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Biocenter Oulu and Department of Clinical Chemistry, University of Oulu, FIN-90220 Oulu, Finland
Address all correspondence and requests for reprints to: Dr. Hellevi Peltoketo, Biocenter Oulu and Department of Clinical Chemistry, University of Oulu, Kajaanintie 50, FIN-90220 Oulu, Finland. E-mail: hpeltoke{at}whoccr.oulu.fi
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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17HSD type 1 is encoded by the HSD17B1 gene (14, 15) (previously also called EDH17B2) localized to loci 17q12–21 (7, 16, 17). The gene contains 2 transcription start points, about 9 and 971 nucleotides upstream from the translation initiation codon, which result in 2 transcripts, 1.3 and 2.3 kb in size, respectively (14). The latter one, whose function remains unknown, is constitutively expressed in several tissues (7, 11, 14) and cell lines with a minor relationship to the presence of the 17HSD type 1 protein (11, 18). On the other hand, the 1.3-kb messenger RNA (mRNA) is expressed in cells producing 17HSD type 1 protein, and its amount largely correlates with the concentration of the protein (11, 18). It is also the 1.3-kb mRNA whose concentration is subject to regulation by growth factors (19, 20), retinoic acids (21), and cAMP (7, 22, 23) in choriocarcinoma cells.
In a recent study, we demonstrated that a cell-specific enhancer in the
region from -661 to -392 and a silencer element between the
nucleotides -392 and -78 in the HSD17B1 gene participate in the
regulation of its transcription. These regulatory areas are suggested
to affect the transcription of the 1.3-kb mRNA of 17HSD type 1. On the
other hand, the fragment -78/+9 is able to drive reporter gene
expression, acting as a basal promoter (24). The region from -78 to +9
contains motifs for binding several transcription factors, such as AP-2
and Sp1 (Fig. 1
). In addition, in the middle part of the
area, which has been suggested to be essential for the function of the
HSD17B1 silencer (24), a binding motif for GATA transcription factors
is located. In this study, we characterized the function of these
motifs in JEG-3 and JAR choriocarcinoma cell lines. JEG-3 cells are
able to synthesize progestins and estrogens from suitable substrates
and are thus commonly used for studying placental steroidogenesis.
17HSD type 1 is moderately expressed in JEG-3 cells, whereas its
expression in JAR cells is scant, but detectable (20). We noticed that
binding to the Sp1 motif is important for the promoter function,
whereas GATA proteins repress transcription derived from the 5'-region
of the HSD17B1 gene in choriocarcinoma cells. In addition, AP-2 seems
to prevent binding to the Sp1 motif and thus also to decrease the
transcription.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-35S]deoxy-ATP (3000 Ci/mmol),
[-32P] deoxy-CTP (3000 Ci/mmol), and
[3H-]acetyl coenzyme A (200 mCi/mmol chloramphenicol
acetyl transferase (CAT) (assay grade) were purchased from Amersham
Life Science (Little Chalfont, UK) and Du Pont NEN (Boston, MA). The
transfection reagent N-[(1-(2, 3-dioleoyloxy)propyl)]-N and
N,-trimethyl-ammoniummethylsulfate (DOTAP) were products of Boehringer
Mannheim (Mannheim, Germany). The antibodies against the transcription
factors GATA-2, GATA-3, AP-2, Sp1, Sp2, Sp3, and Sp4 and the
oligonucleotides for the GATA, AP-2, and Sp1-binding sites were
obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), and the
AP-2 protein and HeLa nuclear extracts and the ß-galactosidase enzyme
assay kit were from Promega Co. (Madison, WI). All the media, buffers,
supplements, and reagents for cell culture were purchased from Gibco
BRL-Life Technologics (Grand Island, NY) and Sigma Chemical Co. (St.
Louis, MO). Other reagents not mentioned in the text were obtained from
the Sigma Chemical Co., Boehringer Mannheim, New England Biolabs
(Beverly, MA) and Merck A.G. (Darmstadt, Germany).
Preparation of plasmid constructs for reporter gene analyses
The nested deletion constructs, pCAT-EY-349, pCAT-EY-228,
pCAT-EY-113, pCAT-EY-97, and pCAT-EY-78, were generated from the parent
plasmid pCAT-EY-859 as described earlier (24), using the procedure
developed by Henikoff (25) or the PCR. The overlap-extension technique
described by Ho et al. (26) was used to introduce mutations
into the AP-2 site
(5'-GCCCGCAGGC-3'
5'-GCTTGCAGGC-3') and the Sp1
site (5'-GGGGCGGGGC-3'5'-GGGGCTTGGC-3') in
fragment -97/+9 to obtain the mutants (mAP-2)-97 and (mSp1)-97. The
mutations into the GATA-binding site of fragment -113/+9
(5'-TTATCC-3'5'-TTAAGC-3' and 5'-TATTCC-3')
were included directly in the 5'-end primer of the PCR reaction to
achieve the mutated fragments (m1GATA)-113 and (m2GATA)-113,
respectively. The mutants were then inserted into the pCAT-EY vector,
and the resulting constructs are called p(mAP-2)-97, p(mSp1)-97,
p(m1GATA)-113, and p(m2GATA)-113. All of the plasmid constructs, both
wild-type and mutated ones, were verified by sequencing, and at least
two individual preparations of each plasmid were mixed together for
transfection experiments.
Cell culture, transient transfection, and analysis of reporter gene
expression
The human choriocarcinoma cell lines JEG-3 and JAR were obtained
from the American Type Culture Collection (Rochville, MA) and were
maintained according to the instructions of the supplier. For reporter
gene analyses, both JEG-3 and JAR cells were plated onto 60-mm dishes
(8.5 x 105 cells) 20–24 h before transfection. Then
6.0 µg of each construct were transfected into JEG-3 cells, whereas
4.0 µg of each plus 2.0 µg of the ß-galactosidase control vector
pCMVß (Clontech Laboratories Inc, Palo Alto, CA) were transfected
into JAR cells using the transfection reagent DOTAP (6.0 µg/ml).
After 20 h, the media were replaced, and the cells were cultured
for a further 52 h before collection. The harvested cells were
then subjected to four freeze-thaw cycles and, further, to heat
inactivation at 65 C for 20 min, after which the CAT activity of the
samples was measured by fluor diffusion assay (27, 28). The
ß-galactosidase activities were used to normalize the transfection
efficiency of JAR cells. CAT expression was assessed by comparing of
the CAT activity in the samples with a CAT standard curve, and the
final values were calculated as picograms of CAT protein per milligram
of total protein. All the transient transfection experiments were
performed in duplicate, each series was repeated independently two to
three times, and the CAT activity of each sample was measured
twice.
Electrophoretic mobility shift assays (EMSAs)
For the EMSAs, nuclear extracts from cultured cells were
prepared by the method of Dignam et al. (29), as described
by Ausubel et al. (30), and oligonucleotides were labeled by
a filling-in reaction using [-32P] deoxy-CTP and
Klenow fragments of DNA polymerase I. The EMSAs were performed as
described previously (24), with some modifications. Briefly, 4.0–8.0
µg of nuclear extract was first incubated at RT for 10 min in 20 µl
of binding buffer containing 10% glycerol, 20 mM HEPES (PH
7.9), 50 mM NaCl, 5 mM MgCl2, 0.1
mM EDTA, 1 mM dithiothreitol, 0.1
mM phenylmethylsulfonylfluoride, 0.05% NP-40, and 2.0 µg
poly(dI-dC). Then 0.25–0.5 ng of a probe was added to the reaction
mixture, and the incubation was further continued at RT for 20 min. For
the competition experiments, a 100-fold excess of the competitor was
added together with the probe. Unrelated DNA, ßRARE01 fragments (31),
were applied to some reactions to investigate the specificity of
binding. For the supershift assays, 100 ng of appropriate antibody was
added to the reaction mixture subsequent to the addition of a
32P-labeled probe, and the reaction was continued at 4 C
for 60 min. The sequence and position of each oligonucleotide used are
illustrated in Table 1.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The shortest fragment, -78/+9, behaved
similarly to fragment -97/+9, suggesting that the region from -113 to
-97 may contain a negative control element(s). In JAR cells, on the
other hand, the fragments -113/+9, -97/+9, and -78/+9 promoted
similar reporter gene expression, and extending the fragment upstream
to -228 and -349 decreased transcription.
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). To test the capability of these putative silencer areas
of the HSD17B1 gene to bind nuclear proteins, especially GATA proteins,
a labeled fragment -114/-77, called HSD-GATA, was incubated together
with the JEG-3 nuclear extract. As shown in Fig. 3A, HSD-GATA formed two specific DNA-protein complexes with the JEG-3
nuclear extract, and they are described as complex 1 and complex 2 in
Fig. 3, A and B. The formation of these two complexes was inhibited by
unlabeled HSD-GATA and GATA consensus oligonucleotide, but not by
unrelated DNAs, a fragment -69/-36 called HSD-AP-2/Sp1, or ßRARE01
(for complete sequences, see Table 1). As a consequence of mutations in
the GATA motif (5'-TTATCC-3'5'-TTAAGC-3' and
5'-TTATCC-3'5'-TATTCC-3'), HSD-m1GATA and HSD-m2GATA were
not able to prevent the formation of the two complexes either, pointing
to involvement of the GATA site in the formation of the complexes (Fig. 3A).
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). In addition to the JEG-3 nuclear extract, HSD-GATA formed similar
DNA-protein complexes (complexes 1 and 2) with nuclear extracts
prepared from JAR and T47D cells, and the Supershift 1 complex was
generated when anti-GATA-3 was present in the binding reaction. Adding
antibody against GATA-2 to the binding reaction created a weak complex
(Supershift 2), which was detected in the sample containing a nuclear
extract from JEG-3 cells. Although Supershift 2 was not observed with
JAR nuclear extract, probably because of low concentration of GATA-2 in
JAR cells, the intensity of complex 2 was reduced to some extent by the
addition of anti-GATA-2. The nuclear extract from BT-20 cells resulted
in two DNA-protein complexes with HSD-GATA fragments, but they differed
from the ones formed with nuclear extracts from JEG-3, JAR, and T47D
cells. Furthermore, they were not supershifted by anti-GATA-2 or
anti-GATA-3 (Fig. 3B).
Functional analysis of the GATA motif in the HSD17B1 gene by
reporter gene analysis
The role of the GATA motif in the function of the HSD17B1 promoter
was further investigated by reporter gene analysis, in which intact and
mutated -113/+9 fragments were analyzed. In JEG-3 cells, two GATA
mutation constructs, p(m1GATA)-113
(5'-TTATCC-3'5'-TTAAGC-3') and p(m2GATA)-113
(5'-TTATCC-3'5'-TATTCC-3'), resulted in increased CAT
expression, about 170% and 160%, compared with that generated by
intact pCAT-EY-113 (Fig. 4). In fact, the mutation in
the constructs raised reporter gene expression to the level driven by
the shorter fragment -78/+9. The result confirmed that the reduced
promoter activity of fragment -113/+9 compared with fragment -78/+9
is caused by the binding of GATA proteins or related factors to the
GATA-binding motif at a position from -102 to -98. In addition, the
results verified that the difference between the activities of the
fragments -113/+9 and -78/+9 is not a result of dissimilar distances
between the SV40 enhancer and the HSD17B1 promoter in the corresponding
constructs. In JAR cells, mutations of the GATA response element only
slightly raised the promoter activity of fragment -113/+9 (Fig. 4),
which was in line with the fact that there was no notable difference in
the promoter activities of the fragments -113/+9, -97/+9, or -78/+9
(Fig. 2).
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). As shown in Fig. 5A, the 32P-labeled
fragment, termed HSD-AP-2/Sp1, covering the region from -69 to -36 in
the HSD17B1 gene, formed a pronounced complex (complex 3) with the
JEG-3, JAR, and T47D nuclear extracts and the AP-2 extract. The HeLa
extract formed with the fragment a major complex (complex 4) and two
weak complexes, one of which drifted similarly to complex 3, whereas
the other was termed complex 5. Mutation of the AP-2 consensus site in
the fragment resulted in a disappearance of complex 3 and in a
strengthening of the complexes 4 and 5 in all the nuclear
extract tested, including JEG-3, JAR, and T47D (Fig. 5A, lanes 6–9). Instead, mutation of the Sp1 consensus binding site
inhibited or reduced remarkably the formation of the complexes 4 and 5
(Fig. 5A, lanes 11–14). To sum up, the AP-2 motif was likely to be
involved in the formation of complex 3, and the Sp1 motif in the
formation of the complexes 4 and 5.
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, lanes 4 and 6), whereas the fragment with the mutated AP-2
motif was not cabable of such reduction (Fig. 5B, lane 5). The
formation of the complexes 4 and 5 was correspondingly inhibited by
HSD-Sp1 and Sp1 consensus fragments (Fig. 5B, lanes 7 and 9) but not by
the mutated form of HSD-Sp1, HSD-mSp1 (lane 8). The formation of
complex 4 was not clearly detectable when binding to the AP-2 motif was
effective, but again, mutation of the AP-2 site led to enhanced
formation of complex 4, as well as complex 5 (Fig. 5B, lane 12).
Unlabeled HSD-AP-2/Sp1 abolished the formation of all complexes,
whereas unrelated DNA, HSD-GATA, did not affect the formation of any of
the complexes, showing the specificity of the binding reactions (Fig. 5B, lanes 3 and 10). Finally, antibodies against AP-2 were used in
EMSAs to confirm the binding of the AP-2 factor to its motif in the
HSD17B1 promoter. Complex 3, formed from both JAR and JEG-3 nuclear
extracts, was supershifted by anti-AP-2 (Supershift 3), whereas the
formation of the complex was not influenced by anti-GATA-3 used as a
negative control (Fig. 5C).
Because it was obvious that the binding of AP-2 to its motif prevented
the binding of factors to the Sp1-motif (Fig. 5, A and B), a HSD17B1
gene fragment from -69 to -36 with a mutated AP-2 motif
(HSDmAP-2/Sp1) was further incubated with the nuclear extracts JAR,
JEG-3, and HeLa (Fig. 6, lanes 2, 7–9). As a result,
three complexes, marked 4a, 4b, and 5, could be detected in a
high-resolution gel. The formation of all the three complexes was
prevented by the HSD-Sp1 fragment, as well as by the Sp1 consensus
fragment, but not by HSD-mSp1 or HSD-AP-2 (Fig. 6), the latter
demonstrating that none of the complexes formed were caused by the
binding of AP-2 to the mutated fragment.
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, lanes 10–12; Fig. 7), whereas
antibodies against Sp3 prevented the formation of the complexes 4b and
5 and/or slowed down their migration (Supershift 4b/5) (Fig. 7A).
Finally, Fig. 7B shows the binding reactions between the nonmutated
HSDAP-2/Sp1 fragment and the JAR, JEG-3, and HeLa nuclear extracts,
together with the Sp-factor antibodies. Altogether, the results
strongly suggest that both Sp1 and Sp3 can bind to the HSD17B1
promoter, especially in the absence of the AP-2 factor.
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). Mutation of the Sp1-binding site in fragment -97/+9
decreased CAT expression to 30% in JEG-3 cells and to 60% in JAR
cells, compared with that generated by the intact fragment. In
contrast, mutation of the AP-2-binding site in fragment -97/+9
increased promoter activity to 260% in JEG-3 cells, indicating that
transcription can be suppressed by AP-2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The HSD17B1 gene fragment from -78 to +9 is extensive enough to drive reporter gene expression in connection with either HSD17B1 or SV40 enhancer (24), and it contains consensus binding sites for transcription factors Sp1 and AP-2, for example. In the present study we showed that mutation of the Sp1 motif considerably decreased the promoter activity of fragment -97/+9 and that the intact motif was bound by two member of the Sp transcription factor family, Sp1 and Sp3, particularly when the adjacent AP-2 motif was mutated. Both Sp1 and Sp3 are widely distributed transcription factors that bind to the GC-rich Sp1 motif with identical affinities (33). Sp1 activates a wide array of cellular and viral promoters, and it can interact also with several regulatory factors and, consequently, mediate cell- and gene-specific effects on the target promoter (34, 35, 36). For instance, Sp1 is necessary for both basal and enhancer-mediated transcription of the human chorionic somatomammotropin gene (37), a gene which, similarly to HSD17B1, is expressed in JEG-3 cells and in placental syncytiotrophoblasts. Instead, Sp3 has been demonstrated to repress Sp1-mediated transcriptional activation by competing with Sp1 for their common binding site (38, 39, 40) and to be able to increase transcription in only a few cases (40, 41). It has been suggested that some genes have evolved the capacity both to be activated by Sp1 and to be repressed by Sp3, to control the levels of their gene expression more stringently (39). Our present results indicate that binding to the Sp1 motif is needed for full activation of the HSD17B1 promoter. Mutation of the motif prevented effectively the binding of both Sp1 and Sp3, which means that specific roles of Sp1 and Sp3 in controlling the transcription of the HSD17B1 gene and their possible mutual competition in the HSD17B1 gene regulation remain to be clarified.
Sp1 and Sp3 were able to bind their cognate sequence in the HSD17B1 promoter more efficiently when the adjoining AP-2 motif was mutated. The intact motif was bound by AP-2, or an immunologically and functionally similar protein, but in contrast to the Sp1-binding site, mutation of the AP-2 motif increased the transcriptional activity of the reporter gene construct. The results hence suggest that the binding of AP-2 decreases the HSD17B1 promoter activity, which is, at least partially, caused by the prevention of binding of the Sp factor(s). The AP-2 factor is activated by several signaling pathways, such as protein kinase C and cAMP (42), but no further activation/inactivation of the factor apparently is needed for binding of AP-2 to the HSD17B1 promoter, because neither phorbol 12-myristate 13-acetate nor cAMP treatment significantly affected transcriptional activity of the promoter in JEG-3 and JAR cells (data not shown). Therefore, the AP-2 site at the position -62 to -53 may not be involved in mediating the effects of protein kinase C and cAMP pathways on HSD17B1 gene expression, reported previously (7, 20, 21, 22, 23). It seemed that the formation of Sp complexes with intact HSD17B1 promoter was scant in JEG-3, JAR, and T47D nuclear extracts, compared with that in HeLa cells containing a greater abundance of the Sp1 factor in respect to the AP-2 factor. Low amount of Sp factor(s), compared with AP-2 concentration, might explain why fragment -78/+9 alone led to only low reporter gene expression in these cell lines (24).
We previously showed that the promoter fragments -113/+9 and -78/+9, when connected with either HSD17B1 or SV40 enhancer, drove reporter gene expression. Interestingly, however, the promoter activity of the shorter fragment was significantly greater than that of the longer one in JEG-3 cells, as well as in PC-3 prostate cancer and CV-1 kidney cells (24). In the present study, we demonstrated that the GATA-binding motif, which is present in the region between -113 and -78, was able to bind transcription factors GATA-2, and in particular, GATA-3 in JEG-3 cells. Mutation of the motif decreased binding of the GATA proteins and led to increased promoter activity. This suggests that GATA-2 and GATA-3 can repress function of the HSD17B1 gene, at least in the constructs used, and the motif may thus be an essential part of the silencer localized previously (24). In JAR cells, the GATA complexes were formed less than in JEG-3 cells, and deletion or mutations of the GATA site generated only minor increases of promoter activity, which may be caused by low expression of GATA proteins in this cell line.
The GATA family currently comprises six members, GATA-1 to GATA-6,
which recognize the GATA motif WGATAR (see 43 . GATA-1 is mainly
present in erythroid cells, in which it regulates erythroid-specific
gene expression (reviewed in Refs. 43 and 44), whereas other members of
the GATA family are more widely distributed (see 43 . In placental
trophoblasts, GATA-2 and GATA-3 are expressed and are required to
direct trophoblast-specific expression of gonadotropin -subunit and
placental lactogen I genes (32, 45), for example, and it has been
suggested that these transcription factors may play a crucial role in
trophoblast cell differentiation (45). GATA factors are thus important
regulators of placental gene expression. Our present results suggest
that GATA factors may limit the function of the HSD17B1 gene in
trophoblast like cells, thus increasing the number of target genes
under the control of the GATA family in placental tissue.
Altogether, we have identified three binding motifs in the promoter of the HSD17B1 gene that are specifically bound by nuclear factors recognized by antibodies against Sp1, Sp3, AP-2, GATA-2, and GATA-3. Binding to Sp1 motif resulted in increased gene transcription, whereas AP-2 and GATA proteins had an opposite effect. These findings point to complicated regulation of HSD17B1 gene expression, in which a cell-specific enhancer containing several interacting subunits (24), a silencer element with GATA motif, a proximal promoter region with competing Sp1 and AP-2 sites, and possibly still unidentified regions, participate. Mutual interactions of the factors binding to these elements, their concentrations, and activation/inactivation of them finally control the tissue specific expression of HSD17B1 gene.
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Acknowledgments |
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Footnotes |
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Received January 16, 1997.
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References |
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