Karo Bio AB (T.B., S.N.), Department of Medical Nutrition (L.-A.H., J.-A.G.), Novum, S-141 57 Huddinge, Sweden
Address all correspondence and requests for reprints to: Tomas Barkhem, Karo Bio AB, Novum, S-141 57 Huddinge, Sweden. E-mail: tomas.barkhem{at}karobio.se.
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ABSTRACT |
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In the present study, we show estrogen-induced synergistic activity by the p160 coactivator steroid receptor coactivator-1 (SRC-1), mediated via the ERE and the AP1 response element in the pS2 promoter. In addition, we present data that support an interaction between the ERE and the AP1 motif via SRC-1. The related but distinct p160 coactivator, transcriptional intermediary factor-2, was a more potent activator of pS2 gene expression. In addition, transcriptional intermediary factor-2 was less dependent on an intact AP1 response element in the pS2 promoter than SRC-1.
Furthermore, the type of ERE in the pS2 promoter influenced the potentiation by SRC-1, supported by less dependence on the AP1 motif when the natural ERE was substituted for by a consensus ERE.
These results highlight several mechanisms whereby fine-tuning of estrogen responsiveness of an individual gene may be achieved.
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INTRODUCTION |
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The transcriptional response of estrogen target genes is mediated by a specific nuclear receptor, the estrogen receptor (ER). Presently, two subtypes have been described, ER (5, 6) and ERß (7), which appear to have distinct physiological roles and tissue distribution (8).
The ERs are structurally organized in functional domains. These include a DNA binding domain (9, 10), located between the N-terminal region and the C-terminal region, the latter of which has been shown to be organized into -helices forming the ligand binding domain (LBD) (11). ER
harbors two activation domains, activation function (AF)-1 in the N-terminal region of the receptor and AF-2 in the C-terminal region (12).
The transcriptional activity of AF-1 is less well understood, whereas a significant amount of knowledge has emerged as to how AF-2 transactivates target genes. When activated through ligand binding, the ERs bind to specific target sequences on DNA, referred to as estrogen response elements (ERE) (13). In concert with associated factors, termed coactivators, the ERs stimulate the transcriptional activity of target genes. Subsequent to ligand binding, the ER LBD is structurally remodeled so that the AF-2 core within helix 12 together with helices 35 form a hydrophobic cleft that serves as an interaction surface for coactivators (11, 14). Among the best-studied components of coactivator complexes that have been demonstrated to interact with the ERs (15, 16) are the p160 proteins, which are thought to be specific for nuclear receptors (16).
The p160s are divided into three distinct but related family members, which include steroid receptor coactivator-1 (SRC-1)/nuclear coactivator 1 (NCoA-1), transcription intermediary factor-2 (TIF-2)/glucocorticoid receptor interacting protein (GRIP)-1/nuclear coactivator 2 (NCoA-2) and p300/CBP cointegrator-associated protein (pCIP)/activator of thyroid and retinoic acid receptors (ACTR)/amplified in breast cancer 1 (AIB1). They all contain, in their central region, multiple copies of the signature motif LxxLL [nuclear receptor (NR) box] (17). Through these motifs, the p160 proteins interact with the hydrophobic patch within AF-2 of the ER, thus tethering the coactivator complex to the receptor (14).
In addition to the structural studies that have revealed similar ligand-induced conformational changes in the LBD of several nuclear receptors, enabling coactivator interaction, numerous functional studies have been performed to understand how nuclear receptors transactivate target genes. The majority of these mechanistic studies have been done with artificial promoters that basically consist of a hormone response element and core sequences for the general transcription machinery. However, natural promoters, whose activity is controlled by nuclear receptors, are usually considerably more complex. These promoters usually harbor recognition sequences for multiple transcription factors. Interactions between the nuclear receptor and other transcription factors result in transcriptional synergism, which may be a prerequisite for responsiveness to the nuclear receptor and its cognate hormone (18, 19, 20).
We have previously reported that the endogenous pS2 gene is estrogen responsive in ER expressing hepatocarcinoma (HepG2) cells (21). Using pS2 gene promoter constructs, the effect of estrogen was found to be dependent on a transcriptional synergy between the ERE and an adjacent AP1 response element in the pS2 promoter (22). Moreover, the potentiation of the pS2 promoter activity through the AP1 motif was dependent on the LBD of ER
. Our data suggested an interaction, direct or indirect, between factors associated with the respective response element.
The p160 coactivator SRC-1 constitutes a potential bridging factor between the ERE and the AP1 response element through its competence to interact via its NR box with the AF-2 of ER and via its C-terminal region (23) with the factors forming the AP1 complex (24).
In this report, we have investigated the role of p160 coactivators in the estrogen-induced activation process of the pS2 promoter. We show strong transcriptional synergy on the pS2 promoter mediated by SRC-1 or the related p160 coactivator TIF-2 via the ERE and the AP1 response element. In addition, data are presented that further support an interaction between the ERE and the AP1 motif via SRC-1. Furthermore, we show qualitative and functional differences between SRC-1 and TIF-2. Finally, we report that the type of ERE modulates the activity of SRC-1 in the context of the pS2 promoter.
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RESULTS |
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We have preferred to use the synthetic estrogen analog moxestrol rather than estradiol (E2) in the hormone induction experiments because moxestrol appeared to be more stable than E2 in liver cells. Moxestrol exhibited the same characteristics as E2 regarding induction of the pS2 gene in the HepG2 cells except for an approximately 10-fold leftward shift in the EC50 value relative to E2 (data not shown).
We next evaluated whether the distance between the ERE and AP1 response elements and also whether their relative phase on the DNA-helix influenced the transcriptional activity of the pS2 gene in response to estrogen. The distance between the response elements was decreased by 15, 20, or 25 nucleotides, respectively (Fig. 2A). The helical structure of DNA repeats after approximately ten residues. By decreasing the distance between the response elements using intervals of five nucleotides, the relative phasing of the ERE and the AP1 motifs on the DNA-helix was either preserved [pS220-alkaline phosphatase (ALP)] or altered (pS215-ALP and pS225-ALP). The transcriptional activity of the pS215-ALP reporter, in which the relative phase position of the response element on the DNA helix was altered, was decreased to approximately 30% of the wild-type (wt) promoter (pS2-ALP) (Fig. 2B
). The pS220-ALP, in which the relative phase of the ERE and the AP1 response element was preserved, showed a significantly increased activity. The pS225-ALP displayed a somewhat decreased transcriptional activity as compared with the pS2-ALP reporter but a significantly higher activity compared with the pS215-ALP. The decreased distance between the ERE and the AP1 motif thus appears to compensate for the change of their relative phase position on the DNA helix in the pS225-ALP construct. In conclusion, the transcriptional activity of the pS2 promoter was dependent not only on the distance between the ERE and the AP1 motif but also on their relative phase on the DNA helix. This supports the notion that an interaction between factors at the respective response element occurs.
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The Transcriptional Activity of the pS2 Promoter Is Inhibited by the Isolated C-Terminus of SRC-1
We postulated that SRC-1 may act as a bridging factor between the ERE and the AP1 motif of the pS2 promoter, interacting via its C-terminal domain with AP1 and its central NR box containing domain with the AF-2 of ER.
To further test this concept, we overexpressed the C-terminal fragment encoding amino acids 1101 to 1441 of SRC-1 (SRC11011441), which has been reported to interact in vitro with either c-jun or c-fos (23). We expressed the SRC11011441 fragment as a fusion protein with the DNA binding domain of the yeast protein Gal4, to ensure nuclear translocation of the chimeric protein. Intact or AP1 mutated pS2 promoter-reporter was cotransfected with increasing amounts of the vector expressing SRC11011441. The responses were normalized to the activity of a CMV-luciferase (Luc) reporter. We found that the estrogen-induced activity of the pS2 promoter was suppressed in a dose-dependent fashion when the SRC11011441 construct was overexpressed (Fig. 5), whereas the basal activity was not affected (data not shown). Interestingly, the intact pS2 promoter-reporter was significantly more sensitive than the AP1-mutated pS2 promoter variant (mutAP1) to suppression by SRC11011441 (Fig. 5
). Furthermore, the SRC-1 potentiated pS2 promoter that lacks a functional ERE was more sensitive to overexpression of the dominant negative SRC11011441 fragment than SRC-1 potentiated pS2 promoter in which the AP1 response element was mutated (data not shown). These results suggest that the intact pS2 promoter is destabilized by SRC11011441, presumably competing with endogenous full-length p160 coactivator, e.g. SRC-1, for binding to the AP1 complex.
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The Type of ERE in the pS2 Promoter Determines the Response to SRC-1
We next examined the impact of two different EREs on the potentiation by SRC-1 of estrogen-induced pS2 promoter activity. Recent data suggest that different types of EREs may induce distinct conformational changes of the ER structure that could influence receptor-coactivator interaction (25, 26).
The natural ERE of the pS2 promoter binds ER with low affinity as compared with the ERE derived from the vitellogenin promoter (27), which is regarded as the consensus ERE (EREvit) (13). The presence of EREvit in the context of the pS2 promoter results in approximately 5-fold higher estrogen-induced reporter gene expression as compared with estrogen-induced reporter gene activity from the wt pS2 promoter-reporter (22). We evaluated the effect of SRC-1 on intact and AP1-mutated pS2 promoter-reporter constructs where the natural ERE had been substituted for by the EREvit [pS2(EREvit)-ALP or pS2(EREvit) mutAP1] (Fig. 7A
). The effect of SRC-1 was expressed as the potentiation of reporter protein expression from the respective reporter constructs relative to reporter protein expression in the presence of estrogen only. The responses evoked by SRC-1 on the EREvit containing pS2 promoter variants were compared with responses of SRC-1 on the wt pS2 promoter (pS2-ALP) as well as pS2 promoter containing its natural ERE but lacking a functional AP1 response element (mutAP1). The presence of a functional AP1 element has been shown to potentiate also the activity of the pS2 promoter that was substituted with the EREvit in the absence of exogenous SRC-1 (22). However, in contrast to the wt pS2 promoter, the pS2 promoter-reporters containing the EREvit showed a similar relative potentiation in response to overexpression of SRC-1 either in the presence or absence of an intact AP1 motif (Fig. 7B
). Thus, the type of ERE, in the context of the pS2 promoter, modulated the activity of the coactivator SRC-1. However, in contrast to SRC-1, TIF-2 showed similar effects on the relative potentiation of the pS2 promoter activity regardless of the type of ERE present in the promoter (Fig. 6B
; data not shown).
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DISCUSSION |
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In the present study, we observed that the transcriptional response of the pS2 gene was modulated by the distance and the relative phasing of the ERE and the AP1 motif on the DNA helix (Fig. 2B), supporting a direct or indirect interaction between factors bound to the respective response elements. Thus, decreasing the distance between the ERE and the AP1 motif may facilitate an interaction between proteins that results in increased transcription, whereas changing the relative phase position of the response elements on the DNA helix may disturb the interaction and hamper transcription.
Overexpression of SRC-1 resulted in a synergistic estrogen-induced response of the intact pS2 promoter as compared with overexpression of SRC-1 in cells transfected with pS2 promoter constructs with mutated ERE and/or AP1 motifs (Fig. 3, AD). Furthermore, as shown in Fig. 6A
, the potentiation by SRC-1 of the pS2 promoter was significantly less in terms of relative potentiation when the AP1 response element was mutated.
One potential link between the ER/ERE and AP1 and its response element could be SRC-1, which may interact via its NR-boxes with the AF-2 of ER
and via its C-terminal region with the AP1 complex. We overexpressed the C-terminal fragment of SRC-1 that has been reported to interact with either c-jun or c-fos in GST-pull-down experiments (23). The intact pS2 promoter was more sensitive to overexpression of the SRC11011441 fragment than the AP1-mutated promoter (Fig. 5
), possibly due to binding of the SRC11011441 fragment to the AP1 complex, preventing full-length SRC-1 to act as a bridging factor between the ERE and the AP1 motif. Also, the activity of AP1 mutated pS2 promoter was suppressed by overexpression of C-terminal SRC11011441 fragment, although significantly less than the intact pS2 promoter. The splice variant of SRC-1 (28) from which the sequence encoding the C-terminal SRC-1 fragment was derived contains an LxxLL motif. Thus, overexpression of the LxxLL motif within the SRC11011441 fragment may disturb an interaction between the AF-2 of ER
and the NR boxes in the central region of full-length SRC-1, resulting in repression also of the AP1-mutated promoter.
There are opposing reports on a direct interaction between ER and AP1 (29, 30). Teyssier et al. (30) demonstrated an interaction between the hinge region of ER
and c-jun. Interestingly, this interaction was shown to stabilize a tripartite interaction with the p160 coactivator GRIP-1. We have overexpressed the hinge region of ER
either together with intact or AP1- mutated pS2 promoter. However, we were not able to demonstrate selective suppression of either promoter constructs (data not shown). Thus, we have no indication that a direct interaction between the hinge domain of ER
and the AP1 complex contributes to the potentiation of pS2 promoter activity in the presence of an intact AP1 motif in the promoter.
The related p160 coactivator TIF-2 was a more potent enhancer of the pS2 promoter activity than SRC-1 (Figs. 3 and 4
). The potentiation of pS2 promoter activity in terms of the relative fold increase of reporter gene expression in the presence of TIF-2 was less dependent on the AP1 motif in the pS2 promoter as compared with SRC-1 (Fig. 6
, A and B). Indeed, TIF-2 had a greater relative impact on the AP1 mutated (mutAP1) than the intact pS2 promoter. These differences could be due to the fact that TIF-2 binds to ER
with a stronger affinity than SRC-1. Hence, TIF-2 might be less dependent on stabilizing interactions with nearby factors at the AP1 response element as compared with SRC-1. In fact, a recent report showed that GRIP-1, the mouse ortholog of TIF-2, interacted with higher affinity than SRC-1 with ER
bound to the natural ERE (pS2 ERE) of the pS2 promoter (26).
Interestingly, the type of ERE in the pS2 promoter influenced the degree of potentiation evoked by SRC-1. The pS2 promoter in which the natural ERE was substituted for by a high affinity consensus ERE (EREvit), showed an equal relative potentiation in response to SRC-1 irrespective of the presence or absence of an intact AP1 response element in the pS2 promoter (Fig. 7B). This contrasts to the influence of the AP1 motif in the wt pS2 promoter, which showed significantly decreased relative potentiation in the absence of a functional AP1 motif (Fig. 7B
). One potential explanation could be that allosteric modulation of ER conformation by the respective ERE influences the interaction with coactivator proteins. Previous experiments employing partial protease digestion of ER
bound to various types of EREs revealed distinct cleavage products depending on the individual ERE, suggesting that the response element has an effect on receptor conformation (27, 31). Interestingly, Hall et al. (26) showed that SRC-1 had a higher affinity for ER
bound to the EREvit than for ER
bound to the natural ERE of the pS2 promoter. It seems plausible that ER
bound to the natural ERE of the pS2 promoter recruits SRC-1 less effectively and therefore relies on additional stabilization of the p160 protein, i.e. via the AP1 motif. Furthermore, GRIP-1, the mouse ortholog of TIF-2, was shown to interact with ER
bound to either the EREvit or the pS2 ERE with a similar affinity (26). This is in agreement with our observation that irrespective of the type of ERE, TIF-2 showed similar effects on the relative potentiation of the pS2 promoter activity (Fig. 6B
; data not shown). Furthermore, coactivator binding to ER
has been shown to stabilize the ER
/ERE complex (25). Thus, it is tempting to speculate that an interaction between the AP1 complex and ER
via SRC-1 also stabilizes the ER
/ERE interaction in the pS2 promoter.
Among the estrogen-responsive genes presently known, very few contain consensus EREs. Instead the majority harbors imperfect EREs (32, 33) that bind ER with low affinity. These may induce a receptor conformation that has a reduced affinity for p160 coactivators (25, 26). One potential reason for this scenario from an evolutionary point of view could be that stabilization of the ER-coactivator interaction by adjacent transcription factors provides a mechanism allowing a more exact tuning of gene regulation, permitting additional signal transduction pathways that converge on other specific sequences in the promoter to influence the transcriptional activity mediated through the hormone response element. For instance, we have previously shown that the MAPK signal transduction pathway, which may converge on the AP1 motif in the pS2 promoter, contributes significantly to pS2 gene expression in response to estrogen (22).
Thus, the transcriptional rate of the pS2 promoter is influenced in a variety of ways (Fig. 8). We have shown that the promoter context, i.e. an intact AP1 response element adjacent to the ERE in the pS2 promoter, had a significant effect on the potentiation of the pS2 gene expression by the p160 coactivator SRC-1. However, also the type of p160 had a clear effect on the transcriptional rate, TIF-2 being a significantly more potent activator of transcription than SRC-1 in the context of the pS2 promoter. Furthermore, TIF-2 showed less dependence than SRC-1 on the presence of an intact AP1 response element to mediate transcription via the ERE of the pS2 promoter.
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MATERIALS AND METHODS |
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Plasmids
The pS2-ALP vector consists of a 1100 nucleotide pS2 promoter fragment fused to a secreted alkaline phosphatase reporter gene (ALP) (34) (Fig. 1A). The mutERE, the mutAP1, or the mutERE/mutAP1 reporters are identical to the pS2-ALP, except that the ERE (mutERE), the AP1 motif (mutAP1) or both motifs have been substituted for by nonsense nucleotides (Fig. 1A
). These constructs have previously been described (22).
In the pS2(EREvit)-ALP vector, the natural ERE of the pS2 promoter has been replaced by the consensus ERE derived from the vitellogenin gene (EREvit) (22). The pS2(EREvit) mutAP1 contains the EREvit, whereas the AP1 motif has been mutated into nonsense nucleotides (22).
The pS215-ALP, pS220-ALP, and pS225-ALP were constructed by insertion of oligonucleotides into the SacI-PstI sites at -429 and -328 in the pS2 promoter. The sequences of the ERE and AP1 response elements and intervening sequences (upper strand) of the respective constructs are shown in Fig. 2A.
The HE0 vector that expresses ER mutated at amino acid 400 (Gly to Val) has been described previously (35).
Full-length cDNA encoding SRC-1 or TIF-2 was cloned into the CMV-5 expression vector. The vector expressing the Gal4-SRC11011441 was constructed as follows: the cDNA sequence encoding amino acids 11011441 of SRC-1 was PCR-amplified using the upstream primer; 5'-TCCGAATTCCCCCTGAATGCTCAAATGTTG-3' and the downstream primer; 5'-AATGTCGACTTATTCAGTCAGTAGCTGCTGA-3'. The amplified fragment was digested with EcoRI and SalI and cloned into the corresponding sites of the pM vector (CLONTECH Laboratories, Inc., Palo Alto, CA).
Cell Cultures
HepG2 cells were maintained in MEM containing 10% FCS, 1% nonessential amino acids, 1 µM sodium pyruvate, and 2 mM L-glutamine. The cells were cultured at 37 C in humidified chambers in 5% CO2.
Transient Tansfections
HepG2 cells were transiently transfected using the OptiMEM/lipofectin procedure essentially performed according to recommendations of manufacturer (Invitrogen). Cells (5 x 104) were seeded in 48-well plates in Coons/F12 medium supplemented with 1% FCS (double dextran charcoal stripped), 2 mM L-glutamine, and 50 µg/ml gentamicin. Twenty-four hours after seeding, the cells were transfected for 5 h with 200 ng of the different pS2 promoter ALP reporter constructs, 50 ng HE0, and various amounts (exact amounts are given in the figure legends) of expression vector for the different coactivators or fragment of coactivator. The cells were then rinsed once and induced with 10-9 M moxestrol in Coons/F12 medium. In all transient transfection experiments, cells were exposed to hormone 60 h before being harvested and analyzed for reporter gene expression. In the suppression experiment with the SRC11011441 fragment, a CMV-Luc reporter was cotransfected for normalization of the ALP reporter gene expression (Fig. 5). All transient transfection experiments were performed in triplicates and repeated several times. The data presented in the figures are the mean of several independent experiments with the SEM indicated. The number of performed experiments is indicated in the respective figure legend.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: AF, Activation function; ALP, alkaline phosphatase; AP1, activator protein 1; CMV, cytomegalovirus; E2, estradiol; ER, estrogen receptor
; ERE, estrogen receptor
; EREvit, ERE derived from the vitellogenin promoter; FCS, fetal calf serum; GRIP, glucocorticoid receptor interacting protein; HepG2, hepatocarcinoma cells; LBD, ligand binding domain; Luc, luciferase; mutAP1, mutated AP1; mutERE, mutated ERE; NR, nuclear receptor; Pit-1, pituitary specific transcription factor; rGH, rat GH; rPRL, rat prolactin; SRC-1, steroid receptor coactivator-1; TIF-2, transcriptional intermediary factor-2; TR, thyroid hormone receptor; wt, wild-type.
Received for publication February 4, 2002. Accepted for publication July 23, 2002.
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REFERENCES |
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