Aromatase (cytochrome P-450) is a unique member of the cytochrome P-450 superfamily. It catalyzes the conversion of androgen to estrogen, which is a rate-limiting step in estrogen biosynthesis(1) . Aromatase is expressed in various cells and tissues including ovarian cells(2, 3, 4) , testicular Sertoli and Leydig cells(5, 6) , placenta(1, 7) , adipose tissue(8, 9, 10) , skin fibroblasts(11) , and various parts in the brain including the amygdala and hypothalamus(12, 13) . Recent investigations revealed that aromatase mRNAs expressed in adipose tissue, ovary, fetal liver, and brain have differences in the untranslated exon I from that reported previously for the placenta (14, 15, 16, 17, 18) . The use of alternative transcription start sites that occur as a consequence of employing tissue specific promoters is a unique feature of the aromatase gene expression and seems to be the underlying molecular basis for the complexity of the expression of this gene. However, how individual promoters exert their tissue specificities is not known.

The placenta is the primary site of estrogen synthesis in pregnant women and 16-hydroxydehydroisoandrosterone sulfate derived from the fetal adrenal and liver is the major precursor for placental estrogen production. Human aromatase mRNA expressed in placenta has a unique untranslated exon I, which is designated as exon Ia in this laboratory (16, 17) or I-1 by Mahendroo and others(14, 15) . This type of aromatase mRNA is detected only in placenta and in cultured cells of trophoblast origin. Although cDNAs containing other types of untranslated 5`-region have been isolated from placental cDNA libraries(19, 20) , the 5`-end of exon Ia is the major initiation site in placenta. A choriocarcinoma cell line JEG-3 expresses aromatase mRNA of placental type(14, 21) . This cell line expresses aromatase in unstimulated condition as well as in response to phorbol ester and various reagents that raise intracellular cAMP levels(21, 22) . The aromatase activity in the cells was closely related to the content of aromatase mRNA that was very labile, suggesting transcriptional control is the main regulatory point for the enzyme activity. Thus, this cell line seems to provide a good system to investigate transcriptional regulation of placental type aromatase.

In the present paper, we describe analysis of the 5`-flanking region of aromatase exon Ia for the placenta-specific expression of this gene. We show that the initial -301 upstream of the exon Ia is sufficient for the basal expression of the exon. This region, conferring cell specificity, contains at least three binding domains. Two of them are recognized by the same trans-acting factor that binds to the trophoblast-specific element previously located in the enhancer region of human glycoprotein hormone -subunit(23) . A common transcription factor appears to be involved in the expression of two hormone-related genes that are characteristic of human placenta.

EXPERIMENTAL PROCEDURES

Oligonucleotides

()

Figure 1: Schematic illustration of the aromatase exon 1a 5`-flanking region. Boldarrows indicate the 5`-end of the designated CAT constructs. Numbers are relative positions from the initiation start site of aromatase exon Ia. Thinarrows represent PCR primers used in the amplification of promoter fragments. The fragments used for construction of CAT cassettes and gel mobility shift assays are shown below with their identities indicated on the left. Recognition sequences of potential cis-acting elements Sp1, PEA3, and Ad4 are boxed.

Reporter Gene Construction

Transfection of Cells

For the transfection experiment, cells were plated at 1 10 cells/90-mm plate 2 days before transfection. The medium was changed 4 h before transfection. 8 µg of CAT plasmid and 1 µg of reference plasmid RSV/Luci (26) with 0.1 mg of O,O`-didodecyl-N-[p-(2-trimethylammonioalkyloxy)benzoyl]-L-glutamate bromide (27) were suspended in 1 ml of culture medium without serum and kept for 30 min at room temperature. Culture dishes were washed once with 8 ml of the culture medium without serum and refed with 4 ml of the culture medium containing 5% fetal calf serum. Transfection was started by dropwise addition of the liposome-DNA suspension. After 12-16 h, the culture medium was changed to medium containing 10% fetal calf serum and gentamicin. Cells were cultured for 48 h before cell lysis.

Luciferase and Chloramphenicol Acetyltransferase Assay

Preparation of Nuclear Extracts

Gel Mobility Shift Assays

RESULTS

Specificity of the Promoter/Regulator Region of Exon Ia

Figure 2: Transcriptional activities of truncated aromatase exon Ia 5`-upstream fragments. Derivatives of pSV00CAT fused to three different lengths of the 5`-upstream region of aromatase Ia were transfected into JEG-3, HepG2, or HeLa cells as described under ``Experimental Procedures.'' p-301ACAT carries the -301 to +22-bp fragment of the upstream region of aromatase Ia. p-212A CAT and p-115ACAT contain the -212 to +22-bp fragment and the -115 to +22-bp fragment, respectively. Transfection efficiency was standardized by cotransfection of the luciferase expression vector pSV/Luci. The average of the duplicates is shown.

These three constructs and a CAT construct with the tk promoter were transfected into two other cell lines: a hepatic cell line, HepG2, and HeLa cells. In HepG2, exon Ib-type aromatase mRNA, a major form expressed in fibroblasts and fetal liver, was found. ()HeLa cells did not express detectable amounts of aromatase mRNA under our culture and assay conditions including 30 cycles of reverse transcription-PCR. When the same luciferase units were applied to CAT assay, CAT constructs with truncated aromatase exon Ia upstream poorly activated transcription in HepG2 and HeLa cells, compared to the nonspecific promoter tk. Therefore, the regulator/promoter activity of the 5`-upstream region of aromatase exon Ia was found specific to trophoblasts.

Determination of Binding Domains

As shown in Fig. 3, a fragment spanning -307 to -142 (designated as FB) gave two specific bands (panelA, lane1), which are indicated with arrows. These bands were diminished with a 400 molar excess of unlabeled FB as a competitor (lane2). As illustrated in Fig. 1, this region contains several potential cis-elements such as Ad4(32) , PEA3(33) , and Sp1(34) . Ad4 sequences exist in regulatory regions of various steroidogenic P-450 genes, and this element dictates expression specific to steroidogenic tissues. Oligonucleotides containing consensus sequences for Ad4 (5`-GGACATACCCAAGGTCCCCTTT-3`) (32) or PEA 3 (5`-TCGAACTTCCTGCTCGA-3`) (33) did not compete for binding (Fig. 3A, lanes6 and 7). The major binding complex formed with labeled FB was also competed by some segments of FB, namely, FD (-307/-210), FC1 (-307/-240), and HB (-217/-142) (lanes3-5). The smallest fragment that competed for the binding was the 27-bp fragment, C2 (-300/-274), included in the FD fragment (Fig. 3A, lane8). The formation of the minor binding complex was inhibited by the HB and HM (-217/-166) fragments (lanes5 and 9). When the FD fragment was used as a probe (Fig. 3B), only the major band appeared as a specific binding complex (Fig. 3B, lane6). Unlabeled FD, FC1, HB, and C2 (Fig. 3B, lanes1-4) competed for this binding.

Figure 3: Binding of JEG 3 nuclear factors to fragments of aromatase exon Ia promoter regions. Preparation of nuclear extracts from JEG-3 cells and analysis of binding were performed as described under ``Experimental Procedures.'' The arrowheads indicate the position of the major binding complex. The arrows indicate the position of the minor binding complex. Probes used for the assay extended from -307 to -142 (FB) in panelA, from -307 to -210 (FD) in panelB, and from -217 to -142 (HB) in panelC. In panelD, oligonucleotides C2 and C4 were used as probes. In some lanes, a 200-fold molar excess of the designated DNA fragment was added as a competitor for the binding.

In experiments where the HB fragment was used as a radiolabeled probe (Fig. 3, panelC), two specific bands appeared as shown by the arrow and arrowhead. The major band was inhibited by excess amounts of unlabeled HB, FC1, C2, and C4 (-177/-153) (Fig. 3C, lanes2-4 and 8). The restriction enzyme MvaI cuts the HB fragment at -166/-165, and the digested HB probe completely lost the ability to form the major band (data not shown). Both of the resulting fragments of digested HB failed to effectively compete for the binding of major bands (Fig. 3C, lanes6 and 7). The formation of the minor band was not inhibited with C2 or C4 fragments but was inhibited by a segment of HB (-212/-167) (lane6) and a 25-bp fragment encompassing -199/-176 (C3) (lane5). In panelD, mutual competition between C2 and C4 is illustrated. The results of these gel mobility shift assays can be summarized as follows; two sites in the FB region, C2 and C4, bind to the same nuclear factor to form a major band and another factor, which forms minor complex binds to a distinct region between the two domains.

Transcriptional Activity of the Fragments

Figure 4: Specificity of activation of tk-driven transcription by the fragments containing regions for specific binding and C2 binding. A, JEG-3, HepG2, and HeLa cells were transfected with tkCAT(pBLCAT2), pHBCAT, or pFBCAT. Transfections were performed, and the activity was calculated as described under ``Experimental Procedures.'' The average of the duplicates was shown relative to the activity of the pBLCAT2 construct of each cell. B, gel mobility shift assays were carried out with labeled C2 fragment as the probe. Nuclear extracts from either JEG-3 cells (2.7 mg, lanes1, 2, 5, 6), HepG2 cells (3 mg, lane3), HeLa cells (3 mg, lane4), or placenta (3.5 mg, lanes7, 8) were incubated for 30 min at room temperature and subjected to electrophoresis. Samples in lanes1, 5, and 8 contained a 400-fold molar excess of unlabeled C2 fragment as a competitor.

DNA Sequence Required for Binding in the C2 Domain

Figure 5: The effects of mutations introduced into the C2 fragment on competition for binding to C2 or HB. Gel mobility shift assays were carried out as described in the text with nuclear extracts from JEG-3 and radiolabeled C2 or HB. In the designated lanes, the nuclear extract was incubated for 10 min with a 200-fold molar excess of the competitor before the addition of the probe.

As we found that this sequence requirement for C2 and C4 binding was not conflicting with that of TSE, which resides in the upstream region of human chorionic gonadotropin -subunit gene(23, 35, 36) , the 24-bp segment (-182/-159), designated as TSE in the promoter of the -subunit gene(37) , was synthesized and examined for competition with formation of the binding of C2, C4, and HB. As shown in Fig. 6A, TSE effectively inhibited formation of the binding complex of C2, C4 (lanes2 and 5), and the major complex with HB (lane9). The binding factor(s) for TSE is referred to as the TSE-binding protein (TSEB)(23) . Little is known about TSEB except its cell line-specific occurrence and that it cannot bind to a TSE homologue that has a C to T mutation at -172 (TSEµ-172)(37) . The failure of this mutated TSE to compete for the binding of C2, C4, and HB (lanes1, 6, and 10) further confirms that the binding activity to the C2 and C4 regions arises from the same factor with TSEB. As C2 and C4 are recognized by the same factor(s) TSEB, their core binding domains are designated as TSE-C 1 and 2, respectively. Interestingly, the minor complex formed with labeled HB was competed by both TSE and TSEµ-172 (indicated by an arrow) (lanes9 and 10). In panelB, three sequences are aligned to give a consensus sequence for TSEB recognition. The consensus sequence RNCCTNNRG also suffices requirement for the binding shown in Fig. 5. The observation that the C4 fragment with a mutation at -166 and -167 (CC to GG) failed to compete for the binding of C4 (data not shown) also supports this alignment.

Figure 6: Consensus sequence for recognition by TSEB. A, inhibition of specific binding to C2 and C4 fragments by TSE sequences. A gel mobility shift assay was carried out as described in the text with nuclear extracts from JEG-3. The arrowhead and the arrow indicate the position of the major and minor binding complexes, respectively. B, consensus sequence for recognition by TSEB. Underlining of the C2 and C4 sequences indicates the base pairs where introduction of mutation reduced the competitor activity of the fragment. The asterisk in the TSE sequence indicates the -172 C, where mutation to T abolished the binding activity.

In the last experiment transcriptional activity of this region was further examined (Fig. 7). The HB fragment that contains the TSE-C-2 and C3 enhanced transcription when linked to the tk promoter in both directions and placed downstream of tk. The full activity of pFBCAT was not retained when the fragment was reversely placed upstream or downstream of tk promoter. The C2 fragment only marginally activated transcription when single, double, or four copies of the 26-bp fragment were linked to the tk promoter. As the distal TSE core sequence did not enhance transcription by itself, transcriptional activity of a pFBCAT that has two G to T mutations at -283 and -282 (corresponding C2n-type mutation in Fig. 5) was examined. This mutation resulted in marked reduction of the transcription activity.

Figure 7: Transcriptional activity of the fragments containing a specific binding domain. Transfection of JEG-3 cells and subsequent assays for CAT activity were performed as described under ``Experimental Procedures.'' The relative positions of the HSV tk promoter and various fragments are shown on the left of the graph. Transfection efficiency was standardized by cotransfection of luciferase expression vector pSV/Luci. Cell extracts containing equal units of luciferase were subjected to CAT assay. Each data point represents relative activity ± S.E. (relative to the activity of the pFBCAT2 construct, arbitrarily set at 100%) calculated from the result of at least four independent transfections.

DISCUSSION

In the present study, we have shown that DNA sequences within the initial -301 upstream of placenta-specific exon I (exon Ia) are sufficient for basal transcription of placental type aromatase mRNA. Transcription activity directed by this promoter/regulator is cell type specific. Further deletion resulted in gradual loss of the transcriptional activity, suggesting involvement of multiple regulatory elements as shown in other systems(38) . The fragment encompassing the distal half of the region has an activity to enhance transcription driven by a heterologous tk promoter, which was also cell type specific.

Gel mobility shift assay demonstrated three distinct binding domains in this region. Two separate binding domains at -300/-274 (C2) and -177/-153 (C4) form a major complex. The mutual trans-acting factor for C2 and C4 was further found to be the same factor that recognizes the TSE, which resides in the upstream region of the human glycoprotein hormone -subunit gene (Fig. 6)(23, 35, 36, 37) . In human, this gene is expressed in the pituitary as subunits of glycoprotein hormones as well as -subunit of chorionic gonadotropin in placenta. The placental expression of this gene has been extensively studied; it requires a multi-component enhancer composed of tandem cAMP-responsive elements and an adjacent upstream regulatory element. TSE is described as a subdomain of upstream regulatory element and is considered to be a regulatory element that confers tissue-specific expression. Although little is known about its binding protein, TSEB(23, 37) , the cytosine at -172 in TSE is critical for recognition of TSE sequence by TSEB. The observation that TSE with a C to T substitution at -172 also failed to compete for binding to C2 or C4 gives support to the conclusion that TSEB is the factor. The two new TSE-like sequences in the aromatase promoter and the competition analysis of their homologues with sequentially introduced mutations now reveal the sequence required for TSEB recognition, namely, the core sequence of TSE (TSE-C) as RNCCTNNRG.

In the -subunit promoter, TSE is associated with another element, ACT, adjacent to two tandemly repeated cAMPresponsive elements to compose a tissue-specific enhancer. TSE in the -subunit promoter has little independent transcriptional activity when linked to homologous or heterologous promoters, and only when linked to the cAMP-responsive elements does it stimulate basal and cAMP-dependent expression. Likewise, the trophoblast-specific enhancer in the aromatase gene may be composed of multiple cis-elements including two TSE-Cs, although neither of them is associated with cAMP-responsive element or other known cis-elements. The 27-bp DNA fragment containing TSE-C1 was inactive to stimulate tk-driven transcription by itself even when two or four tandem copies were introduced. The binding to TSE-C1, however, seems to make an indispensable part of the aromatase enhancer. A pFBCAT2 analogue that has a mutation which eliminates TSE-C-1 binding loses most of the transcriptional activity that the parent pFBCAT2 shows. At present we do not have the direct evidence of the contribution of the proximal TSE-C or the C3 region to the enhancer activity. The two TSE-Cs seem to be equivalent in terms of recognition by TSEB, but their function may be different because of their position. Indeed, as shown in Fig. 7, the transcriptional activities of FB and HB fragments seem to be affected by their orientation and position in the CAT constructs.

Importance of the region between -242 and -183, which contains the C3 domain, was shown by Toda et al.(39, 40) in transient transfection assays in BeWo cells. They reported that the 89-bp fragment had an enhancer activity when multiple copies were placed with a heterologous promoter. Although the binding to the C3 domain was not competed by the two TSE-C sequences, the TSE derived from the -subunit gene competed for binding to the C3 domain, and the TSEµ -172 that failed to compete for TSE and TSE-Cs is competitive for binding to the C3 region. Pittmann et al.(37) recently described a new binding protein that recognizes the region partly overlapping TSE in the -subunit promoter. This trans-acting element binds to a distinct recognition sequence from that of TSEB and seems to be involved in placenta-specific expression of the -subunit gene as well. It is therefore of particular interest whether this second upstream regulatory element binding protein is also involved in the expression of placental aromatase.

In this study, we demonstrated that TSE-like elements are located in the promoter region of placenta-specific exon I of aromatase and that at least the distal TSE-like element is transcriptionally functional. TSE-like elements in aromatase gene seem to compose a placenta-specific enhancer with other factor(s), as shown in the -subunit gene, though their compositions seem to be quite different. The observation that the element recognized by TSEB, originally found in the -subunit gene, also functions in the promoter regions of unrelated genes to confer placenta specificity emphasizes its functional importance as a placenta-specific transcription element. Although the -subunit and aromatase genes are not closely linked in evolution, both are involved in producing hormones characteristic of the human placenta. Interestingly, the placental expressions of these genes are observed only in some mammals. Nevertheless, the fact that the two hormones are secreted from an early stage of pregnancy and play important roles in development and maintenance of the human placenta highlights the significance of a common trans-acting factor governing their expression. A potential TSE-C sequence is seen in the immediate upstream of human chorionic gonadotropin -subunit genes(41) , though their functional significance has not been tested.

Although various types of transcription factor have been found in trophoblasts so far, only a few are specific to this cell type(42) . Recently, transcription factors of the basic helix-loop-helix family that are important cell lineage determinants in many cell types have been identified with limited occurrence in extra-embryonic structures (43, 44) . At present, however, their immediate target genes have not been shown. TSEB is likely to be a immediate regulator of the genes in the later stage of placental differentiation. Purification of TSEB and cDNA cloning should clarify its identity as a trophoblast-specific transcription factor and provide the basis for further investigations of its roles and temporal and causal relationships with other factors that determine the differentiation to trophoblast lineage.

FOOTNOTES

*

This work was supported in part by a grant-in-aid for research from Fujita Health University and a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence and reprints requests should be addressed. Tel.: 81-562-93-9376; Fax: 81-562-93-8833; kyamada@fujita-hu.ac.jp.

()

The abbreviations used are: PCR, polymerase chain reaction; bp, base pair(s); CAT, chloramphenicol acetyltransferase; tk, thimidine kinase; TSE, trophoblast-specific element; TSEB, TSE-binding protein.

()

K. Yamada, N. Harada, S. Honda, and Y. Takagi, unpublished observation.

ACKNOWLEDGEMENTS

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