Human Molecular Genetics, 2002, Vol. 11, No. 7 723-731
© 2002 Oxford University Press
Oestrogenic repression of human coagulation factor VII expression mediated through an oestrogen response element sequence motif in the promoter region
Division of Genomic Medicine, Floor M, Royal Hallamshire Hospital, Glossop Road, University of Sheffield, Sheffield S10 2JF, UK and 1Department of Vascular Pharmacology and Medicine, Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, Santa Maria Imbaro, 66030, Italy
Received October 9, 2001; Revised and Accepted January 15, 2002.
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ABSTRACT |
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Reporter gene analysis of two regions of the human factor VII (FVII) gene promoter (residues –658 to –1 and –348 to –1, where +1 is the start site of translation) in the mammalian liver-derived cell line HepG2 showed reduced transcriptional activity in the presence of oestrogenic factors. This effect was independent of promoter polymorphic haplotype. Similar analysis using a smaller region of the promoter spanning residues –187 to –1 failed to show any evidence of oestrogenic suppression. Electrophoretic mobility shift assays and supershift assays using recombinant oestrogen receptor
and anti-oestrogen receptor antibody localized the sequence motif to which oestrogen receptor was binding to residues –225 to –212 of the FVII promoter. The lack of oestrogenic suppression in a reporter gene construct spanning residues –658 to –1 modified to abolish oestrogen receptor binding at this site, confirmed the functional significance of this motif. Although superficially similar to the classical oestrogen response element (ORE), comprising two half sites separated by three spacer nucleotides, the FVII ORE represents an alternative type of ORE in which the two half sites are separated by just two spacer nucleotides. EMSAs indicated that increasing spacer nucleotide number from two to three in the FVII ORE, or decreasing it from three to two in a consensus ORE sequence motif, had a small effect on the binding affinity for oestrogen receptor. These data correlate with and provide a plausible mechanism for the inverse relationship between FVII and oestradiol levels observed during the menstrual cycle. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Coagulation factor VII (FVII) is a zymogen of a vitamin K-dependent serine protease that plays an important role in blood coagulation. It is synthesized principally in the liver and secreted as an inactive single-chain glycoprotein of 406 amino acids (molecular weight
50 kDa) (1). The initial stages of coagulation are triggered by the interaction of activated FVII with tissue factor at a site of blood vessel injury. The gene encoding human FVII is 12.8 kb in length (2) and located in close proximity to the factor X gene locus on chromosome 13 (13q34-ter) (3). The FVII gene and protein structures show striking similarities to those for factor IX (FIX), factor X and protein C, other vitamin K dependent serine proteases which also play an important role in the pro-coagulant and anticoagulant processes (4). These similarities also extend to the promoter region with the finding that in common with FVII, these other three genes also lack a definite ‘TATA’ consensus sequence (5–8). Consistent with this finding, the FVII gene has been found to have multiple transcription initiation sites including two minor transcription start sites 13 and 18 bp upstream of the translation initiation site in addition to the major reported start site of transcription 51 bp upstream of this first amino acid codon (5,9). As in the FIX gene, the liver-specific transcription factor HNF4, a member of the nuclear receptor superfamily of transcription factors, would seem to play an important role in regulating FVII gene transcription (10,11). Indeed, patients with the congenital bleeding disorders caused by deficiencies of FVII or FIX (haemophilia B) have been identified with mutations in the characterized HNF4 binding sites located within the FVII and FIX gene promoters, respectively (12,13). The ubiquitous transcription factor SP1 has also been shown to be important in the regulation of the FVII promoter (14).
FVII plasma levels are known to be determined by both environmental (15,16) and genetic factors (17–19) and by their interplay (20–22). To date, six polymorphisms have been described in the proximal region of the FVII promoter: a decanucleotide insertion/deletion dimorphism at position –323 (0/10 bp), a T
C transition at position –122 (–122T/C), a GT transversion at position –401 (–401G/T), a GA transition at –402 (–402G/A), an AG transition at –630 and an AC transversion at residue –670 (5,23–25). The presence of the polymorphic 10 bp insertion at position –323 has been shown to correlate with lower plasma levels of both FVII clotting activity (FVII:C) and FVII antigen levels (FVII:Ag) (26,27). Furthermore, in vitro studies demonstrated a 33% reduction in promoter activity of the –323 allele with the decanucleotide insertion in comparison to the more common allelic sequence without the decanucleotide insertion (5). There is complete linkage disequilibrium between the –323 0/10 bp dimorphism and the polymorphisms at –122 and –401, which are also significantly associated with FVII:C and FVII:Ag levels (28). However, differential binding of two nuclear proteins according to –401 and –402 polymorphic genotype has recently been described and this locus may therefore be the important polymorphic determinant of transcriptional efficacy (24). A gender-dependent regulation of FVII levels, possibly involving steroid hormones, is suggested by the observation that both FVII:C and FVII:Ag are lower in pre-menopausal women than in males (29). The physiological importance of oestrogenic control of the FVII promoter is implied by the inverse relationship which exists between FVII antigen and oestradiol levels at different stages of the menstrual cycle in women (30). To determine whether there is any direct effect of oestrogens on FVII levels, the present study investigates whether they are capable of binding to and modulating the transcriptional activity of the FVII gene promoter.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Oestrogenic factors reduce the transcriptional activity of the FVII promoter
Two common allelic variants of the human FVII gene promoter have been described previously (28), classified according to polymorphic genotype at residues –122, –323 and –401 where residue +1 is the start site of translation. Previous work (5) has identified a region spanning residues –225 to –212 protected by DNAase I footprint analysis, which shows homology to a hormone response element (HRE). Closer inspection indicates that this sequence (5'-GGTCANNTGACC-3') closely resembles the canonical oestrogen response element (ORE) sequence (5'-GGTCANNNTGACC-3'), differing only by the number of spacer nucleotides (N) within the palindromic element (Fig. 1A) (31). Luciferase reporter gene constructs corresponding to the two common polymorphic haplotypes, –323(0 bp), –122(T), –401(G) (haplotype 1) and –323(10 bp), –122(C), –401(T) (haplotype 2), were made which contained this putative HRE within a 658 bp fragment of the 5' flanking region of the FVII gene (residues –658 to –1; Fig. 1A). The effect of oestrogenic factors on this region of the promoter was then monitored by transfection into the human liver-derived cell line, HepG2, in the presence or absence of 17ß-estradiol and an expression vector encoding the human oestrogen receptor (pSG5-HEO) (32). The results of 36 replicates, expressed as the fold activity relative to unstimulated construct, are shown in Figure 1B and demonstrate that reporter gene constructs corresponding to haplotypes 1 and 2 of the FVII promoter each show decreased levels of transcription in the presence of oestrogenic factors compared to those in their absence (P < 0.0001, haplotype 1; P < 0.0001, haplotype 2). Similar results were obtained when testing the effect of oestrogenic factors on reporter gene constructs spanning residues –348 to –1 (P < 0.0001, haplotype 1; P < 0.0001, haplotype 2; Fig. 1B). To test the effect of oestrogenic treatment in the absence of the putative ORE between residues –225 to –212, reporter gene constructs spanning residues –187 to –1, again corresponding to the common haplotypes 1 (–122T) and 2 (–122C), were made (Fig. 1A). Comparison of the promoter activity of each construct in the absence and presence of oestrogenic factors revealed no significant difference in either instance (P > 0.05; Fig. 1B). These data suggest that the fragment –188 to –348 of the FVII promoter is sufficient to confer oestrogen-mediated regulation of FVII gene expression.
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Oestrogen receptor binds to the FVII promoter
To determine whether the putative ORE site (residues –225 to –212) within the –188 to –348 region of the promoter is able to bind oestrogen receptor, electrophoretic mobility shift assays (EMSAs) were performed with double-stranded oligonucleotide probes corresponding to residues –204 to –233. Recombinant oestrogen receptor in either the presence or absence of HepG2 nuclear extract showed slightly lower levels of binding to the FVII promoter probe than to one corresponding to the consensus sequence for an ORE (Fig. 2A). Specificity of binding was confirmed by the absence of these specific bands when a non-specific oligonucleotide was used as probe. Supershift assays using an antibody to oestrogen receptor gave additional confirmation that oestrogen receptor binds to this region of the FVII promoter (Fig. 2B).
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Mutation of the ORE in the FVII promoter abolishes oestrogen receptor binding and oestrogenic repression
Gel mobility shift assays were carried out to compare the binding affinity of the double-stranded oligonucleotide probe containing a mutated FVII ORE site (5'-GGATC-CCCCGAACT-3'; mutated nucleotides highlighted in bold type; palindromic half sites underlined) to that obtained with the probe having the wild-type sequence (5'-GGGTCACCTGACCT-3'), both spanning residues –228 to –187 (Table 1). As shown in Figure 3A, the FVII ORE mutated in this fashion almost completely prevented oestrogen receptor binding to this site.
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To confirm the functional significance of this site, the wild-type FVII ORE sequence in the reporter plasmids pGL3-FVII -1/-658 h1 and pGL3-FVII -1/-658 h2 was altered to resemble the mutated FVII ORE motif in the gel shift probes used above. Analysis of the data from reporter gene assays in HepG2 cells showed that whilst wild-type constructs spanning residues –1 to –658 again showed evidence of oestrogenic repression, this effect was abolished in their mutated counterparts, with no significant difference in activity being observed in the presence and absence of oestrogenic factors (P > 0.05 in both haplotypes; Fig. 3B). As expected, a positive control reporter gene plasmid spanning residues –181 to +51 of the human factor XII (FXII) gene promoter, including a previously identified ORE spanning residues –43 to –31 (33), showed significantly enhanced transcriptional activity in the presence of oestrogenic factors compared to that seen in their absence (P < 0.0001; Fig. 3B). Hence, our data demonstrate the functional role of an ORE in the FVII gene promoter, which binds oestrogen receptor in a specific fashion, leading to the observed net negative effect of oestrogenic factors.
Half-site spacing and effect on binding of oestrogen receptor to its target sequence
It was previously noted that the –225 to –212 region of the FVII promoter shows close homology to the two half site sequences within a consensus ORE (31). However, this homology is only apparent assuming two, rather than the normal three spacer nucleotides between the palindromic half sites (Fig. 1A). Accordingly, EMSAs were carried out using probes corresponding to either the wild-type FVII ORE (containing two spacer nucleotides) or a ‘mutated’ form with the addition of an extra nucleotide between the two half sites, to determine the effect, if any, of spacer nucleotide number on binding affinity. Probes corresponding to the consensus ORE half sites separated by either two or three spacer nucleotides were analysed in a similar fashion. Our data indicates in both cases a slight reduction in binding affinity in the ORE containing two spacer nucleotides relative to that in the ORE with three, this effect being slightly more pronounced and reaching statistical significance in the case of the consensus ORE (P = 0.02, n = 5; see Fig. 4 for representative result).
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An additional protein factor is able to bind to the FVII ORE
Incubation of whole cell protein extracts from HepG2 cells with double stranded oligonucleotide probes spanning residues –228 to –187 of the FVII promoter corresponding to wild-type and mutated FVII ORE sequence indicated the formation of a DNA–protein complex whose intensity was reduced in the mutated FVII ORE sequence (Fig. 5B). Failure of this novel complex to either co-migrate with the DNA–protein complex formed with recombinant oestrogen receptor or to supershift with anti oestrogen receptor antibody, coupled with the more pronounced binding of both wild-type and mutated FVII ORE sequences in whole-cell extract than in nuclear extract (Fig. 5A) suggests that an alternative member of the nuclear and cytoplasmically located nuclear hormone receptor family may also be binding to the FVII ORE.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We present evidence supporting the hypothesis that oestrogenic factors repress the transcriptional activity of the FVII promoter by binding to a region encompassing residues –225 to –212. This effect is independent of promoter polymorphic haplotype.
Reports in the literature suggest that the promoters of two other genes, FIX and FXII, which participate in the pro-coagulant process are also regulated by steroid hormones (10,33). Indeed, it has been proposed that the clinical recovery seen in patients with the haemophilia B Leiden phenotype is due to the activation of an androgen response element at puberty, which partially compensates for the deleterious effect of a mutation in another transcription factor binding site in the FIX gene promoter of these patients (10). Similarly, the FXII promoter has been shown to be stimulated by oestrogenic factors, with the degree of oestrogenic stimulation being reduced in the presence of HNF4, another member of the nuclear hormone receptor superfamily (34). Analysis of the FXII promoter demonstrated the presence of nuclear factor binding sites in close proximity to the ORE whose sequences showed close homology to that of the consensus binding sequence for HNF4. The reduced binding of another protein, not oestrogen receptor, to the mutated FVII ORE sequence compared to the wild-type FVII ORE sequence strengthens the hypothesis that in addition to oestrogen receptor, another nuclear protein may also be capable of binding to this region of the FVII promoter. HNF4 is also known to be an important regulator of FVII expression (11). Indeed, homozygous disruption of a characterized HNF4 site between residues –63 and –58 (Fig. 1A) has been found in a severe FVII-deficient patient with a FVII level <1% of the normal value (12). It is noteworthy that inspection of the FVII ORE region (5'-TGACCTTTCTCCT-3'; 3' half site of FVII ORE underlined) with the Matinspector software analysis programme indicates that it too shows close homology on the antisense strand (12/13 match) to the HNF4 binding site (5'-TGAC/ACTTTGNCCC/T-3'; antisense strand) in the TRANSFAC database (35,36). Hence, by a similar mechanism it is plausible that oestrogen receptor is binding to the FVII ORE in competition with another transcription factor leading to the observed net inhibitory effect of oestrogenic factors on promoter activity.
The classical ORE, to which both forms ( and ß) of the oestrogen receptor are known to bind, comprises the 13 bp (or often quoted 15 bp) consensus sequence 5'-(A)GGTCANNNTGACC(T)-3' (31). However, an oestrogen responsive region comprising four direct repeats (DRs) of the half site sequence 5'-TGACC-3' separated by >100 bp has been identified in the chicken ovalbumin gene (37). In vitro experiments have demonstrated that the spacing of these DRs is crucial to transcriptional stimulation, no oestrogen responsiveness being observed when the DRs are separated by <5 bp (38). The a form of the receptor is also capable of binding to the response element for the orphan nuclear receptor SF1, essentially a single ORE half site preceded by a TCA trinucleotide (39). Finally, another mode of oestrogenic modulation has been documented in which oestrogen receptor interacts with proteins which form the protein complex at AP1 sites, generally eliciting an enhancement of transcriptional activity (40). However, this type of protein–protein interaction in the lipoprotein lipase gene promoter has recently been shown to inhibit transcription (41). Inspection of the oestrogen receptor binding sequence within the FVII promoter indicates that, although to the best of our knowledge it represents a novel promoter element with which the oestrogen receptor is capable of interacting, differing from all those described above, it corresponds most closely to a classical ORE with the two half sites forming the inverted repeat separated by not three but just two spacer nucleotides. In agreement with our findings, in vitro band shift assay experiments using synthetic oligonucleotides have previously demonstrated a reduced binding affinity of oestrogen receptor to a classical ORE ‘mutated’ in this fashion (42). However, the inherent effect, be it positive or negative, on transcriptional activity of oestrogen receptor binding to this type of sequence motif, independent of any effect due to competition for overlapping binding sites with other transcription factors alluded to above, remains to be elucidated. Transactivatory and transrepressive functions have been described for glucocorticoid receptors (GRs) which, like their oestrogenic counterparts, have been shown to bind as homodimers at specific sites, glucocorticoid responsive elements (GREs), located in the promoter regions of target genes (31,43–45). These GREs, like OREs, classically comprise two inverted palindromes separated by three spacer nucleotides (31). There is evidence in a number of different genes repressed by glucocorticoids that the repressive activity is related to steric hindrance by the GR preventing the binding of positively acting transcription factors to adjacent binding sites (44–46). However, inspection of the sequences to which GR binds and mediates this repressive effect has led to the suggestion that these nGREs show subtle differences in nucleotide sequence to the traditional positively acting GREs and that as a consequence of binding to these nGRE sequences, receptor conformation is altered and the transactivatory function masked (31,46,47). Indeed, a similar mechanism has been proposed to explain the inhibitory effect on transcription of thyroid hormone receptor binding to the vitellogenin A2 ORE (48). In this model, the binding of thyroid receptor to an ORE (which differs from the inverted palindromic recognition sequence seen in thyroid response elements only by the presence of the three spacer nucleotides) induces a conformational change upon binding which ablates its transactivatory function. Hence, one possible mechanism to explain the observed suppression of FVII activity in the presence of oestrogenic factors could be that oestrogen receptor binding to the FVII ORE prevents a positively acting transcription factor binding at this site, whilst the unusual nature of this ORE may induce a conformational change in the bound oestrogen receptor protein configuration which prevents it from displaying any transactivatory properties.
Our findings are consistent with previously reported in vivo data showing an increased level of FVII in post-menopausal compared to pre-menopausal women of a similar age and also the inverse relationship between FVII and oestradiol levels during the menstrual cycle (29,30). The increased levels of oestrogens during pregnancy and an associated increase in FVII levels appear at first paradoxical and at variance with these findings (49). However, the predominance of oestriols over oestradiols produced during pregnancy may be critical. The importance of oestrogenic ligand sub-type is further implied from data analysing the effect of exogenously administered oestrogenic formulations in women using oral contraceptives and those on hormone replacement therapy. For instance, oral contraceptive formulations using synthetic oestrogenic compounds are generally accepted to result in an increased, not decreased, level of FVII antigen in the plasma (50,51). Contrary to expectation, increased, unaltered and decreased levels of FVII antigen have been observed in women on hormone replacement therapy; these conflicting data may again reflect differences in oestrogenic and progestogenic formulation, together with route and frequency of administration (29,52,53). In conclusion, an understanding of the basic mechanisms by which steroid hormones influence plasma levels of proteins, including FVII, and their relationship to risk of cardiovascular disease, may prove to be a key consideration in the future development of oral contraceptive and hormone replacement therapy formulations.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Construction of FVII promoter reporter gene plasmids
A fragment of the 5' flanking region of the FVII gene spanning nucleotides –658 to –1 (where +1 is the start site of translation in accordance with previous nomenclature) (5) was amplified by PCR (54) from the DNA of individuals homozygous for the two common allelic forms of the FVII promoter (28): haplotype 1, –122 (T), –323 (0 bp) and –401 (G); and haplotype 2, –122 (C), –323 (10 bp) and –401 (T); –122 (C/T), –323 (0 bp/10 bp) and –401 (G/T) indicate genotype at the –122, –323 and –401 polymorphic loci, respectively. Briefly, 100 ng of genomic DNA was amplified in 1x reaction buffer [67 mM Tris–HCl pH 8, 16 mM (NH4)2SO4, 2 mM MgCl2, 0.2 mM dNTPs, 10% v/v DMSO, 100 µg/ml bovine serum albumin (BSA), 10 mM ß-mercaptoethanol] in a final volume of 50 µl. Following the addition of 2.5 U Pfu DNA polymerase (Promega) and 100 ng each primer (FVII Pr sense and FVII Pr antisense) (Table 1), amplification was carried out by incubation at 95°C for 7 min followed by 30 repeated cycles of 91°C for 1 min, 62°C for 1 min and 72°C for 3 min. PCR products corresponding to haplotypes 1 and 2 were then subcloned into the multiple cloning site of the pCR2.1 TA cloning vector (Invitrogen). Digestion of these recombinant plasmids with XmaI and BglII allowed the FVII promoter fragments spanning residues –658 to –1 to be cloned into the corresponding sites of the promoterless firefly luciferase reporter plasmid, pGL3 Basic (Promega). The plasmids pGL3-FVII -1/-348 haplotype 1 [–323 (0 bp), –122T] and pGL3-FVII -1/-348 haplotype 2 [–323 (10 bp), –122C] were made by cloning DraI/BglII fragments from the corresponding pGL3-FVII -1/-658 parent vectors into pGL3 Basic digested with SmaI and BglII. The plasmids pGL3-FVII -1/-187 haplotype 1 (–122T) and pGL3-FVII -1/-187 haplotype 2 (–122C) were constructed by SacI digestion and religation of the corresponding pGL3-FVII -1/-658 parent vectors. To make constructs with a mutated version of the FVII promoter, XmaI/NsiI fragments spanning residues –658 to –162 of the FVII promoter from the plasmids pGL3-FVII -658/-1 haplotype 1 and pGL3-FVII -658/-1 haplotype 2 were subcloned into XmaI/NsiI-digested pRL-Null
Sac [pRL-NullSac was prepared from pRL-Null (Promega) which had been digested with SacI and then religated after treatment of the digested vector with the Klenow fragment of DNA polymerase I in the presence of all four dNTPs]. Fragments corresponding to residues –229 to –187 of the FVII promoter were then removed by digestion of each plasmid with PvuII and SacI and replaced with FVII ORE Mut (Table 1) spanning the same residues but containing a mutated form of the putative ORE between residues –225 and –212. The 471 bp XmaI/NsiI fragment (containing the mutated ORE on a background sequence corresponding to haplotypes 1 and 2) from each of the resultant plasmids was then ligated back into XmaI/NsiI-digested pGL3-FVII -658/-1 haplotype 1 and pGL3-FVII -658/-1 haplotype 2, respectively. The correct orientation and authenticity of the insert sequences in all the plasmids was verified by DNA sequencing using the Thermo Sequenase Cycle Sequencing Kit (Amersham) and the plasmid derived sequencing primers, GL primer 2 and RV primer 3 (Promega).
Construction of a FXII promoter reporter gene plasmid
A fragment of the 5' flanking region of the FXII gene spanning nucleotides –181 to +51 (where +1 is the transcription initiation site in accordance with previously described nomenclature) (55) was amplified from 500 ng genomic DNA using 2 U Taq polymerase (Bioline) and 500 ng each primer FXII Pr S and FXII Pr AS (Table 1) in 50 µl 1x buffer [16 mM (NH4)2SO4, 67 mM Tris pH 8.8, 2 mM MgCl2]. After an initial denaturation at 95°C for 7 min, the sample was subjected to 30 rounds of denaturation at 91°C for 1 min, annealing for 1 min at 62°C and primer extension at 72°C for 2 min. Cloning of the PCR product into the TA cloning vector pCR2.1 (Invitrogen) permitted its subsequent excision by digestion with Asp718 and XhoI and cloning into the cognate sites in PGL3 Basic. Authenticity was confirmed using the same primers and sequence analysis procedure described for the FVII plasmids.
Cell culture, transfection and reporter gene assays
HepG2 cells were cultured and maintained in modified Eagle’s medium (MEM) containing 10% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine and 1x non-essential amino acids (Life Technologies). Transfections were carried out in 10 cm2 wells using a previously described calcium phosphate procedure (56). Briefly, 2.5 µg of firefly luciferase reporter gene plasmid was transfected into HepG2 cells in the presence or absence of 1.25 µg of the expression vector pSG5-HEO encoding the human oestrogen receptor (32). Included in each overnight transfection at 37°C/5% CO2 was 1.25 µg of pRL-Null (Promega) encoding renilla luciferase, to act as an internal control for cell number, transfection efficiency and harvesting efficiency. Where appropriate, 1.25 µg non-specific DNA was added to maintain a constant level of 5 µg total DNA (2.5 µg DNA/ml MEM culture medium) in each transfection. After transfection the cells were incubated for a further 48 h prior to harvesting in phenol red-free MEM containing charcoal-stripped fetal calf serum. ß-Oestradiol (1 x 10–8 M final concentration; Sigma) was added to the incubation medium of those cells that had been transfected with the oestrogen receptor expression plasmid. Cells were harvested into 500 µl reporter lysis buffer (Promega). Firefly and renilla luciferase activities were then determined using the Dual-Luciferase Assay Reporter System (Promega) essentially according to the manufacturer’s instructions. Mean peak luminescence levels were measured using an ML3000 luminometer (Dynex Laboratories).
Electrophoretic mobility shift assays
Nuclear or whole-cell extracts were prepared from HepG2 cells using previously described extraction procedures (57,58) and total protein concentration in the extracts estimated by the method of Bradford (59) using the DC Protein Assay Kit (BioRad). Oligonucleotide probes used in this study (Table 1) were radiolabelled with 
32P ATP (NEN). Binding reactions were performed in a final volume of 20 µl 1x binding buffer (25 mM Tris–Hcl pH 7.5, 100 mM KCl, 0.0625 mM EDTA, 4.75% v/v glycerol, 500 µg/ml BSA) containing 0.5 µg poly dI-dC (Pharmacia) and 0.2 pmol radiolabelled probe and 10 µg nuclear extract, whole cell extract or BSA. Human recombinant oestrogen receptor- (2 pmol) (Panvera) was also included where appropriate. Reactions were incubated at room temperature for 5–10 min. Alternatively, in supershift assays 3 µg anti mouse ER (F10) monoclonal antibody or 3 µg control mouse IgG (Santa Cruz Biotechnology, Inc.) was added and incubated at 0°C for 60 min. Samples were then loaded onto a 4% acrylamide gel (120:1 acrylamide:N,N’ methylene bis acrylamide) and size-fractionated at 4°C for 3 h at 4 V/cm in 1x TBE (89 mM Tris–HCl, 89 mM Boric acid, 2.5 mM EDTA). The gel was dried at 80°C under vacuum and exposed to autoradiographic film.
Statistical analysis
In reporter gene assays, logarithmic transformations were carried out to obtain normal distributions of the continuous variables allowing statistical computation by parametric methods. The statistical significance of any difference in activity of the test constructs in the presence or absence of oestrogenic factors was assessed using 2-tailed, paired t-tests. In gel shift assays, the significance of any differences seen in the mean percentage of radiolabelled probe bound to oestrogen receptor between different gel shift probes (estimated by densitometric analysis of autoradiograms) was assessed by 2-tailed, unpaired t-tests. All statistical analyses were carried out using the Microsoft Excel 2000 software package.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +44 114 271 3213; Fax: +44 114 272 1104; Email: p.r.winship@sheffield.ac.uk
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REFERENCES |
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