized by autoradiography. The arrow indicates the supershifted complex. No protein was incubated with the probe in lanes 1 and 5 (free probe).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The syncytiotrophoblast layer of the human placenta, which is formed upon the fusion of the underlying proliferative, mononuclear cytotrophoblast cells, synthesizes both steroid hormones and steroid-metabolizing enzymes, including aromatase. Previous studies from our laboratory indicate that human syncytiotrophoblast P450arom mRNA transcripts contain 5'-untranslated sequences encoded by exon I.1, which lies at least 35 kb upstream of the translation start site in exon II of the aromatase gene (16). The molecular events that promote and maintain syncytiotrophoblast differentiation and culminate in expression of the gene encoding P450arom are not clearly understood.

In the present study, we have used a primary cell culture system using cytotrophoblasts isolated from midgestation human placenta for functional analysis of the genomic sequences involved in syncytiotrophoblast-specific expression of aromatase. The levels of aromatase activity and of P450arom mRNA in freshly isolated cytotrophoblasts from midterm placenta are very low or undetectable; however, upon differentiation to syncytiotrophoblast, there is a marked induction of aromatase activity and P450arom gene expression. This is the first time that regulatory regions of a human placenta-specific gene have been mapped using primary cultures of trophoblast cells. To define the regulatory regions of the P450arom gene that mediate placenta-specific expression, trophoblast cells in primary culture were infected with recombinant adenoviruses containing P450aromI.1-hGH fusion genes comprised of genomic sequences flanking the 5'-end of exon I.1 of the human P450arom gene. Replication-defective recombinant human adenoviruses have been used as vehicles for efficient transfer of exogenous genes into slowly dividing or nonproliferating cells (38, 39). In the present study, we have used recombinant adenoviruses for transfer of P450arom I.1-hGH fusion gene constructs into midgestation cytotrophoblast cells immediately after plating. In this manner, expression of the fusion genes can readily be assessed over time as the cells differentiate to form syncytiotrophoblast that expresses high levels of aromatase.

Our findings indicate that expression of P450arom-hGH fusion genes containing 125-2400 bp of exon I.1 5'-flanking DNA increases in concert with syncytiotrophoblast differentiation and P450arom gene expression. By deletional analysis, we observed that expression of fusion genes containing 2400 bp of exon I.1 5'-flanking sequence was lower than expression of P450aromI.1-923-hGH fusion genes in the transfected trophoblast cells. Similarly, hGH production levels in placental cells infected with adenoviruses carrying the -501 bp containing fusion gene construct were lower than those of the -246 bp containing fusion gene construct. These results suggest the presence of silencer elements between -2400 bp and -923 bp, and also between -501 bp and -246 bp upstream of P450arom exon I.1. The highest levels of fusion gene expression were observed in trophoblast cells infected with P450aromI.1-hGH fusion genes containing -246 bp of exon I.1 5'-flanking sequence. Further deletion of the 5'-flanking region resulted in a gradual decrease in fusion gene expression, suggesting the presence of enhancer elements within the 5'-flanking sequence between -246 bp and -42 bp of exon I.1. These findings support those of Toda et al. (14), which suggested that 5'-flanking sequences between -500 bp and -243 bp, and between -242 bp and -138 bp, contained cis-acting elements that repressed and activated expression, respectively, of P450arom I.1-CAT reporter gene constructs in BeWo choriocarcinoma cells.

Aromatase mRNA transcripts containing 5'-untranslated sequences encoded by exon I.1 also have been found in the human choriocarcinoma cell line, JEG-3 (18). A number of investigators have used this cell line for gene transfection studies because of its ability to proliferate and readily take up DNA. However, we have observed that aromatase activity of JEG-3 choriocarcinoma cells is ~0.02 that of the midgestational trophoblast cells in primary culture (Fig. 2AGo). The low levels of aromatase expression in choriocarcinoma cell lines is not surprising since choriocarcinomas consist of mitotically active cytotrophoblast cells with moderate degrees of differentiation (21, 27, 40). Hence, these cells secrete high levels of CG, characteristic of cytotrophoblasts, and only modest amounts of hormones, characteristic of syncytiotrophoblast, such as chorionic somatomammotropin (21). In the present study, the P450arom I.1-hGH fusion genes were found to be expressed at considerably lower levels in the JEG-3 cells than in primary human trophoblast cultures. Additionally, the pattern of expression of the different fusion gene constructs in the JEG-3 cells was not comparable to that observed in the midgestational placental cells in culture. Hence, although the JEG-3 cells share many characteristics of normal trophoblast cells, our studies indicate that they may not provide a suitable system for mapping of genomic regions required for placenta-specific expression of the human P450arom gene.

To assess the importance in placenta-specific expression of regulatory elements present within -2400 bp of sequence flanking the 5'-end of P450arom exon I.1, we compared the expression of the P450aromI.1-hGH fusion gene constructs in primary human trophoblast cultures with expression in cell lines that do not express aromatase (the human lung adenocarcinoma-derived cell line A549, the canine kidney cell line MDCK, and the rat hepatoma cell line H4IIE), as well as in the R2C rat Leydig cell line, which expresses aromatase from a different promoter. We observed that whereas expression of the P450aromI.1-hGH fusion genes containing >=501 bp of exon I.1 5'-flanking DNA were expressed at relatively high levels in trophoblast cells in primary culture, they were expressed at extremely low or undetectable levels in kidney (MDCK), lung (A549), liver (H4IIE), or Leydig (R2C) cell lines. Interestingly, in addition to being expressed at relatively high levels in the human syncytiotrophoblast, fusion genes containing 246 bp, 201 bp, and 125 bp of exon I.1 5'-flanking sequence were found also to be highly expressed in A549 and MDCK cells. In conjunction with the deletional analysis in primary cultures of trophoblast cells, these findings suggest that 501 bp of exon I.1 5'-flanking DNA contains binding sites for transcription factors that mediate enhanced levels of trophoblast-specific expression of the P450arom gene. The finding that P450aromI.1-hGH fusion genes containing <=246 bp of exon I.1 5'-flanking DNA also were expressed at relatively high levels in other cell types suggests that the 5'-flanking region between -501 bp and -246 bp contains silencer elements that may bind inhibitory transcription factors expressed in other cell types.

By use of site-directed mutagenesis, we have identified two potential enhancer elements present within 501 bp of P450arom exon I.1 5'-flanking DNA: a GC box (5'-GGCGGG-3') at -233 bp and two Hex sequences (5'-ATTCCAGAGGAGGTCATGC-3', shown in bold) between -191 bp and -183 bp. Mutation of either the GC box or Hex sequences reduced fusion gene expression, suggesting their functional significance in mediating high levels of aromatase gene expression in placenta. Sp1 is known to bind to a consensus GC box (GGGCGG) and direct tissue-specific, developmental, and hormonal regulation of a number of genes (41, 42). Although Sp1 is a ubiquitous transcription factor, it is likely that it does not function alone but acts in concert with other regulatory factors (43). In electrophoretic mobility shift assays using human syncytiotrophoblast nuclear proteins and Sp1 antibody, we have observed that Sp1 comprises one of the components that binds to the GC box at -233 bp. It should also be noted that GC-rich sequences serve as binding sites for other members of the Krüppel family of transcription factors, several of which are expressed in a tissue-selective manner (44). Studies are now in progress to characterize the other placental nuclear proteins that bind to the GC box in the 5'-flanking sequence of P450arom exon I.1.

The transcription factor, Ad4BP/SF-1, which binds to the sequence PuPuAGGTCA and regulates gonadal and adrenal development (30, 31, 45), has been reported to participate in the transcriptional regulation of P450arom in the ovary of humans (46) and rodents (36, 47). However, the findings that SF-1 is expressed at very low levels in human placenta (48) and that the placenta develops and functions normally in SF-1 ‘knock-out’ mice (49) suggest that this transcription factor may not play an important role in placenta-specific gene expression. It is possible that another member(s) of the nuclear receptor superfamily could bind to the Hex sequence upstream of exon I.1 and activate expression of P450arom promoter in the human placenta. Recently, Sun et al. (50) have found that an imperfect palindromic sequence between -183 bp and -172 bp upstream of P450arom exon I.1, which contains the downstream Hex sequence (5'-GGAGGTCA-3') binds a heterodimer of RXR{alpha} and VDR in JEG-3 cells.

A unique combinatorial interaction of ubiquitously expressed and cell-selective transcription factors has been reported to mediate placenta-specific expression of a growing number of eukaryotic genes, including those encoding the {alpha}-subunit of the glycoprotein hormones (51), leptin (52), and placental lactogen I (53). Expression of the {alpha}-subunit of the glycoprotein hormones in placental cells requires the cooperative interaction of proteins bound to five different regulatory elements, including a TSE, an {alpha}-activating element ({alpha}-ACT), a tandem pair of cAMP response elements (CREs), a junctional regulatory element (JRE), and a CCAAT box (54). Our findings that P450arom gene expression is induced in concert with syncytiotrophoblast differentiation suggest that transcription factors involved in trophoblast differentiation may also serve a role in induction of P450arom expression. In a number of recent reports, it has been suggested that trophoblast differentiation is regulated by changes in expression of both positive and negative transcriptional regulators that belong to basic helix-loop-helix, POU domain, zinc finger, and nuclear receptor transcription factor families (55, 56, 57, 58, 59).

In summary, in the present study we have observed that sequences between 42 and 501 bp upstream of exon I.1 of the human aromatase gene contain an enhancer(s) that mediates elevated levels of expression in primary cultures of human trophoblast cells. Furthermore, we have found that sequences between -246 and -501 bp suppress P450arom promoter I.1 expression in nonplacental cell types. Studies currently are in progress to further define the roles of the GC box, the Hex elements, and other cis-acting regulatory elements within the 501 bp 5'-flanking region involved in the enhanced levels of promoter I.1 activity in placental trophoblasts and in inhibition of promoter I.1 activity in other cell types. In parallel, transgenic mice are being used to define the minimal genomic region and the cis-acting elements that are required to mediate appropriate placenta-specific, developmental, and regulated expression of human P450arom gene.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Isolation and Culture of Human Placental Cells
Placental tissues from midtrimester human abortuses were acquired in accordance with the Donors Anatomical Gift Act of the State of Texas after written consent was obtained. In all cases, consent forms and protocols were approved by the Human Research Review Committee of the University of Texas Southwestern Medical Center at Dallas. A placental cell culture system developed by Kliman et al. (25) was modified for isolation and culture of cytotrophoblast cells from human midgestation placenta. Briefly, the placental tissues were washed with HBSS, pH 7.4 (GIBCO, Grand Island, NY) before being finely minced and digested in HBSS containing 0.125% trypsin at 37 C for 30 min. The trypsin digestion was repeated three times; at the end of each 30-min digestion period, the supernatant was collected, layered over serum, and then briefly centrifuged at 1000 x g for 10 min. The pellet was suspended in DMEM (GIBCO), filtered, and then layered over a Percoll gradient (70%–5%). The gradients were centrifuged at 1200 x g for 20 min at room temperature; the cells in the middle layer (density 1.045–1.062), which comprised the cytotrophoblasts, were collected, washed, and counted. The cells were resuspended in DMEM supplemented with 10% FBS and 1% antibiotic/antimycotic solution, plated at a density of 2 x 106 cells/2 ml on dishes coated with extracellular matrix (ECM) and incubated overnight at 37 C in a humidified atmosphere of 95% air-5% CO2. After 24 h, the medium was aspirated and replaced with DMEM containing 2% FBS. The ECM-coated dishes were prepared from confluent monolayers of Madin-Darby canine kidney cells (ATCC CRL 6253) (2–5 x 106 cells per dish) that were treated with 1% deoxycholate for 5 min. The dishes were then washed three times with HBSS and stored at 37 C until use.

Preparation and Maintenance of Cell Lines
The human choriocarcinoma cell line, JEG-3 (ATCC HTB-36), was maintained in RPMI medium supplemented with FBS (10% vol/vol), while the rat R2C Leydig cell line (ATCC CCL-97) was maintained in Ham’s F-10 medium containing horse serum (15% vol/vol) and FBS (2.5% vol/vol). The human lung adenocarcinoma cell line, A549 (ATCC CCL-185), the canine kidney cell line, MDCK (ATCC CRL-6253), and the rat hepatoma cell line, H4IIE (ATCC CRL-1548) were maintained in Waymouth’s medium supplemented with FBS (10% vol/vol). The adenovirus-transformed human embryonic kidney cell line, 293 (ATCC CRL-1573), was cultured in DMEM containing FBS (10% vol/vol). The various media used contained antibiotic/antimycotic solution (1%).

Tritiated Water Assay of Aromatase Activity in Placental and JEG-3 Cells
Aromatase activity was assayed in placental and JEG-3 cells in monolayer culture by the tritiated water assay as described previously (60). Briefly, [1ß-3H]androstenedione (New England Nuclear, Boston, MA) was added to the culture media for 1 h; 2-ml aliquots were then removed and combined with 1 ml of ice-cold 30% (wt/vol) trichloroacetic acid. The incorporation of tritium from [1ß-3H]androstenedione into water was assayed in aqueous scintillation fluid after extraction with 4 volumes of chloroform and 1 volume of dextran-charcoal suspension. The cell monolayers were rinsed twice in PBS and stored at -20 C for subsequent assays of protein content by the method of Lowry et al. (61).

Northern Blot of Placental mRNA
Total RNA was isolated from cells by the method of Chirgwin et al. (62). Briefly, monolayer cultures were washed with PBS, lysed in 4 M guanidinium isothiocyanate, followed by centrifugation through a cesium chloride cushion (5.7 M). The RNA samples were then extracted with phenol/chloroform and precipitated with ethanol, and the pelleted RNA was resuspended in water. Total RNA (20 µg) was electrophoresed, transferred to nitrocellulose, and probed using a 32P-labeled human aromatase cDNA probe (63). The relative levels of aromatase mRNA were assessed by autoradiography.

Construction of P450aromI.1-hGH Fusion Genes and Preparation of Recombinant Adenoviruses
Genomic sequences comprised of 2400, 923, 501, 246, 201, 125, and 42 bp of 5'-flanking DNA and 103 bp of untranslated exon I.1 of the human aromatase gene (P450aromI.1) were subcloned into the Sal/BamHI sites of the plasmid pACsk2OGH to obtain various P450aromI.1:pACsk2OGH plasmids. pACsk2OGH contains the promoterless hGH structural gene and the left 17% of human adenovirus 5 genome (39, 64). In this manner, various amounts of exon I.1 5'-flanking DNA and the first 103-bp segment of untranslated exon I.1 of the aromatase gene were fused to the first exon of the hGH structural gene. A fusion gene containing 952 bp of 5'-flanking DNA and +29 bp of ovary-specific exon II of human aromatase gene linked to the hGH structural gene also was constructed and used as a control. As an alternative control, a TK-hGH fusion gene was constructed in which the hGH structural gene was ligated to the herpes simplex virus thymidine kinase promoter.

Fusion genes containing mutations in the GC box at -233 bp [5'-CCCAAGGCGGGACTCT-3' (wild-type sequences are shown with the GC box in bold and underlined)] or the hexameric (Hex) sequences at -183 bp and -191 bp [5'-ATTCCAGAGGAGGTCATGC-3' (wild-type sequences are shown with the Hex sequences in bold and underlined)] were created by site-directed mutagenesis according to the method out-lined in Bio-Rad Mutagene Kit (Bio-Rad Laboratories, Richmond, CA). Briefly, pBlaromI.1-501 [pBluescript II KS vector (Stratagene) containing 501 bp of sequence flanking the 5'-end of P450arom exon I.1 and 103 bp of exon I.1] and helper phage, M13K07, were used to transform CJ236 Escherichia coli strain deficient in dUTPase and uracil-N-glycosylase. The isolated single-stranded uracil-containing phage DNA was used as a template for site-directed mutagenesis using oligonucleotide Sp1 mutrev [5'-ATCAGATTATAGAGAAAGATCTTGGGTGGATGAA-3' (mutated nucleotides in italics and bold)] or Hexmutrev [5'-GGTATGGGGCATGGGGTCGACCGGGATGAGGGTCTTATG3' (mutated nucleotides in italics and bold)] as primers. The resulting plasmids, pBlaromI.1-501Sp1 mut and pBlaromI.1-501Hexmut, were used to obtain P450aromI.1-501Sp1 mut: pACsk2OGH and P450aromI.1-501Hexmut:pACsk2OGH plasmids, respectively. The SacI/EcoRI fragment (-923 bp to -501 bp) from P450aromI.1-923:pACsk2OGH plasmid was then subcloned into P450aromI.1-501Sp1 mut:pACsk2OGH to obtain P450aromI.1-923Sp1 mut:pACsk2OGH plasmid. Sequences of each fusion gene construct were confirmed by double-stranded sequencing using the dideoxy chain termination method and a Sequenase kit (US Biochemical, Cleveland, OH).

To generate recombinant adenoviruses, 293 cells, a permissive human embryonic kidney cell line, were cotransfected with recombinant pACsk2OGH plasmids containing the various fusion gene constructs and with pJM17. The pJM17 plasmid is too large to be packaged into viral particles, since it contains the entire adenovirus genome and a 4.3-kb insert of pBR322 plasmid. Infectious viral particles of appropriate size for packaging are obtained by in vivo homologous recombination of the plasmids that results in the formation of a recombinant viral genome with the release of the pBR322 insert (35, 65). Viral DNA was analyzed to confirm the presence of the fusion genes by restriction endonuclease digestion, Southern analysis, and DNA sequencing (Sequenase 2.0, U.S. Biochemical). The recombinant viruses were then titered in 293 cells at least twice to determine the number of infectious particles (plaque-forming units).

Infection of Trophoblasts in Primary Culture and Different Cell Lines with Recombinant Adenoviruses
Freshly isolated cytotrophoblast cells, plated at a density of 2 x 106 cells per dish in DMEM containing 10% FBS, were infected within 1 h of plating with 1 x 106 recombinant infectious viral particles, resulting in a multiplicity of infection of ~0.5. In this manner, the same number of cells (1 x 106) was infected in each experiment. After an overnight incubation, media were collected and replaced with DMEM containing 2% FBS. Media from infected cells were then collected at 24-h intervals and replaced with fresh media containing 2% FBS. The collected media were assayed for hGH by RIA, using an Allegro hGH kit (Nichols Institute Diagnostics, San Juan Capistrano, CA).

Different cell lines used in this study were also infected with 1 x 106 recombinant adenoviral particles containing various P450aromI.1-hGH fusion genes. hGH secreted into the media was assayed by RIA, as described above for primary trophoblast cultures.

EMSA
Nuclear extracts were prepared from placental cells on day 4 of culture by the method of Dignam et al. (66). Protein concentrations were determined by a modified Bradford assay (Bio-Rad, Richmond, CA). Double-stranded oligonucleotides corresponding to a region from -241 bp to -217 bp upstream of the translation start site in P450arom exon I.1 containing a GC box [GCaromI.1, 5'-CCACCCAAGGCGGGACTCTATAATC-3' (wild-type sequences are shown in bold and underlined)] or a mutated GC box [GCaromI.1 mut, 5'-CCACCCAATATGTTTCTCTATAATC-3' (mutated sequences are shown in bold and italics)] were synthesized. An oligonucleotide containing a consensus GC box (GCcon, 5'-ATTCGATCGGGGCGGGGCGAG-3') was also synthesized. For competition EMSAs, double stranded oligonucleotides were end-labeled with T4 kinase and [{gamma}-32P]ATP, incubated with syncytiotrophoblast nuclear proteins (3 µg) for 30 min at room temperature in binding buffer (20 mM HEPES, pH 7.6, 12% glycerol, 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol) in the absence or presence of nonradiolabeled double-stranded oligonucleotides and 1 µg poly(dI-dC)-poly(dI-dC) (Pharmacia, Piscataway, NJ) as nonspecific competitor. The DNA-protein complexes were resolved on a 5% polyacrylamide gel and visualized by autoradiography. For supershift EMSA, the nuclear proteins were incubated for 1 h at 4 C in binding buffer in the absence or presence of Sp1 antibody (1 µg) (Santa Cruz, Biotech, Santa Cruz, CA) and poly(dI-dC)-poly(dI-dC). The nuclear protein mix was then incubated with 32P-labeled probe for an additional hour at 4 C before separation by PAGE.


    ACKNOWLEDGMENTS
 
The authors are grateful to Margaret Smith for her expert help with Northern blot analysis and cell culture.


    FOOTNOTES
 
Address requests for reprints to: Carole R. Mendelson, Ph.D., Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9038. E-mail: cmende{at}biochem.swmed.edu

This research was supported by NIH Grant 5 RO1 DK-31206. A.K. was supported in part by NIH Training Grant 5-T32-HD-07190–16.

Received for publication May 4, 1998. Revision received June 24, 1998. Accepted for publication July 16, 1998.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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