(Received for publication, July 24, 1995; and in revised form, August 8, 1995)
From the
The aromatase (cytochrome P-450) gene contains
multiple untranslated exons I that are differentially transcribed in a
tissue-specific manner. DNA sequences within the initial -301
upstream of placenta-specific exon I (exon Ia) are sufficient for
placenta-specific expression of aromatase. In gel mobility shift assay,
three separate domains in this region form specific binding complexes
with proteins extracted from choriocarcinoma JEG-3 nuclei. A fragment
containing these domains activates transcription driven by a
heterologous promoter in a cell type-specific manner. Two of the
binding domains that form major complexes in gel shift assay compete
with each other and with a DNA fragment containing the
trophoblast-specific element (TSE), which is derived from the enhancer
region of the human chorionic gonadotropin
-subunit gene and is
believed to confer placenta-specific expression of the gene. The core
sequence RNCCTNNRG is sufficient for recognition of the TSE-binding
protein, which is detected only in nuclear extracts prepared from
placenta and choriocarcinoma. A mutation introduced in the distal TSE
core in aromatase promoter resulted in marked reduction of
transcriptional activity, although TSE region by itself did not show
enhancer activity as that in human chorionic gonadotropin
-subunit
gene.
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.
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.
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.
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.
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.
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.
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.
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-C
1 was inactive to stimulate
tk-driven transcription by itself even when two or four tandem copies
were introduced. The binding to TSE-C
1, 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-C
s 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.