Introduction

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Conjugation with glucuronic acid represents one of the major phase 2 detoxification pathways in mammals, and the liver represents one of the primary sites of this reaction. This most likely is due to differential expression within hepatocytes of the enzymes that catalyze glucuronidation, the UDP-glucuronosyltransferases (UGTs). Two related families of UGTs are known, UGT1 and UGT2 (Mackenzie et al., 1997); many from each family are expressed in liver. Although the mechanisms underlying their predominant expression in liver remain poorly understood, recent studies investigating the mechanisms of regulation of the UGT1A1 and UGT2B1 genes suggest the involvement of two different liver-enriched transcription factors: hepatic nuclear factor-1 (HNF1) and CCAAT-enhancer binding protein- (C/EBP; Hansen et al., 1997, 1998; Bernard et al., 1999).

In the rat UGT family, one of the major UGTs expressed in liver for the glucuronidation of bulky planar substrates is UGT1A7. The 1A7 isoform is active toward several metabolites of benzo(a)pyrene, a well-established environmental carcinogen. These include the major phase 1 metabolites, 3-hydroxy-, 9-hydroxy-, and 3,6-dihydroxy-benzo(a)pyrene, and the toxicologically significant proximate carcinogen 7,8-dihydrodiol (Grove et al., 1997). UGT1A7 appears unique among UGTs in its ability to form diglucuronides of both chrysene and benzo(a)pyrene-3,6-diphenol (Bock et al., 1999). UGT1A7 from other species has been shown to catalyze the glucuronidation of dietary phenols such as octyl gallate (Bruck et al., 1997), steroid hormone metabolites such as catechol estrogens and 4-hydroxyestrone, tertiary amine drugs such as imipramine (Bruck et al., 1997), and the environmental carcinogen, 2-hydroxyamino-1-methyl-6-phenylimidazo-(4,5)-pyridine (Strassburg et al., 1999). UGT1A7 also catalyzes the glucuronidation of 7-ethyl-10-hydroxycamptothecin (SN-38; Ciotti et al., 1999), the active metabolite of the anticancer drug irinotecan, which is implicated in the severe gastrointestinal toxicity observed in some irinotecan-treated patients.

In adult rats, the expression of UGT1A7 in liver appears to be tightly controlled. Control rats exhibit low levels of hepatic microsomal UGT1A7 protein levels as well as low glucuronidating activities toward benzo(a)pyrene-7,8-dihydrodiol, a marker substrate of UGT1A7. UGT1A7 mRNA is virtually undetectable by standard Northern blot analysis (Grove et al., 1997). However, on treatment with the aryl hydrocarbon receptor (AHR) agonists 3-methylcholanthrene (3-MC) and -naphthoflavone, UGT1A7 mRNA levels are markedly increased (Emi et al., 1995; Kobayashi, 1998; Metz and Ritter, 1998). UGT1A7 is also highly induced by oltipraz, a candidate chemopreventive agent in the phase 2 detoxifying enzyme inducer subclass (Grove et al., 1997). Interestingly, this pattern of expression is unique to the liver. The two tissues showing the highest constitutive expression of UGT1A7, intestine and kidney, exhibit lower or no inducibility by oltipraz and -naphthoflavone (Grove et al., 1997; Kobayashi et al., 1998). Other tissues, such as the spleen, lung, and ovary, express UGT1A7 at slightly higher constitutive levels compared with liver and show no inducibility by oltipraz.

Data from nuclear runoff assays support a role for transcriptional activation in the mechanism of the observed increase in liver UGT1A7 mRNA levels by inducing agents (Metz and Ritter, 1998). Although the locations of elements mediating transcriptional activation remain unknown, the existence of a promoter in the region flanking the UGT1A7 transcription start sites determined by primer extension and RNase protection analysis (Metz and Ritter, 1998) was supported in gene reporter experiments. Primary rat hepatocytes transfected with the UGT1A7 reporter plasmid p(965/+56)1A7-Luc exhibited an increase in luciferase expression compared with cells transfected with the promoterless reporter plasmid pGL3-Basic. Sequence analysis of the promoter indicated the possible contribution of several liver-enriched transcription factors, HNF1, HNF3, HNF4, and C/EBP, in the detected basal expression of the UGT1A7 promoter.

To test the hypothesis that one or more of these factors contribute to the function of the UGT1A7 promoter, we investigated the effect of 5'-terminal deletions on the activity of the promoter using primary rat hepatocytes transiently transfected with various UGT1A7 reporter plasmids. Unexpectedly, the full activity of the promoter was found to reside in the region between 157 and +76 (respective to the 5'-most transcriptional start site), suggesting that neither HNF3, HNF4, nor C/EBP is required for the basal function of the promoter. Analysis by DNase footprinting revealed two sites of binding by rat liver nuclear proteins. One of these corresponds to the HNF1-like binding site located between bases 52 and 38, and the other is a region normally associated with TATA protein and RNA polymerase II binding. Evidence is presented that the HNF1-like site is functional and that HNF1 plays a role as a general enhancer of constitutive and induced UGT1A7 gene expression in rat liver.

	Experimental Procedures

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Materials. Adult male Sprague-Dawley rats used in this study for the preparation of rat liver nuclei and primary hepatocyte cultures were maintained on a standard 12-h light/dark cycle and allowed free access to water and standard laboratory rodent chow. All uses and handling of the animals required for this study were approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University.

Plasmids. The construction of p(965/+56)1A7-Luc and p(+56/965)1A7-Luc was described previously (Metz and Ritter, 1998). p(619/+71)1A7-Luc and p(157/+71)1A7-Luc were generated by polymerase chain reaction amplification using either 5'-CTA AGG GGC ATC ACA GT-3' or 5'-CAC ATT GTG TGA TTC TG-3', respectively, as the forward primer, 5'-ACC AAG GCA AGA CAG CTG CTG AGA CTG T-3' as the common 3' primer, and p1.4XB (cloned UGT1A7 genomic DNA; Metz and Ritter, 1998) as template. Each amplicon was initially cloned into pCR2.1, excised with EcoRI, and blunt-ended with Klenow DNA polymerase and then cloned into SmaI cut pGL3-Basic, forming p(619/+71)1A7-Luc and p(157/+71)1A7-Luc. p340XP contains a 340-bp XbaI/PstI fragment from p1.4XB corresponding to bases 253 to +96 cloned into the XbaI/PstI sites of pBluescript SK⁺.

Cell Culture. Primary rat hepatocytes isolated by collagenase-EDTA perfusion were allowed to adhere to gelatin-coated plastic tissue culture plates (0.8 × 10⁶ cells/35-mm dish) at 37°C under a humidified atmosphere containing 5% CO₂. The plating medium was Williams E (Life Technologies) without fetal bovine serum, supplemented with 0.6 µg/ml insulin, 100 µg/ml transferrin, 1 µM dexamethasone, fungizone, and gentamicin.

RNA Isolation and Northern Analyses. Total RNA from cultured cells was isolated and analyzed by Northern blotting as described previously (Grove et al., 1997), using rat HNF1 cDNA labeled with ³²P-dCTP by random priming as the probe. After exposure of the hybridized blot to x-ray film for 6 days, the blot was rehybridized with radiolabeled rat cyclophilin cDNA to demonstrate similar loading and quality of the RNA samples.

Transient Transfection. Primary rat hepatocytes were transfected 6 h after plating by removing the medium and adding 1.5 ml of fresh Williams E (without supplements) containing freshly mixed LipoFECTIN (3.75 µg/dish) and UGT1A7 reporter plasmid (1.5 µg). For normalization of interplate differences in transfection efficiency, a small amount (0.5 µg) of control plasmid, pRSV-Gal, was included in each transfection. In the cotransfection studies, the HNF1 expression plasmid pRHNF1 or pRHNF1 was included (0.03-1 µg/dish as indicated). After overnight incubation, the medium was removed and replaced with fresh serum-free medium. At 24 h later, the medium was replaced with fresh medium. Cells were harvested 24 h later in 150 µl of 1× cell culture lysis reagent (Promega), and luciferase activity was measured using the Luciferase Assay kit and a Berthold Lumat LB9501 luminometer. -Galactosidase activities were determined as described by Sambrook et al. (1989).

Isolation of Rat Liver Nuclear Extract. The preparation of liver nuclear extract from adult male Sprague-Dawley rats was based on the method of Gorski et al. (1986) with the following modifications. Sodium fluoride was included in the homogenization and nuclear lysis buffers at 10 and 5 mM, respectively. Protease inhibitors in addition to phenylmethylsulfonyl fluoride were added to the nuclear lysis and nuclear dialysis buffers, including pepstatin A, chymostatin, antipain, and leupeptin at 25 µg/ml and aprotinin (Trasylol) at 10 µg/ml (0.1 mM final concentration). The second centrifugation step was modified as follows: the initial nuclear pellet was resuspended in 25 ml of nuclear lysis buffer (containing 100 mM KCl, 10 mM HEPES, pH 7.4, 0.1 mM EDTA, 3 mM MgCl₂, 1 mM dithiothreitol, and 10% glycerol in addition to the phenylmethylsulfonyl fluoride, NaF, and protease inhibitors) and immediately recentrifuged at 9000g for 10 min at 4°C. For precipitating protein and chromatin material, 5 M NaCl was used in place of 4 M (NH₄)₂SO₄ (final NaCl concentration 0.4 M), and the solid salt addition step was omitted. The protein concentrations of the final dialyzed extracts were determined using a BCA protein assay kit (Pierce Chemical, Rockford, IL) and adjusted to a final concentration of ~1 µg/µl with nuclear dialysis buffer. The extracts were stored in 0.1-ml aliquots and stored at 86°C.

DNase I Footprinting Assay. The probe used for DNase I footprinting was a ~390-bp fragment corresponding to bases 253 to +96 of UGT1A7 (described by Metz and Ritter, 1998). The probe was labeled by digesting p340XP with HindIII, filling in with dNTPs containing [³²P]dCTP, and then digesting with SacI to release a ~390-bp fragment labeled specifically at the 3' end of the top (coding) strand. The fragment was purified by electrophoresis through low melting point agarose followed by purification using Promega PCR Wizard. Binding reactions and DNase I digestions were performed as described by von der Ahe (1991). Briefly, a binding reaction containing 1× buffer B (10 mM HEPES, pH 7.9, 0.5 mM EDTA, 1 mM dithiothreitol, and 6% glycerol), 60 µg of nuclear extract, 3 × 10⁴ cpm of the probe, 5 µg of poly(dI/dC), and water to a final volume of 20 µl was incubated for 25 min at 25°C. DNase I was added where applicable to a final concentration of 0.03 µg/ml, and the samples were incubated for 2 min at 25°C. The reactions were stopped by the addition of 75 µl of DNase stop solution (30 mM EDTA, 0.25% SDS, 0.5 mg/ml proteinase K) and incubated for 30 min at 42°C. Samples were phenol-extracted and ethanol-precipitated overnight at 20°C. Pellets were dissolved in 3 µl of formamide loading buffer (95% formamide, 0.5× Tris-Borate-EDTA (TBE), 0.1% xylene cyanol, 0.1% bromophenol blue), denatured at 95°C for 3 min, and run on a 7 M urea-0.5× TBE-6% polyacrylamide sequencing gel.

EMSA. Oligonucleotides corresponding to the top and bottom strands of PR-1, mutPR-1, and the rat albumin HNF1 sequences (Fig. 2B) were annealed by heating to 70°C and slow cooling to room temperature. Oligonucleotide probes (PR-1 or the rat albumin HNF1 sequence as indicated in each figure) were 5'-end-labeled in T4 polynucleotide kinase reactions containing 2 pmol annealed PR-1 oligonucleotide and 20 nmol [-³²P]ATP. Binding reactions (20 µl total volume) contained 1 µg of nuclear extract protein, 10 fmol (~20,000 cpm) of radiolabeled probe, 2 µg of poly(dI/dC), 1 µmol of KCl, 100 nmol of MgCl₂, 0.4 µmol of Tris-HCl, pH 7.9, 4 nmol of EDTA, 800 µg of Ficoll 400, and 50 µmol of dithiothreitol. After a 20-min incubation on ice, 2 µl of 0.02% bromophenol blue tracking dye was added, and the samples were loaded onto a 20 × 25 cm 0.5× TBE-4.5% polyacrylamide slab gel (1.5 mm thick). The samples were electrophoresed at 150 V for 2 h (until the tracking dye migrated two thirds of the way down the length of the gel). The gel was then dried and exposed to film.

	Results

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The transfection of primary rat hepatocytes with the UGT1A7 promoter-luciferase reporter plasmid p(965/+56)1A7-Luc resulted in ~5-fold greater luciferase activity compared with cells transfected with the promoterless control plasmid pGL3-Basic (Fig. 1). This activity, as expected, was dependent on the orientation of the UGT1A7 promoter fragment. Luciferase activity was very low in hepatocytes transiently transfected with p(+56/965)1A7-Luc, even compared with pGL3-Basic. The promoter activity of p(965/+56)1A7-Luc appeared weak compared with the SV40 early promoter, which exhibited >100-fold higher activity (data not shown). To define the sequence or sequences contributing to basal expression, the effect of introducing 5'-terminal truncations on the function of the promoter was examined. Hepatocytes transfected with either p(619/+71)1A7-Luc or p(157/+71)1A7-Luc exhibited luciferase activities that were similar to that of p(965/+56)1A7-Luc-transfected cells (Fig. 1). From these observations, the potential regulatory elements for HNF4 (bases 365/353, 5'-AGGGGCAGGTTCA-3'), HNF3 (bases 248/238, 5'-AAATGTAGGCT-3'), and C/EBP (bases 181/171, 5'-TTTCCTCACA-3') appear not to contribute significantly to the basal UGT1A7 promoter activity in rat hepatocytes.

View larger version (41K): [in this window] [in a new window]   Fig. 1.   Effect of 5' deletions of the UGT1A7 promoter on expression of luciferase in primary rat hepatocytes transfected with UGT1A7 reporter constructs. Indicated reporter plasmids were transfected with pRSV-Gal into primary cultures of rat hepatocytes (three wells/group). Cells were harvested at 48 h post-transfection, and luciferase activities of the cellular lysates were determined. Data (mean ± S.E.) were normalized for transfection efficiency by dividing by the relative -galactosidase activity.

To characterize the rat liver nuclear factors recognizing the proximal region of the UGT1A7 promoter, DNase I footprint analysis was performed using a probe representing bases 253 to +96. Two regions were found to be protected from DNase I cleavage, which were designated PR-1 and PR-2 (Fig. 2). PR-1 extends from 50 to 40 and is centered over the HNF1-like binding site identified previously (Metz and Ritter, 1998). Specific sites protected from DNase cleavage include the cytosine and guanine residues at 40 and 42, respectively. Two other protected sites not visible in the figure corresponding to cytosine 46 and adenine 50 were apparent after longer film exposures. The binding was not dependent on whether the liver nuclear extract was prepared from control or inducer-treated rats. Binding to the PR-1 and PR-2 positions was also apparent when the noncoding strand was used in the analysis (data not shown).

View larger version (37K): [in this window] [in a new window]   Fig. 2.   DNase I footprint analysis of the rat UGT1A7 promoter. Left, probe corresponding to bases 252 to +96 of UGT1A7 was incubated without liver nuclear extract ("None") or with liver nuclear extracts from oltipraz (OTP)- or -naphthoflavone (BNF)-treated rats. After limited DNase I digestion, DNA was isolated and analyzed by polyacrylamide gel electrophoresis. Two regions of DNA, PR-1 and PR-2 (indicated by vertical lines), were protected from DNase I digestion in the presence of liver nuclear extract. PR-1 and PR-2 were flanked by sites that were hypersensitive to DNase I digestion in the presence of liver nuclear extracts (indicated by dots). A DNA sequence ladder was used as a standard to determine fragment sizes (STD). Right, sequence of the UGT1A7 gene surrounding PR-1 and PR-2 and their relationship to the 5'-most transcription start site (indicated by the arrow at base +1). The HNF1- and Sp-1-like binding sites are indicated by the solid and dashed underlining, respectively.

PR-2 is located immediately 3' of PR-1 and features a cluster of protected bands between positions 9 and 25. This region includes a potential TATA element, TATGT, from bases 30 to 26 and a 10-base sequence with 6 of 10 bases matching the consensus Sp-1-binding site sequence 5'-GGGGCGGGGC-3', located between bases 22 to 13. In addition, several DNase hypersensitive sites were evident in the samples containing rat liver nuclear extract (indicated by the dots in Fig. 2). These were located in positions immediately flanking the protected regions, PR-1 and PR-2, and likely reflect changes in the DNA configuration induced by binding of rat liver nuclear extract, thereby increasing accessibility by DNase I. There were no other sites of binding detected in the region from 252 to +94 by DNase I footprinting.

To further characterize the binding to PR-1 and assess the possibility that it represents a functional HNF1 binding site, EMSA analysis was performed using radiolabeled PR-1 probe corresponding to bases 61 to 30 as shown in Fig. 3. The formation of the DNA-protein complex indicated by the arrow was completely prevented in binding reactions containing a 50-fold excess of cold PR-1 but not mutPR-1 probe (Fig. 3). mutPR-1 contained three base substitutions in the higher conserved 5' half of the HNF1 binding sequence. The binding was significantly competed using a competitor probe corresponding to the rat albumin HNF1 site (Lichtsteiner et al., 1987).

View larger version (36K): [in this window] [in a new window]   Fig. 3.   EMSA analysis of UGT1A7 PR-1 binding site. Liver nuclear extract from a control rat was mixed with radiolabeled double-strand DNA corresponding to PR-1 and incubated in either the absence (None) or presence of a 50-fold excess of cold PR-1 (PR-1) probe, mutant PR-1 probe (Mut PR-1), or rat albumin promoter HNF1 probe (AlbHNF1) (sequences indicated). The arrow indicates the specific complex formed between the probe and rat liver nuclear protein. FP, free probe.

To determine whether HNF1 is present in the protein complex recognizing PR-1, EMSAs were carried out using specific antibodies to HNF1 and HNF1 (Fig. 4). For comparison, the rat albumin HNF1 site probe (see Alb HNF1 in Fig. 3) was ³²P-end-labeled and run in parallel with the PR-1 probe. Both probes yielded similar profiles in the EMSA assay. In the absence of antibody, each probe exhibited a similarly migrating DNA-protein complex. The addition of the HNF1 antibody to the binding reaction retarded the mobility of the complex, whereas the HNF1 antibody had no apparent effect. These results support the presence of HNF1 in the protein complex recognizing PR-1 and are consistent with the dominant expression of HNF1 in liver (Rey-Campos et al., 1991).

View larger version (58K): [in this window] [in a new window]   Fig. 4.   EMSA using antibodies to rat HNF1 and HNF1. Liver nuclear extract from a control rat was preincubated in either the absence or presence of antibody to HNF1 (lanes 2 and 5) or HNF1 (lanes 3 and 6) followed by the addition of either radiolabeled PR-1 (lanes 1-3) or rat albumin HNF1 (lanes 4-6) probe. Complexes were resolved by polyacrylamide gel electrophoresis and visualized by autoradiography. The closed arrow and open arrowhead indicate the unshifted and shifted complex, respectively. NS, nonspecific complex. FP, free probe.

To investigate the in vivo significance of the PR-1 site in UGT1A7 transcription, the effect of mutating PR-1 on the expression of the UGT1A7 gene reporter in transiently transfected primary rat hepatocytes was determined. A mutant version of p(965/+56)1A7-Luc, p(965/+56)1A7 mutPR1-Luc, was constructed that contained the same three base substitutions in the PR1 site as the mutPR-1 oligonucleotide (Fig. 3). The mutation was associated with a marked reduction in luciferase expression directed by the UGT1A7 gene reporter in primary rat hepatocytes. The relative luciferase/-galactosidase ratios were 1.00 ± 0.15 for p(965/+56)1A7-Luc- transfected cells, 0.12 ± 0.024 for p(965/+56)1A7 mutPR1-Luc, and 0.022 ± 0.006 for pGL3-Basic.

Experiments were conducted to examine the effect of overexpressing the two major HNF1 isoforms, HNF1 and HNF1, on UGT1A7 reporter expression. Using primary rat hepatocytes as a cellular model, cotransfection of pRHNF1 produced a marked decrease in luciferase activity (data not shown). This effect was potent (with detectable inhibition only at 0.01 µg of pRHNF1) and was concentration-dependent and may be due to a transcriptional squelching phenomenon, based on the observation that the expression of cotransfected pRSV-Gal was also highly attenuated.

In HepG2 cells, which were used by Hansen et al. (1997) to demonstrate the responsiveness of the rat UGT2B1 promoter to HNF1 overexpression, cotransfection of pRHNF1 had no apparent effect on p(965/+56)1A7-Luc-directed expression of luciferase, although pRHNF1 cotransfection produced up to a ~3-fold stimulation (Fig. 5A). Similar effects were observed (Fig. 5A) using a 5' deleted UGT1A7 reporter plasmid, p(157/+79)1A7-Luc, which is consistent with the presence of the HNF1-binding site between bases 52 to 38. For comparison of our data with those of Hansen et al. (1997), a control experiment was also carried out using a rat UGT2B1-luciferase reporter plasmid, p(303/+13)2B1-Luc. In contrast to the findings of Hansen et al. (1997), we were unable to observe the expected stimulation by pRHNF1. These findings are likely explained by high endogenous HNF1 expression in HepG2 cells as demonstrated by Northern blot analysis (Fig. 5C).

View larger version (64K): [in this window] [in a new window]   Fig. 5.   The effect of overexpressing HNF1 or HNF1 on luciferase activity in various cell lines cotransfected with UGT1A7 or UGT2B1 reporter plasmids. HepG2 cells (A) or HEK cells (B) were cotransfected with the indicated UGT1A7- or UGT2B1-luciferase reporter plasmids in the presence of pRSV-Gal and increasing amounts of HNF1 or HNF1 expression plasmid. For controls, cells were cotransfected with the blank expression vector pCDNA 3.1. Cells were harvested for the determination of luciferase and -galactosidase activities. Data represent the mean ± S.D. of three determinations per group normalized for the relative -galactosidase activity. C, Northern analysis of the HNF1 mRNA in primary rat hepatocytes, HepG2, and HEK 293 cells. Total RNA (10 µg/lane) was prepared and analyzed as described previously (Grove et al., 1997) using rat HNF1 cDNA excised from pRHNF1 as probe. The blot was reprobed for cyclophilin mRNA using rat cyclophilin cDNA to evaluate the quantity and quality of the RNA.

Experiments were therefore carried out using a model cell line that does not express significant levels of endogenous HNF1, HEK 293 cells (Fig. 5C). The latter were used by Bernard et al. (1999) to demonstrate the responsiveness of the UGT1A1 promoter to coexpressed HNF1. In contrast to HepG2, HEK cells showed strong responsiveness to coexpression of HNF1 or HNF1 (>40-fold maximal increase in each case; Fig. 5B). The positive control UGT2B1 reporter also showed much greater responsiveness in HEK cells. Consistent with the hypothesis that PR-1 mediates the transactivational effect of HNF1 or HNF1, the stimulatory effect of pRHNF1 or pRHNF cotransfection was blocked in HEK cells transfected with p(965/+56)1A7 mutPR-1 (Fig. 6).

View larger version (44K): [in this window] [in a new window]   Fig. 6.   Mutation of UGT1A7 PR-1 site prevents HNF1 and HNF1 stimulation of luciferase activity in p(965/+56)1A7-Luc-transfected HEK cells. HEK cells were cotransfected with either p(965/+56)1A7-Luc or p(965/+56)1A7 mutPR-1-Luc in the absence (pCDNA) or presence of HNF1 expression vector (as indicated) and pRSV-Gal. At 48 h after transfection, the cells were harvested for the determination of luciferase and -galactosidase activities. Data represent the mean ± S.D. of three determinations normalized for -galactosidase activity.

To further investigate the role of the HNF1 site in the function of the UGT1A1 promoter, an experiment was carried out using primary rat hepatocytes transfected with the "basal" UGT1A7 reporter plasmids [p(965/+56)1A7-Luc and p(965/+56)1A7 mutPR1-Luc] and two 3-MC-responsive UGT1A7 plasmids designated p(965/+56)1A7-Luc-3.7-B and p(965/+56)1A7 mutPR1-Luc-3.7B. The latter two plasmids contain a 3.7-kbp BamHI fragment from the UGT1A7 intron (Fig. 7, A and B; Metz and Ritter, 1998), which encompasses a 3-MC-responsive element or elements that may contribute to 3-MC-induced expression of the endogenous UGT1A7 gene. In agreement with the finding that the HNF1 mutation significantly reduces basal expression from the base UGT1A7 reporter plasmids p(965/+56)1A7-Luc, a similar effect on basal expression was apparent using the 3.7B-containing reporter plasmids [Fig. 7C, compare vehicle-treated p(965/+56)1A7-Luc-3.7B- and p(965/+56)1A7 mutPR1-Luc-3.7B-transfected cells]. The reduced expression was also apparent after induction with 3-MC. Although the fold of induction appeared not to be significantly affected by the HNF1 site mutation [4.7- versus 3.4-fold induction ratio for p(965/+56)1A7-Luc-3.7B and p(965/+56)1A7 mutPR1-Luc-3.7B-transfected cells, respectively], the maximal induced activity of the reporter was significantly decreased by this mutation (Fig. 7C, compare luciferase/-galactosidase activity ratios for the two 3-MC treatment groups). These results are consistent with a general role of HNF1 as an enhancer of UGT1A7 gene transcription in rat hepatocytes under either basal or induced conditions.

View larger version (29K): [in this window] [in a new window]   Fig. 7.   The effect of mutating the UGT1A7 PR-1 site on basal and 3-MC-induced UGT1A7 reporter expression in primary rat hepatocytes. Primary rat hepatocytes were transfected with the indicated reporter plasmids and treated for 48 h posttransfection with either 0.1% DMSO (VH) or 2.5 µM 3-MC before harvesting the cells for the determinations of luciferase and -galactosidase activities. A, structure of the UGT1A7/UGT1A6 locus and relationship of fragments used for the construction of UGT1A7 reporter plasmids. B, schematic showing the general structures of the UGT1A7 reporter constructs. C, data represent the mean ± S.D. expressed as the ratio of luciferase to -galactosidase activity.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

This report describes evidence that the UGT1A7 gene is under the control of HNF1, a transcription factor associated with hepatocyte-specific gene expression and differentiation. HNF1 occurs in two different forms, HNF1  and HNF1, which are expressed in a tissue-specific manner (Rey-Campos et al., 1991). The three cell types recognized as having the highest HNF1 expression are hepatocytes, enterocytes, and renal proximal tubular epithelial cells. Hepatocytes preferentially express the HNF1 form, whereas enterocytes and proximal tubular cells express HNF1 and HNF1 in a more equal ratio. Because the kidney, intestine, and liver correspond to the tissues with high or (in the case of liver) potentially high UGT1A7 mRNA expression, these data suggest a role for HNF1 in tissue-specific expression of UGT1A7.

The binding site for HNF1 in UGT1A7 is located immediately upstream of the UGT1A7 transcription start site region between positions 52 to 38. This context for HNF1 binding is typical of liver-expressed genes under HNF1 control. These include the two other known UGT genes controlled by HNF1, UGT2B1 and UGT1A1, which feature HNF1 binding sites at positions 55 to 42 and 75 to 63, respectively (Hansen et al., 1997; Bernard et al., 1999). Furthermore, it appears that a number of additional UGT genes are under the control of HNF1, based on the presence of HNF1-like binding sites in their flanking sequence. Several of these were identified by Tronche et al. (1997), who applied a consensus HNF1 site sequence deduced from 21 known functional HNF1 binding sites [5'-GGTTAAT(A/T)ATTA(A/C)CA-3']. Proposed HNF1 sites are located in the intronic sequence 3' of UGT1A1 exon 1 and 5' of UGT1 exon 2, in the promoter region of UGT1A4, and in the human and rat UGT1A6 promoter regions. In addition, the human UGT1A7 gene (GenBank entry HSU39570) contains an almost identical HNF1 site to that of rat (11 of 12 bases match) in its putative proximal promoter region.

A unique feature of the UGT1A7 gene compared with other UGT genes under HNF1 control is its weak constitutive expression in livers of untreated rats (Metz and Ritter, 1998). These data appear to contrast with the fact that HNF1 is abundantly expressed and constitutively active in this tissue. One explanation is that the UGT1A7 promoter is intrinsically weak and that HNF1 alone is not sufficient for high-level expression. In this study, we analyzed the UGT1A7 promoter function in primary rat hepatocytes, a cell model in which low UGT1A7 expression is maintained (in contrast to rat hepatoma and SV40-immortalized rat hepatocyte cell lines). The UGT1A7 promoter activity assessed by the transiently transfected gene reporter method appeared weak in hepatocytes, with luciferase expression only ~5- to 10-fold higher than that in control hepatocytes transfected with a promoterless control reporter (pGL3-Basic). In contrast, the UGT1A7 reporter was highly expressed in HEK cells driven to overexpress HNF1. This observation suggests that HEK cells contain additional factors not found in hepatocytes or HepG2 cells that act in concert with HNF1 to promote UGT1A7 promoter expression. The transformation of hepatocytes may also result in the up-regulation of additional factors that result in the overexpression of UGT1A7. Indeed, we have observed high UGT1A7 reporter expression in RALA tsA25510G LCS-3 cells, an immortalized rat liver-derived cell line that exhibits high UGT1A7 mRNA levels and its associated benzo(a)pyrene-7,8-dihydrodiol UGT activity (Grove et al., 1997). These findings support a general correlation between the level of UGT1A7 mRNA expression and the apparent activity of the UGT1A7 reporter.

This study suggests that the full basal UGT1A7 promoter activity is contained in a short segment between 157 and +56. This observation, together with the finding that mutation of PR-1 nearly abolishes expression of the promoter in primary rat hepatocytes, supports a principal role of HNF1 in maintaining basal expression of UGT1A7 in primary rat hepatocytes. However, the data suggest that HNF1 binding to a single site is not sufficient in itself for attaining high-level UGT1A7 expression in liver. Indeed, most liver-expressed genes are controlled by multiple liver-enriched transcription factors or by multiple binding sites for a single factor. The human albumin gene, for example, is under the control of three distinct HNF1 binding sites (Hardon et al., 1988; Frain et al., 1990; Hayashi et al., 1992) in addition to binding sites for C/EBP and several other transcription factors (Friedman et al., 1989). In the case of UGTs, the constitutively expressed UGT2B1 gene has been shown to be regulated by both C/EBP and HNF1, which may act cooperatively to confer high-level expression. For UGT1A7, it is likely that high expression requires HNF1 acting either additively or synergistically with an additional transcription factor or factors. The identities of these factors remain currently unknown; however, one possibility is that one or more of these factors are drug-regulated. The known inducibility of UGT1A7 by polycyclic aromatic hydrocarbons suggests that this factor is the AHR, a ligand-dependent transcription factor in the PAS-basic helix-loop-helix domain transcription factor family (Whitlock, 1999), although this remains to be definitively established. AHR mediates the induction of certain xenobiotic-metabolizing enzymes, including CYP1A1, CYP1A2, and CYP1B1, by various receptor agonists, such as 3-MC. The agonists bind to AHR, triggering its movement from the cytosol to the nucleus, where it dimerizes with AHR nuclear translocator, a second member of the PAS basic helix-loop-helix domain family. The AHR-AHR nuclear translocator complex binds to xenobiotic response elements usually found in the 5' flanking regions of responsive genes to activate transcription of nearby genes. Although the identities and positions of elements mediating UGT1A7 inducibility by 3-MC in vivo are not yet known, a 3.7-kbp BamHI fragment representing a portion of the UGT1A7 coding exon and intron flanking region has been found to confer a limited extent of 3-MC inducibility on the UGT1A7 promoter. We therefore tested the impact of mutating the HNF1 site on the extent of 3-MC-inducibility conferred by the 3.7-kbp BamHI fragment. Although our data clearly show that HNF1 is not required for inducibility by 3-MC [measured as fold of induction; Fig. 7C, 4.7-fold induction for p(965/+56)1A7-Luc-3.7B and 3.4-fold induction for p(965/+56)1A7-Luc-3.7B], the absolute extent of expression in the 3-MC-treated cells was clearly decreased (Fig. 7C, Luc/gal ratio of 3.5 for p(965/+56)1A7-Luc-3.7B versus 0.7 for p(965/+56)1A7-Luc-3.7B). The latter decrease was roughly in the same proportion (80% decrease) as was observed for the basal UGT1A7 reporter construct containing the HNF1 mutation (85-90% decrease). These data suggest that HNF1 serves a general role to enhance expression from the UGT1A7 promoter to comparable extents under basal and induced conditions. A similar conclusion was reached in the case of the phenylalanine hydroxylase gene, which requires functional HNF1 and C/EBP binding sites for basal activity as well as a maximum induction response to glucocorticoids (Faust et al., 1996).

It is interesting to speculate about the role of HNF1 in the species-specific pattern of hepatic UGT1A7 expression. Similar to in the rat, the human liver expresses very low levels of UGT1A7 (Strassburg et al., 1997), but in contrast, the level of UGT1A7 mRNA is not significantly increased by 3-MC or oltipraz in cultured primary human hepatocytes (J. K. Ritter, F. K. Kessler, and R. A. Fisher, unpublished observations). Because the human UGT1A7 gene shows conservation of the HNF1 binding sequence, it may be hypothesized that the difference between the human and rat UGT1A7 gene is the absence of drug-regulated responsive elements in the human gene. In contrast to human and rat, the mouse and rabbit express high basal UGT1A7 mRNA levels in liver and do not show significant inducibility by AHR agonists (Bruck et al., 1997). The mechanism of these differences remains to be established.

The identity of the protein(s) recognizing the second UGT1A7 promoter site (i.e., PR-2) currently remains unknown. However, the general position of binding suggests that PR-2 may correspond to a binding site for factors involved in the recruitment of a functional RNA polymerase II initiation complex. We have previously described the rat UGT1A7 gene as a TATA-independent gene (Metz and Ritter, 1998), based on the utilization of >25 distinct transcription start sites, one hallmark of TATA-less promoters (Smale, 1997). However, it is interesting that PR-2 overlaps a potential TATA element, 5'-TATGT-3', which is located in an appropriate position (30 to 26) for initiation from the 5' most transcription start site or sites. This could indicate that TATA-binding factors represent the proteins recognizing PR-2 and that transcription of UGT1A7 represents a combination of TATA-dependent and TATA-independent mechanisms. An additional possibility is that PR-2 represents a low-affinity binding site for Sp-1. The core region of PR-2 includes a 10-bp sequence with 6 of 10 bases matching the consensus Sp-1 binding site, 5'-GGGGCGCCCC-3'.

In summary, this study demonstrates an enhancer function for HNF1 in basal regulation of UGT1A7 in rat hepatocytes. UGT1A7 represents a third UGT gene demonstrated to be under the control of a functional HNF1 binding site. HNF1 is proposed to be required to obtain maximum UGT1A7 induction responses to various inducing agents, such as the polycyclic aromatic hydrocarbons and oltipraz. Furthermore, our data suggest that HNF1 is likely to contribute to the high expression of UGT1A7 observed in other HNF1-enriched tissues, such as intestine and kidney.