(Received for publication, May 4, 1995; and in revised form, July 31, 1995)
From the
The antioxidant response element (ARE) found in the 5`-flanking
region of the rat quinone reductase gene has been further characterized
by mutational and deletion analysis. The results indicate that the
31-base pair ARE, which contains a 13-base pair palindromic sequence,
can be further separated into three regions, all three of which are
required for elevated basal level gene expression. These three regions
include the proximal and distal half-sites as well as a 3`-flanking
region consisting of 4 adenine nucleotides. Neither the proximal nor
the distal half-site alone mediates transcriptional activation by
-naphthoflavone. However, when placed together the two half-sites
restore responsiveness to the inducer. Interestingly, the presence of
only 1 of the 4 adenine nucleotides in the 3`-flanking region of the
proximal half-site is required for responsiveness to the inducer. Point
mutations within the ARE indicate that several nucleotides in both the
proximal and distal half-sites are required for basal level gene
expression. Electrophoretic mobility shift analysis using the ARE as
the probe indicates that enhancers found in the glutathione S-transferase Ya and P genes recognize a similar trans-acting factor(s) found in crude nuclear extracts from
human Hep G2 cells. Further, this complex can be detected in nuclear
extracts from rat liver and rat hepatoma cells but not in mouse Hepa
1c1c7 cells or in human HeLa cells. The ARE-nucleoprotein complex can
also be detected in F9 cells which lack significant levels of Jun/Fos
proteins. Although the rat ARE resembles the human quinone reductase
ARE which contains a consensus TRE, the 2-nucleotide change in the core
sequence (TGACTCA versus TGACTTG) eliminates the high affinity
TRE motif in the rat ARE. The rat ARE forms a nucleoprotein complex in
Hep G2 and other cells with different properties than AP-1.
NAD(P)H:quinone oxidoreductase (DT-diaphorase, quinone
reductase) is a phase II drug-metabolizing enzyme found in liver and
other tissues and is inducible by a wide variety of compounds including
phenolic antioxidants, planar aromatic compounds and
TCDD()(1, 2, 3) . In order to
further understand the molecular mechanisms underlying the constitutive
and inducible expression of this gene, two response elements found in
the 5`-flanking region of the rat gene have been
identified(4) . One of these elements is the xenobiotic
response element (XRE) found in the CYP1A1 and rat glutathione S-transferase Ya subunit genes (5, 6) .
The second element is the antioxidant response element (ARE) which
is responsible for basal and inducible expression of the
gene(4) . We have demonstrated that diverse xenobiotics such as
-naphthoflavone (
-NF), tert-butylhydroquinone (t-BHQ), hydrogen peroxide, and
12-O-tetradecanoylphorbol 13-acetate (TPA) can
transcriptionally activate gene expression through the ARE (4, 7) . A high affinity DNA-protein binding complex
exists in crude nuclear extracts of Hep G2 cells based on DNase I
footprinting, methylation interference and protection assays, and
electrophoretic mobility shift analyses(7) .
The ARE for the human quinone reductase gene has been identified and shown to contain a consensus TRE sequence within the ARE(8, 9) . However, the rat ARE does not contain a high affinity binding site for in vitro synthesized c-Jun and c-Fos, nor does the ARE bind Jun/Fos proteins found in crude nuclear extracts from HeLa cells(7) .
Similar studies have been performed for the rat glutathione S-transferase Ya subunit gene, and ARE and XRE motifs have also been identified and characterized as well(6, 10, 11) . Thus, the coordinate induction of these two genes may be at least partially explained by the presence of these two elements.
The mouse glutathione S-transferase Ya subunit gene is regulated by an electrophile responsive element (EpRE) which resembles the rat Ya ARE in 39 of 41 nucleotides(12) . A second glutathione S-transferase, glutathione transferase P, has also been identified which is induced by phenolic antioxidants(13) . A response element (GPEI, glutathione transferase P enhancer I) in the 5`-flanking region of this gene mediates constitutive expression in hepatoma cells (14, 15) . Analysis of this enhancer reveals a significant degree of sequence identity with segments of the AREs found in the rat quinone reductase and the rat and mouse glutathione S-transferase Ya subunit genes (4, 6, 12, 15).
In this communication, we determine the specific nucleotides within the rat quinone reductase ARE sequence that are required for high basal level expression and xenobiotic responsiveness. We demonstrate that a high affinity DNA-binding protein complex is present in nuclear extracts of Hep G2 cells, rat liver, a rat hepatoma cell line (H4IIEC3), and mouse F9 cells, but not in extracts from HeLa or Hepa 1c1c7 mouse hepatoma cell lines. Finally, we demonstrate that the GPEI enhancer of the rat glutathione S-transferase P gene and the rat quinone reductase ARE recognize a similar nucleoprotein(s), as judged by electrophoretic mobility shift assays, found in nuclear extracts from rat liver, Hep G2, rat H4IIEC3, and mouse F9 cells.
Nuclear extracts from all five cell lines were prepared according to the procedure of Dignam et al.(16) . After dialysis for 4-5 h, the extracts were clarified by centrifugation and frozen at -80 °C. The extracts were stable for at least 6 months when stored under these conditions.
Transfection of Hep G2 cells was
performed by a modification of the calcium phosphate-glycerol shock
procedure(4, 17) . To correct for transfection
efficiency, the plasmid pTK (Clontech) was cotransfected with each
CAT construct.
-Galactosidase assays were performed as described
previously(7) .
Figure 1:
Functional analysis of mutants of the
rat quinone reductase ARE. Hep G2 cells were cotransfected with each
construct and with pTK- and either treated with Me
SO (solid bars) or with 50 µM
-NF (striped
bars) for 20 h. CAT activity is the mean ± S.D. of three
separate transfections and is reported relative to
-galactosidase
activity to correct for transfection efficiency. (In one typical
experiment, the wild type pARE-CAT construct yielded 395 ± 87
pmol of acetylated chloramphenicol/µg of protein/h.) MP,
minimal promoter (p-164CAT). A, mutations were introduced in
the proximal half-site (M1), the distal half-site (M2), or in the terminal ``GC'' residues in the
proximal half-site. (The mutated sequences are underlined.) B, deletion analysis of the 3`-terminus of the ARE. C, deletion analysis of the 5`-terminus of the ARE. D, analysis of constructs containing multiple copies of either
the proximal or distal half-sites.
Deletion mutants in the 3`-terminus of the ARE were prepared in
order to determine the minimum sequence required for functional
activity. Deletion of 5 nucleotides at the 3`-end of the pARE-CAT
construct (i.e. pARE26) results in a construct which retains
high basal and inducible CAT expression (Fig. 1B).
Deletion of one, two, or three additional adenine nucleotides at the
3`-end (pARE25, pARE24, and pARE23) results in constructs which
progressively lose basal level gene expression. However, all three
constructs retain inducible activity. Deletion of the final adenine
nucleotide results in a construct (pARE22) that is only slightly
responsive to -NF (1.3-1.5-fold). Thus, the terminal adenine
in the sequence 5`-GTGACTTGGCA-3` represents the 3` boundary of the
minimal sequence required for inducible activity. In contrast, the
highest basal level expression occurs when all 4 adenines are present.
pARE23 is 5-7-fold inducible with
-NF while the other
constructs (pARE24, pARE25, and pARE26) are only about 3-fold
inducible. However, the absolute level of inducible activity in cells
transfected with pARE23 is lower than that observed in cells
transfected with the wild type pARE-CAT construct.
A deletion
analysis of the 5`-end of the ARE was performed using the minimum
sequence required for inducible activity as the 3`-end. Deletion of 2
or 5 nucleotides in the 5`-end results in constructs (pARE21 and
pARE18) which retain inducible expression of CAT activity (Fig. 1C). Deletion of 8 or 9 nucleotides in the 5`-end
yields constructs containing only the complete downstream half-site.
When these constructs (pARE15 and pARE14) are transfected into Hep G2
cells no increase in basal level or inducible activity is detected.
Therefore, -NF responsiveness requires the presence of several
residues found in the 5`-region flanking the proximal ARE half-site.
Since a single copy of the proximal half-site was not responsive to
-NF, a construct containing two copies of this sequence in a head
to tail orientation was made in order to determine if
-NF
responsiveness could be restored. When this plasmid (pARE-2X15) is
transfected into Hep G2 cells, no increase in basal level expression of
CAT activity is found (Fig. 1D). However, transfected
cells treated with
-NF have a 2.5-3.0-fold higher level of
CAT activity. Thus, the presence of two copies of the proximal ARE
half-site restores
-NF responsiveness. Interestingly, the level of
induction is lower for this construct than for pARE23 (i.e. 2.5-3.0-fold compared to 5-7-fold induction). Since
the wild type ARE exists as a palindrome, additional constructs were
made which contained two copies of either the distal or proximal
half-sites arranged in a palindromic orientation. The distal half-site
arranged as a palindrome does not mediate an increase in basal level or
inducible gene expression (Fig. 1D). However, a
palindromic orientation of the proximal half-site results in an
increase in basal level CAT expression (6-10-fold) as well as
-NF responsiveness (5-7-fold).
Figure 2: Single point mutant analysis of the quinone reductase ARE. The nucleotide sequence of the ARE is numbered from -11 to +19 with 0 being the adenine nucleotide which is at the center of the palindrome. Mutations were made as follows: A and T were changed to G; C and G were changed to A. Mutant constructs were analyzed in Hep G2 cells as described in Fig. 1.
In contrast, several mutations
made in the downstream half-site abolish basal level expression and
-naphthoflavone responsiveness (Fig. 2). When nucleotides
at positions T
, G
, or A
are
mutated, both basal level and inducible expression is completely
abolished. Mutations at positions C
and C
result in constructs which also have decreased basal activity
relative to the minimal promoter (22 and 13% of the wild type ARE
activity). However, both mutants C
and C
are
responsive to
-naphthoflavone (2.5- and 2.0-fold, respectively).
Additional mutations made at positions G
, G
,
G
, A
, and C
result in constructs
with varying degrees of basal level expression all of which retain
responsiveness to the inducer (Fig. 2). Although both half-sites
are required for full basal level gene expression, only 3 residues in
the downstream half-site appear to be absolutely required for high
basal and inducible expression, 5`-gTGActtggca-3`.
Figure 3:
Electrophoretic mobility shift analysis
using mutant ARE oligonucleotides as competitors. A 150-fold molar
excess of synthetic double stranded oligonucleotides containing the
point mutants described in Fig. 2were incubated with nuclear
extract from untreated Hep G2 cells as described under
``Experimental Procedures.'' The arrow points to the
specific ARE-nucleoprotein complex. Lanes 1-15 contained
15 µg of Hep G2 nuclear extract. Lanes 2-15 contained the following double-stranded oligonucleotides: lane
2, wild type ARE; lane 3, M; lane 4, M
; lane 5,
M
; lane 6, M
; lane 7,
M
; lane 8, M
; lane 9,
M
; lane 10, M
; lane 11,
M
; lane 12, M
; lane 13;
M
; lane 14, M
; lane 15,
M
.
Figure 4:
The GST-P enhancer GPEI recognizes the
same nucleoprotein complex in Hep G2 cells and is functionally an ARE. A, electrophoretic mobility shift analysis of the ARE and
competition with GPEI was performed as described in Fig. 3. Lanes 3-7 contained a 150-fold molar excess of the
following double-stranded oligonucleotides: lane 3, quinone
reductase ARE; lane 4, glutathione S-transferase Ya
ARE; lane 5, human collagenase TRE; lane 6,
glutathione transferase GPEI; lane 7, random oligonucleotide. B, CAT assays of lysates from Hep G2 cells transfected with
constructs containing the ARE from the rat quinone reductase gene, the
ARE from the rat glutathione S-transferase Ya gene, and the
GPEI from the glutathione transferase P gene. See Fig. 1for
details. Cells were treated for 20 h with either MeSO (solid bars), 60 µMt-BHQ (striped
bars) or 100 nM TPA (stippled bars). CAT
activity is reported relative to
-galactosidase
activity.
Figure 5: Electrophoretic mobility shift analysis comparing the ARE and TRE. A, nuclear extracts from each cell line were incubated with either the ARE or TRE as described under ``Experimental Procedures.'' Lanes 1-6, ARE probe; lanes 7-12, TRE probe. Lanes 1 and 7, no extract; lanes 2 and 8, Hep G2 nuclear extract (30 µg); lanes 3 and 9, rat hepatoma H4IIEC3 nuclear extract (20 µg); lanes 4 and 10, mouse hepatoma 1c1c7 nuclear extract (40 µg); lanes 5 and 11, mouse embryonal carcinoma F9 nuclear extract (30 µg); lanes 6 and 12, human epitheloid carcinoma HeLa nuclear extract (10 µg). B, competition studies were performed using nuclear extracts from H4IIEC3 cells and a 150-fold molar excess of the following double stranded oligonucleotides: lane 1, nuclear extract; lane 2, rat quinone reductase ARE; lane 3, rat glutathione S-transferase Ya subunit ARE; lane 4, glutathione transferase GPEI; lane 5, random oligonucleotide; lane 6, TRE; lane 7, ARE-M1; lane 8, ARE-M2; lane 9, human quinone reductase ARE. C, as in B except the nuclear extract was from 1c1c7 cells. D, as in B except the nuclear extract was from F9 cells. E, as in B except the nuclear extract was from HeLa cells. F, as in B except the nuclear extract (10 µg) was from rat liver.
Interestingly, the human collagenase TRE was also found to compete (partially or completely) with the ARE in all instances including the F9 mouse embryonal carcinoma cell nuclear extracts (Fig. 4A and 5, B, C, D, and F). Since this cell line has been shown to contain low levels of AP-1(18, 19) , nuclear extracts from F9 cells were tested in the electrophoretic mobility shift assay using the TRE probe. As expected, very little binding to the TRE could be detected in extracts from F9 cells. Nuclear extracts prepared from Hep G2, H4IIEC3, Hepa 1c1c7, and HeLa cells all contain detectable TRE complexes. The retarded band from each cell line is specific since it is competed by an excess of unlabeled TRE, but not by the ARE or a random oligonucleotide (data not shown). The migration of the TRE complex in Hep G2 and H4IIEC3 cells is considerably different than in the Hepa 1c1c7 and HeLa cells (Fig. 5A).
Figure 6: Electrophoretic mobility shift analysis of the ARE/TRE probe. Hep G2 nuclear extract from untreated cells was incubated with the radiolabeled ARE/TRE probe (lanes 2-8). Lane 1 contained only the probe, 5`-TCTAGAGTCACAGTGACTCAGCAAAATCTGA-3`. The following oligonucleotides, at a 150-fold molar excess, were added prior to addition of the probe: lane 3, quinone reductase ARE; lane 4, human collagenase TRE; lane 5, mutant ARE/TRE; lane 6, rat Ya ARE; lane 7, GPEI; lane 8, random oligonucleotide.
The antioxidant response element found in the rat quinone reductase gene has been shown to be responsible for both basal level and inducible transcription in hepatoma cells(4) . It is composed of a palindromic sequence in which the two half-sites are separated from each other by a single nucleotide. Footprinting assays indicate that both the proximal and distal half-sites are protected from DNase I digestion by crude nuclear extracts from Hep G2 cells(7) . However, we previously showed by electrophoretic mobility shift competition assays and by methylation protection analysis that only the proximal half-site is required for high affinity binding of the ARE to a protein found in crude Hep G2 nuclear extracts(7) .
In this communication we have extended our
analysis of this enhancer by mutagenesis of nucleotides in the
``core sequence'' previously defined for the ARE found in the
rat glutathione S-transferase Ya subunit gene(11) .
Mutational analysis of the ARE indicates that neither the proximal nor
the distal half-site alone is sufficient for high basal level gene
expression. However, together in the intact enhancer they function
synergistically. Furthermore, additional adenine residues are required
in the 3`-flanking region for high level basal expression in Hep G2
cells. Mutations in the distal half-site decrease basal activity,
although some level of inducible activity is conserved as long as the
proximal half-site remains intact. In the absence of the distal
half-site, however, the proximal half-site alone does not function as
an enhancer in Hep G2 cells. In contrast, mutations in the proximal
half-site result in a complete loss of basal and inducible activity.
The organization of the rat quinone reductase ARE is similar to the
mouse glutathione S-transferase Ya subunit EpRE in that both
half-sites are required for constitutive and inducible
expression(20) . Results presented here indicate that mutations
in 3 residues found in the proximal half-site,
5`-gTGActtggca-3`, completely eliminate basal and inducible activity. However, a number of other residues in both
the distal and proximal half-sites are important for maximum basal and
inducible activity. Synthetic oligonucleotides containing mutations of
these three residues are no longer able to compete with the wild type
ARE sequence for the binding protein. Additionally, mutating C+10
which decreases basal expression to background also eliminates
competition with the wild type ARE. Taken together, these results
indicate that while the proximal half-site is essential for induction
by -naphthoflavone, the intact enhancer is required for maximum
basal and inducible activity.
These results are similar to results previously obtained for the ARE found in the glutathione S-transferase Ya subunit gene(11) . The slight functional differences between the quinone reductase and glutathione S-transferase Ya subunit gene ARE mutants may arise from the differences in the surrounding sequences both proximal and distal to the core sequences. The rat quinone reductase ARE is a more potent enhancer than the glutathione S-transferase Ya ARE (Fig. 4B), although the fold induction is the same(4, 11) . Regardless, both enhancers appear to bind to a similar nuclear factor(s) and both enhancers are responsive to the same stimuli, i.e. phenolic antioxidants, metabolizable planar aromatic compounds, and hydrogen peroxide. Similarly, the human quinone reductase ARE has also been shown to consist of two half-sites, both of which are required for functional activity(21) .
Based on results from in vitro assays such as DNase I and
methylation protection and interference footprinting assays as well as
functional assays deploying mutations of the wild type ARE, several
observations about the structure and function of the enhancer may be
made. Although only the proximal half-site is required for binding, at
least part of the distal half-site is required for maximal functional
activity. Further, point mutations of residues in the same relative
position in each half-site affect basal and inducible activities in
different ways, suggesting that the two half-sites are not equivalent.
Results presented here indicate that both basal level expression and
-NF responsiveness are greater when the two half-sites are
arranged in a palindromic orientation. Interestingly, using two copies
of a consensus TRE sequence, Okuda et al. (22) showed
that the palindromic orientation was 3-7 times more active in F9
and HeLa cells than the tandem repeat orientation.
It is interesting
to note that in F9 cells, which lack AP-1 activity and c-Jun, an
ARE-nucleoprotein complex with similar properties to the complex found
in Hep G2 cells can be detected. Furthermore, in F9 cells the mouse
glutathione S-transferase Ya EpRE (ARE) and the human quinone
reductase ARE are responsive to t-BHQ or -NF,
respectively(20, 21, 23) . Thus, the ARE is
responsive to inducers in the absence of AP-1. Electrophoretic mobility
shift assays conducted with nuclear extracts from treated Hep G2 cells
revealed no complexes that would be consistent with Jun/Fos binding to
the ARE(7) . Based on these results, we have concluded that
induction is mediated by a nuclear protein(s) expressed constitutively
which is distinct from Jun/Fos(7) .
In undifferentiated F9 cells, the only jun family member expressed is Jun-D(24, 25) . Jun-D and Jun-B have been shown, along with c-Fos, to be part of the human quinone reductase ARE-nucleoprotein complex in mouse Hepa 1 cells(9, 26) . All three Jun proteins contain a conserved cysteine residue in the DNA-binding domain which has been implicated in the redox regulation of Jun and Fos binding activity(25, 27) . The human ARE contains a perfect TRE motif and has been shown to bind Jun/Fos proteins in vitro and in nuclear extracts(7, 9, 26) . We and others have shown that the rat ARE does not contain a high affinity site for Jun/Fos proteins nor does it compete with the human ARE for Jun/Fos proteins(7, 28) . Therefore, the 2-nucleotide difference in the core sequence of the rat ARE is sufficient to abolish high affinity binding to Jun/Fos.
Recently, a number of transcription factors have been identified which recognize sequences similar to the ARE(29, 30, 31, 32, 33) . One of these, Maf, has been reported to bind to the AREs found in the rat and human quinone reductase, the rat glutathione S-transferase Ya, and the rat glutathione transferase P genes(34) . However, it is not known if any of these factors are present in rat liver or the cell lines which show nucleoprotein binding activity to the ARE.
A number of structural and functional similarities exist between the rat quinone reductase ARE and the glutathione transferase P GPEI enhancers(7, 15) . Both enhancers contain palindromic sequences which are required for full basal level gene expression. Electrophoretic mobility shift analyses indicate that along with the ARE from the rat glutathione S-transferase Ya gene, the GPEI also competes the nucleoprotein complex formed with the quinone reductase ARE. Constructs containing the GPEI enhancer inserted upstream from the minimal promoter CAT fusion gene are responsive to t-BHQ and TPA when they are transfected into Hep G2 cells. Recent evidence indicates that although Jun and Fos proteins bind GPEI, another binding factor also exists which represents the main mechanism for transactivation of the glutathione transferase P gene(35) . These results may provide a common link between these three rat genes which are elevated in persistent hepatocyte nodules(13, 36) . A transgenic rat model containing the upstream regulatory sequence of the GST-P gene fused to the CAT reporter gene has recently been described(37) . Liver foci and nodules produced by chemical carcinogens were found to express high CAT enzyme levels indicating that during hepatocarcinogenesis a trans-acting mechanism activates the expression of the GST-P gene. Preliminary results indicate that it is the GPEI motif which is required for tumor-specific expression of the GST-P gene(38) .
In summary, we have characterized further the quinone reductase ARE by identifying nucleotides that are required for basal and inducible activity. These nucleotides are similar to those previously identified for the rat glutathione S-transferase Ya subunit ARE. We have also demonstrated that nuclear extracts from human and rat hepatoma cell lines as well as from rat liver contain a similar nucleoprotein(s) which interact(s) with the rat ARE. The ARE-binding protein can be distinguished from AP-1 by its different mobility in gel shift assays, its presence in F-9 cells which lack AP-1 activity and by its unique nucleotide requirements for binding and functional activity. Finally, competition assays indicate that the enhancers from three rat genes (i.e. quinone reductase and the glutathione S-transferase P and Ya subunits) which are elevated in liver preneoplastic nodules recognize a similar nucleoprotein complex in rat liver, human Hep G2, and rat H4IIEC3 nuclear extracts. The purification, identification, and cloning of this factor will lead to an understanding of how this factor regulates gene expression as a response to the oxidative stress produced by a large number of chemical inducers.