From the Department of Pharmacology and Toxicology, Virginia Commonwealth University, Medical College of Virginia, Richmond, Virginia 23298
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ABSTRACT |
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UDP-glucuronosyltransferase UGT1A7 catalyzes the
glucuronidation of benzo(a)pyrene metabolites and other
bulky aromatic compounds. Both UGT1A7 mRNA and an associated enzyme
activity (benzo(a)pyrene7,8-dihydrodioltransferase activity) are markedly increased in livers of rats treated with -naphthoflavone or
4-methyl-5-pyrazinyl-3H-1,2-dithiole-3-thione (oltipraz).
Nuclear runoff assays show that the effects of both inducers are
primarily due to transcriptional activation. A 27-kilobase region that
included the UGT1A7/UGT1A6 promoter regions was cloned. Primer extension and RNase protection studies indicated
30
transcription start sites in five clusters between bases
85 and
40
respective to the translation start codon. There was no recognizable
TATA box, but the promoter region is TA-rich. Sequence analysis
revealed potential binding sites for CCAAT enhancer-binding protein,
activator protein 1, and hepatic nuclear factors 1, 3, and 4, but no
xenobiotic response elements or antioxidant response elements,
implicated in the regulation of other genes by
-naphthoflavone or
oltipraz, were found. A UGT1A7 gene reporter plasmid
directed strong constitutive expression in transient transfection
assays using primary rat hepatocytes. Treatment with
3-methylcholanthrene or oltipraz had no effect compared with similarly
treated pGL3-Basic-transfected cells. These results suggest that the
regulatory elements controlling xenobiotic inducibility of
UGT1A7 transcription are located either 5' or 3' of bases
1600 to +54. One possibility is that the polycyclic aromatic-mediated
regulation of UGT1A7 occurs via the xenobiotic response
element flanking the UGT1A6 locus 7 kilobase pairs
downstream.
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INTRODUCTION |
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UDP-Glucuronosyltransferases
(UGTs)1 (EC 2.4.1.17)
catalyze the reaction of UDP-glucuronic acid with substrates bearing a variety of different functional groups, including OH, NH2,
SH2, and COOH. Because the glucuronides are usually less
biologically active, more polar, and readily excreted from organisms,
the UGTs are considered important for the general detoxification and
elimination of a host of endogenous and exogenous substances, such as
bilirubin, steroids, environmental pollutants, carcinogens, and dietary
substances. Molecular cloning evidence indicates the existence of two
large families of UGTs, UGT1 and UGT2 (1). UGT1 family members are encoded by a unique gene complex, UGT1, which utilizes exon sharing to
generate 12 isozyme forms with homologous but unique amino-terminal (
287 amino acids) and identical carboxyl-terminal sequences (245 amino acids) (2, 3). UGT2 family members, in contrast, appear to be
encoded by independent genes. Each UGT exhibits its own unique profile
of substrate and tissue specificity and regulation by exposure to
endogenous or xenobiotic compounds (1).
A recent focus of our studies has been the UGT isozymes responsible for
the glucuronidating activity of rat liver microsomes toward
benzo(a)pyrene metabolites. Benzo(a)pyrene is a
prototype of the polycyclic aromatic hydrocarbons (PAHs), a class of
carcinogens introduced into the environment from the partial combustion
of organic material (e.g. tobacco). Although metabolism is a
fundamental requirement for their carcinogenicity (reviewed in Ref. 4), their mutagenic properties are also subject to the activity of detoxification enzymes, such as the UGTs (5-7). Using
benzo(a)pyrene 7,8-dihydrodiol as substrate, we observed a
marked increase in UGT activity in liver microsomes from rats treated
with -naphthoflavone or oltipraz
(4-methyl-5-pyrazinyl-3H-1,2-dithiole-3-thione (8)). A
cDNA (clone LC14) was isolated from an adult rat hepatocyte-derived cell line encoding a UGT active in the glucuronidation of 14 different benzo(a)pyrene metabolites, including 7,8-dihydrodiol (9). Sequencing of LC14 revealed identity with the product encoded by the
UGT1A7 locus2 (3). UGT1A7
mRNA is markedly increased in rat liver by PAHs and oltipraz. An
identical pattern of mRNA regulation is observed for UGT1A6 (3, 9,
11, 12), the better known PAH-inducible UGT with activity toward
4-nitrophenol and other simple phenols but not
benzo(a)pyrene phenols (13, 14). Because PAHs and oltipraz
are known to induce transcription of other metabolizing enzymes by
mechanisms involving the arylhydrocarbon receptor and/or oxidative
stress (15-17), our findings suggest that UGT1A6 and UGT1A7 may each be under the control of cis
elements implicated in these responses, i.e. the xenobiotic
response element (XRE) for arylhydrocarbon receptor mediated effects
and the antioxidant response element (ARE) for oxidative
stress-associated effects.
To characterize the mechanism of PAH inducibility of rat UGT1A6, Emi
et al. (18) have cloned and characterized the
UGT1A6 gene promoter. The structure of the transcription
unit was unusual in that it was composed of six rather than the five
exons that are typical of other UGT1 transcription units (2, 3). A putative XRE with the core sequence characteristic of all known XREs,
5'-GCGTG-3', was identified between 134 and
129 upstream from the
transcriptional start site. This sequence was shown to be a functional
XRE, mediating 9-15-fold inducibility of a chloramphenicol acetyltransferase reporter enzyme. These observations suggest that this
particular sequence motif underlies the in vivo regulation of UGT1A6 by PAHs.
In this paper, we describe a study aimed at characterizing the mechanism of the increase in steady state UGT1A7 mRNA level by PAH and oltipraz. Nuclear runoff data indicate that increased gene transcription is the primary mechanism of mRNA accumulation in response to these agents. Characterization and sequencing of the UGT1A7 promoter and 5'-flanking region indicated several differences from UGT1A6, including its basic exon composition, the existence of >30 transcription start points, and the absence of any functional XRE or AREs in its immediate 5' flanking sequence.
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EXPERIMENTAL PROCEDURES |
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Materials--
Rats used in this study were male Sprague-Dawley
rats purchased from Harlan Laboratories (Indianapolis, IN). Animal
handling and experimentation for this study were carried out with the
approval of the Institutional Animal Care and Use Committee at Virginia Commonwealth University. -Naphthoflavone and 3-methylcholanthrene were from Aldrich and Sigma, respectively. Oltipraz was from the Chemoprevention Branch, National Cancer Institute (Bethesda, MD). The
rat genomic DNA library (in bacteriophage EMBL3 SP6-T7) was from
CLONTECH (Palo Alto, CA). Custom oligonucleotides
used in this study for primer extension and gene sequencing were
synthesized by the Nucleic Acid Synthesis and Purification Core
Resource of the Massey Cancer Center (Virginia Commonwealth
University). Cultures of primary rat hepatocytes were prepared by the
Hepatocyte Isolation and Preservation Core Facility of the Liver Center
at the Medical College of Virginia (Virginia Commonwealth University).
LipofectinTM cationic liposomes and Luciferase Assay
SystemTM were purchased from Life Technologies, Inc. and
Promega (Madison, WI), respectively.
Plasmids--
pRUGT1.6 contains a 783-bp fragment from the 5'
end (bases +28 to +810) of the UGT1A6 cDNA (8). The plasmid p1.4XB
contains a 1499-bp XhoI-BamHI fragment from the
extreme 5' end of the bacteriophage clone RPT-6 inserted into the
XhoI and BamHI sites of pSK+. The plasmid p2.0XB
contains the 5' XhoI-BamHI fragment of
VIP-14 cloned into XhoI-BamHI cut pSK+. pLC14 (+9/+697)
corresponds to a 690-bp fragment from the 5' end of the LC14 (UGT1A7)
cDNA cloned into pCR2.1 (Invitrogen, Carlsbad, CA). pRBA-6 is a
pCR2.1-based plasmid containing a 597-bp fragment of the rat
-actin
cDNA amplified from rat liver cDNA using primers 5'-AGA CTA CCT
CAT GAA GAT CCT GAC C-3' and 5'-GGA CTG TTA CTG AGC TGC GTT TTA C-3',
respectively. p340XP is a pSK+ based plasmid containing the 340-bp
XbaI-PstI fragment from p1.4XB. pHCYP1A1B5
represents the 5' BamHI fragment of the human CYP1A1
cDNA (including 18-bases of the 5' untranslated region, the
complete 1563-base open reading frame, and 130 bases of the 3'
untranslated region) (8).
UGT1A7/UGT1A6 Promoter Cloning--
A rat genomic DNA library
(1 × 106 independent plaque-forming units) was first
screened using Escherichia coli K802 as the host strain and
pRUGT1.6 insert labeled to high specific activity (>1 × 109 dpm/µg) by random hexamer priming as described,
yielding clones RPT6,
RPT12, and
RPT19. One of these,
RPT6,
also hybridized with a probe prepared from pLC14 (+9/+697). The library
was rescreened using the pLC14 +9/+697 insert as probe, but a large
number of clones were hybridized. These clones appear to correspond to
UGT1A7-like exons. To narrow the number, the filters were
rehybridized using a probe prepared from the
EcoRV-EcoRI fragment flanking the
UGT1A6 locus, yielding phage clone
VIP-14. Large-scale
phage DNA purification was carried out as recommended by
CLONTECH. The nucleotide sequence of a 1.4-kbp
XhoI-BamHI fragment from
RPT-6 was determined
using the dideoxy chain termination method with a commercially
available kit (SequenaseTM, U.S. Biochemical Corp.) and
custom-made oligonucleotide primers.
RNA Isolation and Primer Extension Analysis--
Total RNA was
isolated by the acid phenol method (19) from livers of rats receiving
either vehicle alone (1 ml/kg 30% polyethylene glycol-1450 (v/v) for 3 days), oltipraz (90 mg/ml, 300 mg/kg/day for 3 days), or
-naphthoflavone dissolved in corn oil (12 mg/ml, 80 mg/kg/day for 3 days). Poly(A)+ RNA was extracted by cellulose poly(dT) affinity
chromatography (Boehringer Mannheim,). Two non-overlapping UGT1A7
antisense primers (6.375 and 6.376) were end-labeled with 32P using [
-32P]ATP and T4 bacteriophage
polynucleotide kinase to
1 × 109 dpm/µg and
purified by centrifugation through Chroma Spin columns (CLONTECH). Primers (
1 × 105
dpm/assay) were hybridized to poly(A)+ RNA (10 µg) from each group
for 16 h at 42 °C in 40 mM PIPES, 1 mM
EDTA, 0.4 M NaCl, and 40% formamide. The primer-annealed
RNA was then precipitated and dissolved in 1 µl of deionized water
prior to extension using AMV-reverse transcriptase (25 units, Promega)
in 10 µl of 1× AMV-reverse transcriptase buffer (Promega) containing
0.2 mM each dNTP, RNasin (20 units, Promega), and 0.5 µg
of actinomycin D. The products were analyzed on a 7 M urea,
8% polyacrylamide sequencing gel.
RNase Protection--
A 32P-labeled riboprobe
corresponding to the antisense strand of the sequence between the
XbaI and PstI sites (positions 337 and +12 with
respect to the UGT1A7 translation start) was synthesized in a reaction
containing NotI-linearized p340XP (0.25 µg), 1× riboprobe
synthesis buffer (supplied by Promega), 10 mM
dithiothreitol, RNAsin (20 units), ATP/GTP/UTP (5 mM each),
[
-32P]CTP (50 µCi, 12 µM), and T7 RNA
polymerase (20 units, Promega) in a total volume of 20 µl for 60 min
at 37 °C. The reaction was treated with RQ1 DNase (2 units, Promega)
for 30 min, and the riboprobe was purified by centrifugation through a
spin column (5'
3', Inc., Boulder, CO). Riboprobe (5 × 105 cpm) was mixed with yeast tRNA, control rat liver RNA,
or oltipraz-treated rat liver RNA (10 or 50 µg) in 50 µl containing
40 mM PIPES, 1 mM EDTA, 0.4 M NaCl,
and 40 or 80% formamide. The samples were treated with 300 µl of
RNase (300 mM NaCl, 10 mM Tris-Cl, pH 7.4, 5 mM EDTA, 2 µg/ml RNase T1, 40 µg/ml RNase A) for 30 or
60 min. After digestion with proteinase K (100 µg/tube), the products were phenol-extracted and ethanol-precipitated prior to analysis on a 7 M urea, 8% acrylamide, 0.5× Tris-borate-EDTA gel.
Nuclear Runoff Assays--
The procedure used for hepatic nuclei
isolation was based on that of Lee et al. (20). Adult male
Sprague-Dawley rats (n = 3/group) were euthanized
12 h after treatment with a single dose of either vehicle (30%
polyethylene glycol-1450, 3 ml/kg, intragastric), oltipraz (90 mg/ml
suspension, 3 ml/kg, intragastric), or -naphthoflavone (12 mg/ml in
corn oil, 5 ml/kg, intraperitoneal). Livers were perfused in
situ with 50 ml of ice-cold 0.9% saline and removed, and 3-g
samples were minced into small pieces with scissors in 3 volumes of 0.3 M sucrose in Buffer A (15 mM Tris-HCl, pH 7.4, 15 mM NaCl, 60 mM KCl, 2 mM EDTA, 1 mM dithiothreitol, and 0.5 mM
phenylmethylsulfonyl fluoride). After a brief homogenization using a
Polytron homogenizer (set on low speed), samples were completely
homogenized in 50-ml Potter-Elvjeham glass homogenizing tubes with a
drill-driven Teflon pestle (three passes), and nuclei were then
isolated by sucrose gradient centrifugation as described. The final
nuclear pellet was suspended in 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol, and 40%
glycerol, and stored in aliquots of 200 µl at
70 °C. Nuclei
concentrations were between 20 and 26 × 106
nuclei/200 µl. For nuclear runoff assays, nuclei were thawed, mixed
with 50 µl of a 5× buffer containing 25 mM Tris-Cl, pH
7.5, 12.5 mM MgCl2, 0.75 M KCl,
12.5 mM dithiothreitol, 2.5 mM each of ATP,
CTP, and GTP, 100 µCi [32P]UTP (800 Ci/mmol), and
incubated at room temperature for 15 min. RQ1 DNase (10 units) was
added, and reactions were incubated for an additional 5 min. Yeast tRNA
(50 µg) was then added, and labeled nascent RNA was isolated by the
method of Fei and Drake (21). RNA (8 × 106 cpm) was
hybridized to Nytran membranes containing 5 µg of immobilized, XhoI-linearized pRBA-6, pHCYP1A1B5, pLC14 +9/+697, and pSK+
in 7 ml of 10 mM Tris-ethane sulfonic acid, pH 7.4, 10 mM EDTA, and 0.2% SDS for 48 h at 65 °C. Membranes
were washed and exposed to film (7-day exposure at
80 °C with
medium intensifying screen) and then to a Molecular Dynamics
phosphorimaging cassette (1 day). Exposure were quantified using
ImageQuant software (Molecular Dynamics, Sunnyvale,
CA).
Northern Analyses--
Northern analyses of total RNA isolated
from liver tissue (0.3 g) or primary rat hepatocytes (8 × 106 cells/100-mm dish) was carried out as described (8).
Probes were prepared from DNA inserts purified by low melting point
agarose electrophoresis and labeled with [-32P]dCTP by
random priming (22).
Transient Transfection of Primary Rat
Hepatocytes--
Hepatocytes (8 × 105) isolated from
collagenase-perfused livers of male Sprague-Dawley rats were plated on
gelatin-coated plastic tissue culture dishes (35 mm), and 6 h
later, the medium was removed and cells were washed once with
serum-free medium (Williams E containing 0.6 µg/ml insulin, 100 µg/ml transferrin, 1 µM dexamethasone, fungizone, and
gentamicin). LipofectinTM (7.5 µg/dish) mixed with
reporter plasmid (1.5 µg) in 1.5 ml of serum-free medium was then
added to the plate. After overnight (12 h) incubation in a
humidified, 5% CO2 atmosphere, the medium was removed and
replaced with fresh serum-free medium containing the inducing agents at
the indicated concentration. Twenty-four h later, the medium was again
removed and replaced with fresh inducer-containing medium. Cells were
harvested at 48 h following the start of drug treatment.
Luciferase activity was measured using the Luciferase Assay System
(Promega) and a Berthold Lumat LB9501 luminometer.
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RESULTS |
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Effect of Oltipraz and -Naphthoflavone on the Rate of UGT1A7
Transcription--
To investigate the mechanism of the
inducer-associated increases in liver UGT1A7 mRNA level, nuclear
runoff assays were carried out using nuclei isolated from rats 12 h after treatment with vehicle (30% polyethylene glycol), 250 mg/kg
oltipraz or 80 mg/kg
-naphthoflavone. The Northern analysis of
-actin, cytochrome P4501A, and UGT1A7 transcripts at this time point
is shown in Fig. 1A.
Quantitation by phosphorimaging showed that oltipraz and
-naphthoflavone elevated the CYP1A mRNA 10- and 173-fold, respectively, and the UGT1A7 mRNA 19- and 43-fold.
-actin RNA levels were only slightly affected (2.5- and 2.8-fold for oltipraz and
-naphthoflavone, respectively).
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Isolation and Sequencing of the LC14 Unique Exon and Identification
as UGT1A7--
To learn more about the mechanism(s), we isolated four
genomic DNA clones from the UGT1A7/UGT1A6 locus using 5'
fragments of the UGT1A6 and/or LC14 cDNAs as probes as described
under "Experimental Procedures." The overlap relationships of these
clones (designated RPT6,
RPT12,
RPT19, and
VIP14) was
determined by restriction site mapping of XhoI,
BamHI, and EcoRI sites (Fig.
2A) together with Southern
analysis. Two of the clones,
RPT6 and
VIP14, hybridized with both
the UGT1A6 and LC14 probes. Together the cloned inserts form a contig
of
27 kb. A detailed restriction map is included in Fig.
2B to establish the identity of the DNA investigated in this
study. In agreement with the report of Emi et al. (3), the
UGT1A6-amino-terminal coding exon was localized to a 5.1-kbp internal
EcoRI fragment of
RPT6 immediately 5' of two small
EcoRI fragments (0.5 and 0.9 kb). The UGT1A6*
leader exon was localized by the presence of HindIII,
PvuII, and HindIII sites 2 kb 5', 0.1 kb 5', and
0.3 kb 3' of the EcoRI site in the exon, respectively (Fig.
2B).
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Determination of UGT1A7 Transcription Start Point--
To define
the UGT1A7 transcription unit, we carried out primer
extension and RNase protection experiments. For primer extension, two
antisense primers, 6.375 and 6.376, with an offset of 32 bases, were
utilized for analysis of poly(A)+ RNA from the livers of vehicle-,
oltipraz-, and -naphthoflavone-treated rats. The results are
presented in Fig. 4. As expected, the
products were only visible in samples from inducer-treated animals,
especially with
-naphthoflavone. In these samples, similar ladders
of extension products with the expected length differential of 32 bases
were seen. In both reactions, the longest product maps to a guanosine
residue at position
85 from the translation start codon. This residue
is designated as base +1 in Fig. 3 and maps 11 bases upstream of the
first base in one of the cDNA clones isolated from RALA255-10G
LCS-3 cDNA library (clone LC11).
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Induction of UGT1A7 and CYP1A mRNA in Primary Cultures of Rat
Hepatocytes--
Analysis of the mechanism(s) of the inducer-mediated
increases in UGT1A7 transcription requires an appropriate
cellular model. Previous studies carried out with an immortalized
SV40-transformed adult rat hepatocyte cell line (RALA LCS) indicated
high constitutive UGT1A7 mRNA expression compared with in
vivo expression. Fig. 6 compares the
in vivo and in vitro responses to Ah receptor
agonists and oltipraz at the level of mRNA. The low constitutive
expression of UGT1A7 mRNA observed in vivo in livers of
untreated rats (Fig. 6, lane 4) is maintained in untreated
primary rat hepatocytes (lane 1). Treatment with aryl
hydrocarbon receptor agonists and with oltipraz leads to a strong
stimulation of UGT1A7 mRNA both in vivo (Fig. 6,
lanes 6 and 5, respectively) and in primary
hepatocytes (lanes 2 and 3, respectively).
-Actin mRNA was highly expressed both in vivo and in
hepatocytes in vitro but was not affected by either
treatment. Some qualitative differences were observed in the
constitutive expression and inducibility of the CYP1A RNAs as detected
using the human CYP1A probe. Compared with in vivo, oltipraz
was not as effective at inducing the CYP1A1 and CYP1A2 mRNAs in
primary hepatocytes in vitro. Overall, the data lend support
for the usefulness of primary rat hepatocytes as a cellular model for
investigating mechanisms of UGT1A7 transcriptional
control.
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Effect of 3-Methylcholanthrene and Oltipraz on Luciferase
Expression Controlled by UGT1A7--
The above studies predicted that
the DNA flanking the UGT1A7 exon should act as an
orientation-dependent promoter with cis regulatory elements controlling UGT1A7 transcription. To investigate this, an XhoI-PvuII fragment of RPT-6
corresponding to bases
965 to +56 was cloned in either orientation
upstream of the luciferase reporter gene of pGL3-Basic, and the
resulting constructs were transfected into primary rat hepatocytes.
Lysates of cells transfected with the promoter fragment in the correct
orientation exhibited more than 50-fold higher luciferase activity than
cells transfected with the reverse construct. The activity was not
affected by oltipraz treatment, but it was increased 2-3 fold by
3-methylcholanthrene (Fig.
7A). Similar results were
observed for a reporter plasmid containing a 660-base 5' extension
(p-1600/+56 1A7-Luc). However, the base luciferase vector itself
(pGL3-Basic) yielded
3-fold inducibility by 3-methylcholanthrene
(Fig. 7B). Similar "inducibility" was observed with
pGL3-Promoter plasmid (data not shown), suggesting that the
3-methylcholanthrene responsiveness is not dependent on the
UGT1A7 sequence but rather on a sequence present in the vector itself. In contrast, experiments with hepatocytes transfected with an analogous pGL3-based UGT1A6 luciferase reporter
plasmid (pSTM11) showed much greater responsiveness to
3-methylcholanthrene; unlike p-965/+56 1A7 Luc, pSTM11 was responsive
to oltipraz.
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DISCUSSION |
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Work in two different laboratories now conclusively supports the existence of two different PAH- and oltipraz-inducible forms of UDP-glucuronosyltransferase in rat liver (3, 9, 18, 23). The two forms are encoded by the UGT1A6 and UGT1A7 loci and have distinct substrate specificities toward planar phenols. Given the close physical proximity of the two loci and the many parallels in their patterns of mRNA expression (e.g. repressed constitutive expression and high inducibility by both PAHs and oltipraz), we expected that the genes would share certain characteristics involving exon structure and organization and transcriptional initiation. Instead, key differences between the two PAH-regulated UGT genes stand out.
One difference involves the total number of exons composing the
complete transcription unit of each gene: six exons for
UGT1A6 and five for UGT1A7. The difference lies
in the number of exons coding for the unique 5' sequence of each
respective mRNA. UGT1A6 uses two exons for this purpose,
including a short 142-bp non-coding leader exon (labeled
UGT1A6* in Fig. 2) and a longer exon containing the first
845 bp of the UGT1A6 coding region (labeled UGT1A6 in Fig. 2). Our
primer extension and RNase protection data demonstrate that
UGT1A7 differs from UGT1A6 in utilizing only a
single exon to code for its entire unique 5' terminal sequence. This
arrangement predicts that the DNA flanking the long UGT1A7
unique amino-terminal-coding exon functions as a gene promoter. In
accordance with this, a fragment representing bases 965 to +56 of
UGT1A7 promoter drove high expression of a downstream
luciferase reporter when inserted in the correct (5'-3') but not the
reverse (3'-5') orientation. UGT1A7, therefore, possesses
the 5 exon structure that is more typical of UGT1 genes such as
UGT1A1, UGT1A4, and
UGT1A63 (2).
Another key difference between the UGT1A6 and 1A7 loci is the utilization by UGT1A7 of a complex of transcription start sites, clustered in a 45-bp region between 85 and 40 bases upstream of the translation start codon. Regardless of the site of initiation within this cluster, all transcripts are predicted to encode the full-length UGT1A7 protein, with the first AUG codon being the normal translation start site of UGT1A7. The basis for this heterogeneous initiation appears to be related to the lack of an efficient TATA box to direct precise transcriptional initiation. The region flanking the complex of transcription sites is enriched in TA residues, which are postulated to serve as weak TATA factor binding sites. The UGT1A7 promoter does not appear to be TATA-independent (housekeeping type) because there are no Sp1-binding sites and the promoter region overall is low in GC dinucleotide content. Furthermore, similar to other TATA-dependent genes, UGT1A7 possesses a sequence in its near upstream region closely matching a functional CCAAT enhancer-binding protein in the rat apolipoprotein B gene (24). The lack of a strong transcriptional initiating sequence may account for the lower relative abundance of UGT1A7 mRNA compared with UGT1A6 in livers of PAH- and oltipraz-treated rats (9) and the low or undetectable expression of UGT1A7 mRNA in human liver samples (25).
One major goal of research is to identify transcription factors
regulating the expression of rat UGT1A7, because variation in such factors may underlie variability in UGT1A7
expression and thereby alter individual susceptibility to
PAH-associated toxicities. The UGT1A7 promoter contains sequences
closely matching (2 mismatches) known functional or consensus binding
sites for hepatic nuclear factor 1 (rat glutathione
S-transferase Ya gene) (26), hepatic nuclear
factor 3, hepatic nuclear factor 4 (27), and activator protein 1. Further analysis is necessary to determine whether these sites are
functionally involved in the control of UGT1A7 gene
expression. Computer-aided inspection of the sequence failed to uncover
any close matching ARE or XRE sequences; these elements were previously
implicated in the regulation of other phase 1 and/or phase 2 metabolizing enzymes by PAHs (15, 18) and oltipraz (16). Consistent
with these negative findings, no PAH or oltipraz inducibility (above
the 2.5-fold level observed with the control luciferase vector) was
conferred by the UGT1A7 promoter and 5' flanking sequence. Clearly,
UGT1A7 lacks an XRE in an analogous position to that of
UGT1A6.
Because the nuclear runoff assays indicate that the effects of PAHs and oltipraz on UGT1A7 mRNA are due primarily to transcriptional activation, the data suggest that the cis elements mediating this activation are located either 5' or 3' of the region studied in this report. One possibility is that the inducibility of UGT1A7 is controlled by the XRE located 7 kb downstream in the UGT1A6 promoter region. This model is attractive because it could explain the concordant patterns of UGT1A6/UGT1A7 expression in various species. In mice and rabbits (and apparently humans), expression of both UGT1A6 and UGT1A7 is high in the constitutive state, and each is relatively refractory to inducing agents (28, 29). In rats, both UGT1A6/1A7 have low constitutive expression and are highly responsive to the inducing agents. The loss of transcriptional inducibility of two genes, UGT1A6 and 1A7, is most simply explained by a mutation involving only a single regulatory element. Another possibility, however, is that their inducibility by PAHs or oltipraz does in fact occur through distinct regulatory elements. We are currently testing the intronic region of the UGT1A7 gene for evidence of inducer-responsive enhancers or repressors.
In contrast to PAHs, the sequence mediating the responsiveness of UGT1A6/1A7 to oltipraz remains unknown. Our transient transfection analysis with the UGT1A6 luciferase reporter provides the first evidence that the UGT1A6 promoter flanking sequence contains oltipraz-responsive transcriptional enhancer elements. It is not clear whether this element corresponds to the XRE element previously identified by Emi et al. (3) or some other sequence motif (i.e. ARE). Evidence is appearing that oltipraz or perhaps one of its metabolites may be a weak activator of the arylhydrocarbon receptor (30). These findings are further supported by our study, showing transcriptional stimulation of CYP1A genes and increased CYP1A1/1A2 mRNA levels following oltipraz treatment in vivo and in rat hepatocytes in vitro.
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ACKNOWLEDGEMENTS |
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We thank Fay Kessler for the nucleotide sequence of UGT1A7 and Russell Prough and S. McGill for their gift of the pSTM11 plasmid. We also thank Todd Stravitz and Bin Gao for helpful discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health NIEHS Grant 1R29ES07762.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF039212.
To whom correspondence should be addressed: Box 980613, Dept. of
Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA 23298. Tel.: 804-828-1022; Fax: 804-828-1532; E-mail: jritter{at}hsc.vcu.edu.
1 The abbreviations used are: UGT, UDP-glucuronosyltransferase; PAH, polycyclic aromatic hydrocarbon; ARE, antioxidant response element; XRE, xenobiotic response element; PIPES, 1,4-piperazinediethanesulfonic acid; bp, base pair(s).
2 LC14 is encoded by a transcription unit beginning with the seventh unique amino-terminal-coding exon upstream of the shared carboxyl-terminal-coding exons in the UGT1 gene complex; it is therefore named UGT1A7. For an update of the current UGT nomenclature, see Ref. 10.
3 A species difference exists in the number of exons for the UGT1A6 gene (six for rat, five for human).
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