From the Centre de recherche en cancérologie de l'Université Laval, Pavillon L'Hôtel-Dieu de Québec, Centre Hospitalier Universitaire de Québec, Québec G1R 2J6 Canada and Département de biologie, Université Laval, Québec G1K 7P4 Canada
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ABSTRACT |
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Hepatic cytochrome P450s play a critical role in the metabolism of hydrophobic xenobiotics. One of the major unsolved problems in xenobiotic metabolism is the molecular mechanism whereby phenobarbital induces hepatic enzymes, particularly CYP2B1 and CYP2B2 in rat liver. By using primary rat hepatocytes for transfection analyses, we previously identified in the CYP2B2 5'-flank a 163-base pair Sau3AI fragment that confers phenobarbital inducibility on a cat reporter gene and that has the properties of a transcriptional enhancer. Transfection experiments with sub-regions of the Sau3AI fragment now indicate that a central core together with an upstream or downstream accessory element within the fragment can confer phenobarbital responsiveness. One such accessory element, AF1, was identified and localized. DNase I footprinting analysis revealed the presence of a footprint overlapping this AF1 element. It also identified three other major protected regions, two of which are putative recognition sites for known transcription factors. Site-directed mutagenesis indicated that a putative glucocorticoid response element as well as a nuclear factor 1 site and an associated nuclear receptor hexamer half-site are essential for conferring maximal phenobarbital inducibility. Taken together, the results indicate that phenobarbital induction of CYP2B2 requires interactions among multiple regulatory proteins and cis-acting elements constituting a phenobarbital response unit.
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INTRODUCTION |
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Many different cytochrome P450s (CYPs)1 are involved in the hepatic metabolism of a wide variety of xenobiotic substances including drugs, plant metabolites, and chemical carcinogens (1, 2). The genes encoding these enzymes are either expressed constitutively or are induced by various chemicals (3). One of the major unsolved problems in the study of the induction of CYP proteins is the molecular mechanism whereby phenobarbital (PB) induces the closely related CYP2B1 and CYP2B2 forms in rat liver (4). Although it has long been known that PB induces CYP2B1 and CYP2B2 mRNAs by increasing transcription of their genes (5, 6), details of the transcriptional control of CYP2B1 and CYP2B2 have not been forthcoming. This is largely because, until recently, it was not possible to obtain PB induction of the endogenous CYP2B1 and CYP2B2 genes in cultured cells (4, 7, 8). We have transfected reporter gene constructs into cultured adult rat hepatocytes and localized, in the CYP2B2 5'-flanking region, a 163-base pair (bp) Sau3AI fragment that confers PB inducibility on a cat reporter gene (9). The Sau3AI fragment, which is situated between 2155 and 2317 bp upstream of the CYP2B2 transcription start point, in the vicinity of a liver-specific DNase I-hypersensitive site (10), has the properties of a transcriptional enhancer (9). The capacity of the Sau3AI fragment to confer PB responsiveness on a heterologous promoter has been confirmed in a quite different assay system involving in situ DNA injection into rat liver (11). Furthermore, the homologous region of the 5'-flanking region of the PB-inducible mouse Cyp2b10 gene contains a segment 91% identical to the rat CYP2B2 163-bp fragment that also confers PB inducibility on heterologous promoters and possesses the properties of a transcriptional enhancer (12).
The CYP2B2 163-bp Sau3AI fragment contains a functional nuclear factor 1 (NF1) site (9, 13) and recognition sites for other sequence-specific DNA binding factors present in rat liver nuclear extracts (9). In the present study, we sought to define the elements within the 163-bp Sau3AI fragment which confer PB responsiveness. Deletion constructs of the Sau3AI fragment, as well as constructs in which putative recognition sites for DNA binding factors had been mutated, were transfected into adult rat hepatocytes, and their effect in conferring PB responsiveness was analyzed. DNase I footprinting experiments were used to identify potential regulatory elements by defining interactions between rat liver nuclear proteins and the Sau3AI fragment. A putative glucocorticoid response element as well as an NF1 site and an associated nuclear receptor hexamer half-site were found to be essential for conferring maximal phenobarbital inducibility. Taken together, the results indicate that the Sau3AI fragment is a multicomponent enhancer and that multiple regulatory proteins and their cognate recognition sequences within it are critical for obtaining maximal PB responsiveness. Thus, the Sau3AI fragment constitutes a PB response unit (PBRU), analogous to the complex glucocorticoid response unit (GRU) required for glucocorticoid induction of transcription of the rat gene for phosphoenolpyruvate carboxykinase (PEPCK) (14-17).
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EXPERIMENTAL PROCEDURES |
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Materials and Animals--
Chee's medium for hepatocyte culture
as well as restriction and DNA-modifying enzymes were from Life
Technologies, Inc. The double-stranded CTF/NF1 consensus
oligodeoxyribonucleotide (oligo) (5'-CCTTTGGCATGCTGCCAATATG-3') was
from Promega or was purchased as complementary single-stranded oligos
from Life Technologies. Other oligos were also from Life Technologies,
except as noted. [-32P]ATP (6000 Ci/mmol),
[
-32P]dATP (3000 Ci/mmol),
[
-35S]dATP
S (1250 Ci/mmol),
[14C]dichloroacetylchloramphenicol (60 Ci/mmol), and
[3H]dichloroacetylchloramphenicol (30 Ci/mmol) were from
NEN Life Science Products. Male Sprague-Dawley rats (150-180 g) were
from Charles River Canada.
Isolation and Culture of Primary Hepatocytes, Transfection, PB Treatment, and Chloramphenicol Acetyltransferase (CAT) Assays-- The methods for hepatocyte isolation and culture, essentially those of Waxman et al. (18), as well as those for liposome-mediated transfection (19) and PB treatment have been described (9). Plasmids were purified using a plasmid purification kit (Qiagen). CAT activity was assayed by the method of Gorman et al. (20) and, occasionally, by that of Seed and Sheen (21).
Construction of Deletion and Point Mutants--
Sequential 5' or
3' deletions of the 163-bp Sau3AI fragment (2317/
2155)
were generated by restriction enzyme or Bal31 digestion (Fig. 1). The starting point for construction of such deletion mutants
was the pSa-Sa163 plasmid, obtained by subcloning the 163-bp
Sau3AI fragment into the SmaI site of pBluescript
KS (Stratagene). The pSa-Sa163 plasmid or similar pBluescript KS
derivatives containing specified portions of the CYP2B2
5'-flank were used to generate other deletions by polymerase chain
reaction-mediated amplification. To obtain
2257/
2208 (Fig. 1) the
starting pBluescript derivative contained
2257/
2012, and the
primers were KS (Stratagene) and oligo HX
(GATCCACTGTGCCAAGGTCAGGA
2226, lower stand; the
underlined nucleotides were added for convenience). In a series of
amplifications (Fig. 1), primers 2B2Nco (
2257 CATGGTGATTTCAGGCA) and
SK (Stratagene) were used as follows: to obtain
2257/
2207 the
starting pBluescript derivative contained
2317/
2207; to obtain
2257/
2188 it contained
2317/
2188, and to obtain
2257/
2172
it contained
2317/
2172. Point mutations were introduced into the
163-bp Sau3AI fragment using the Altered Sites II in
vitro Mutagenesis System (Promega). In the mutant oligos that
follow, substitutions in the wild-type sequence are indicated in
boldface: GREm (
2253
GTGATTTCAGGCGTGGACTCTGTACTT), NF1m1, synthesized by Michel
Lambert of this Research Center (
2218 TTGGCACAGTGCTTCCATCAACTTGA), NF1dm2 (
2224
CTGACCTTGGTACAGTGCTTC), HXm (
2232
GTACTTTCCTGAGATTGGCACAGT), HXm-NF1dm2 (
2232 GTACTTTCCTGAGATTGGTACAGT). Oligo HXm-NF1dm2
contains, in addition to the HXm mutation, a single mutation in the NF1 sequence; since it was used to mutate a Sau3AI fragment that
already carried NF1 m1, it generated a triple mutant sequence. A 3-bp tandem point mutation was also introduced into the AAAG core of the
Barbie box of the CYP2B2 promoter using oligo BBm (
94
AGTGAATAGCCAGCTCAGGAGGCGTGA). Deletion and point mutants
were subcloned by blunt-ended ligation into the EcoRV site
of the non-PB responsive Ev construct which contains 1681 bp of the
CYP2B2 5'-flanking region cloned upstream of the
cat reporter of pBSCAT (9). The strategy employed to obtain
deletions by Bal31 digestion and by amplification generated an additional 8-nucleotide sequence (5'-GGGGGATC) or, for the deletions
obtained with oligo HX, and additional 12-nucleotide sequence
(5'-GATCGGGGGATC), between the 3' end of the deleted portion of the
163-bp Sau3AI fragment and the EcoRV site used for subcloning. The normal orientation and integrity of the subcloned fragments in the reporter constructs were confirmed by DNA sequence analysis.
Nuclear Extracts and DNase I Footprinting Analyses--
Nuclear
extracts were prepared (22) from pooled livers of two or three
untreated or PB-treated (23) rats. Where noted, nuclear protein
extracts were fractionated by chromatography on heparin-Sepharose using
Hitrap Heparin columns (Amersham Pharmacia Biotech). The fragments used
for DNase I footprinting analyses were prepared from pSa-Sa163, or from
similar plasmids containing a mutated 163-bp Sau3AI
fragment, by releasing a 230-bp fragment with EcoRI and
EagI digestions. After 3' end-labeling using the Klenow
fragment of Escherichia coli DNA polymerase I or 5'
end-labeling using T4 polynucleotide kinase, the fragment was purified
by electrophoresis on a 5% polyacrylamide gel. DNase I footprinting
was performed (22) in 12.5 mM Hepes, pH 7.6, 70 mM KCl, 3.5 mM MgCl2, 5 µM ZnSO4, 1 mM EDTA, 5%
glycerol, 0.5 mM NaMoO4, 0.075 mM
Nonidet P-40, 0.5 mM dithiothreitol, 0.25 mM
phenylmethylsulfonyl fluoride containing 5 µg of poly(dI-dC).
Reaction volumes were typically 70 µl. Na2
-glycerophosphate, when present, was at 10-20 mM.
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RESULTS |
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Functional Analysis of the 163-bp Sau3AI
Fragment--
Transfection analysis was performed with subfragments of
the 163-bp Sau3AI fragment to identify sequences within it
that might confer PB responsiveness. Sequential deletions from the 5'
end up to the internal NcoI site (coordinate 2257) were
all active in conferring PB responsiveness, although the response
conferred by construct
2257/
2155 was reduced by about 2-fold as
compared with that of the full-length fragment (Fig.
1A, constructs with a common
3' end point of
2155 and 5' end points of
2290,
2283,
2273,
2264, or
2257). Further deletion from the 5' end, to an internal
RsaI site (coordinate
2230) or to
2180, led to complete loss of activity (Fig. 1A). Similarly, sequential 3'
deletions up to
2207 were also active, although the response
conferred by constructs
2317/
2188 and
2317/
2207 was again
reduced by about 2-fold as compared with that of the full-length
fragment (Fig. 1A, constructs with a common 5' end point of
2317 and 3' end points of
2172,
2188, or
2207). Further
deletion from the 3' end, to the RsaI site (coordinate
2231; Fig. 1A) or to
2253 (construct
2600/
2253; data
not shown), led to complete loss of activity.
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Functional Evidence for an Accessory Element--
The deletion
analysis presented above suggests the following model to account for
the PB responsiveness conferred by the 163-bp Sau3AI
fragment. A central core, together with an element or elements extending upstream of coordinate 2257 or with an element or elements extending downstream of coordinate
2207, can confer PB
responsiveness. In support of this model, although construct
2257/
2188 was inactive, construct
2257/
2172 was active in
eliciting a PB response (Fig. 1B). This result indicated
that there is a DNA sequence element between coordinates
2188 and
2172 that confers PB responsiveness when combined with the central
core. The element between
2188 and
2172 is insufficient to elicit
PB responsiveness by itself, because it is present in the inactive
2230/
2155 construct (Fig. 1A). Hence, it is an accessory
site for conferring PB responsiveness, and we have designated it as AF1
(Figs. 1B and 2).
Physical Evidence for Proteins Binding to AF1 and to Other Sites
within the 163-bp Sau3AI Fragment--
To ascertain whether an
accessory factor binds to the AF1 site, as well as to look for evidence
of other protein-DNA interactions, the 163-bp Sau3AI
fragment was subjected to DNase I footprinting analysis using rat liver
nuclear extracts. First, the fragment was labeled on the lower strand
at the 3' end (Fig. 3A). Analysis using crude extract
revealed a series of almost continuous footprints over some 85 bp
extending from about 2285 to about
2200 (Fig. 3A). Use
of heparin-Sepharose-fractionated extracts facilitated resolution of
protected regions F1, F2, F3, and F4 within this segment: protected
region F2 was virtually undetectable with the fractionated extracts,
whereas F1, F3 and F4 remained visible (Fig. 3A). An
additional protected region, F0, was revealed after 5' end-labeling of
the lower strand (Fig. 3B). F0 is also visible above the F1
footprint in Fig. 3A (crude extract lane).
Protected regions F1', F2', and F3', corresponding to F1, F2, and F3
plus F4, respectively, were identified by labeling the 5' end of the upper strand (Fig. 3C). Protected region F0' (corresponding
to F0) is also evident above F1' in Fig. 3C. The positions
of the F0 and F0' footprints overlap with the AF1 site defined by
transfection analysis (Fig. 2).
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The 163-bp CYP2B2 Sau3AI Fragment Is a Multicomponent
Enhancer--
The DNA sequence of the 163-bp Sau3AI
fragment (13) (see also Fig. 2) reveals that it contains putative
recognition sites for several transcription factors, only some of which
are shown in Fig. 2. Potential regulatory motifs were identified by
inspection and by application of MatInspector (matrix similarity
threshold, 0.85), a search tool for scanning DNA sequences for matches
to nucleotide distribution matrices for transcription factor binding sites accessible in the TRANSFAC data base (24). Immediately adjacent
to a perfectly symmetrical NF1 site (2217 TGGN7CCA) (25),
previously identified (9, 13), is a perfect hexamer half-site (AGGTCA
2223, lower strand) for orphan members of the nuclear receptor
superfamily (26, 27). We refer to the combined hexamer half-site and
NF1 site (Fig. 2) as the HX·NF1 complex. There are also, in an
unusual everted repeat arrangement with a 7-bp spacing (ER-7), two
other AGGTCA sequences (coordinates
2282 to
2264) (Fig. 2). In
addition, between the internal NcoI site and the HX·NF1
complex, MatInspector finds on each strand a match to a glucocorticoid
receptor binding site (TRANSFAC matrix GR_Q6; coordinates
2244 to
2226 on the upper strand and
2246 to
2228 on the lower strand)
(Fig. 2). The 51-bp central core between
2257 and
2207 defined by
the transfection analyses described above excludes ER-7 but includes
the candidate glucocorticoid receptor binding sites and most of the
HX·NF1 complex.
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PB Responsiveness Is Reduced but Not Eliminated by Mutating Either
or Both Elements of the HX·NF1 Complex--
To investigate the role
of the HX·NF1 complex in conferring PB responsiveness, the NF1 site
was mutated in two steps as follows: first in the distal portion
(NF1m1; Fig. 6) and then in
both the distal and proximal portions (NF1dm2; Fig. 6). The
NF1 m1 mutation created a new DNase I-hypersensitive site at 2207 and
modified the F1 footprint but did not abolish it; furthermore, the
portion of the modified F1 footprint corresponding to NF1 sequences was eliminated by competition with an NF1 consensus oligo (Fig.
5A). With the NF1dm2 mutant sequence, the F1' footprint was
reduced to that seen with the wild-type sequence in the presence of an NF1 consensus oligo competitor (Fig. 5B). Hence, the NF1dm2
mutation completely eliminated detectable NF1 binding, but it left a
footprint on the upstream side, corresponding to the anticipated
binding of a protein or proteins to a sequence including the nuclear
receptor hexamer half-site (Figs. 4B and 5B).
Here, as elsewhere, identical results were obtained using nuclear
extracts prepared from livers of untreated and PB-treated rats (Fig.
5B).
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Mutation of a Candidate Glucocorticoid Receptor Binding Site
Dramatically Reduces PB Responsiveness--
The consensus sequence for
the glucocorticoid response element (GRE) is GGTACAnnnTGTTCT (28). The
two candidate glucocorticoid receptor binding sites found by
MatInspector within the central core defined by transfection analysis
contain putative GREs, one of which, 2244 GGCACAgacTCTGTA on the
upper strand, matches the consensus at 7 of 12 positions. This
sequence was mutated to GGCGTGgacTCTGTA, thereby reducing the match to
4 of 12. This led to virtual abolition of the PB response (Fig. 6).
This suggests that a protein binding in the region of the putative GRE
is required to confer PB responsiveness.
Mutation of the CYP2B2 Barbie Box Does Not Affect PB Inducibility-- It has been suggested that the Barbie box (29) is involved in conferring PB responsiveness on the rat CYP2B1 and CYP2B2 genes (30). However, when the core sequence (5'-AAAG-3') of the CYP2B2 Barbie box was mutated, PB responsiveness was retained (Fig. 6).
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DISCUSSION |
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Our previous work has shown that an upstream enhancer element,
located between 2317 and
2155 in 5'-flanking region, confers PB
responsiveness on the rat CYP2B2 gene (9). This element is
situated in the vicinity of a liver-specific DNase I-hypersensitive site in chromatin (10). Such sites are a hallmark of regulatory regions
(31). Furthermore, in transgenic mice a rat CYP2B2 transgene including only the first 800 bp of the 5'-flank was not PB-inducible, whereas a transgene carrying 19 kb of 5'-flank (and hence the
2317/
2155 segment) was normally inducible (32). The experiments described in our previous report (9) and in subsequent reports from two
other laboratories (11, 12), as well as those described here, provide a
strong body of evidence indicating that upstream control elements
confer PB responsiveness on the rat CYP2B2 and mouse
Cyp2b10 genes. The mouse Cyp2b10 active sequences
are situated between
2426 and
2250 and display 91% sequence
identity to those of CYP2B2 (12). Particularly important in
this context is the observation that generally similar results have
been obtained in three different laboratories using different
experimental approaches and PB-inducible CYP2B genes of two
different rodent species.
The results presented here, showing that at least three and probably more sequence elements within the 163-bp Sau3AI fragment are required to confer maximal PB responsiveness, reveal the surprising complexity of this multicomponent enhancer. According to the now classical model for steroid hormone action, the liganded receptor binds to one or more response elements upstream of the target gene and acts as a transcriptional enhancer (26). However, synergistic interaction of a variety of transcription factors with steroid hormone response elements have been known for some time (33), and in a number of genes several cis-acting elements function in concert to activate transcription (34). The best studied such case is that of the rat PEPCK gene. Full glucocorticoid induction of PEPCK transcription is mediated by a complex GRU consisting of two glucocorticoid response elements, as well as three accessory factor binding sites (14, 17). We originally referred to the CYP2B2 Sau3AI fragment as a PB-responsive element (9), but, in the light of its complexity, we now refer to it as a PBRU (35, 49). The homologous mouse Cyp2b10 sequence has been termed a module (12, 36). Proteins that can serve as accessory factors in the PEPCK glucocorticoid response include hepatocyte nuclear factor-3 (16) and the orphan receptors chicken ovalbumin upstream promoter transcription factor (15, 17) and hepatocyte nuclear factor-4 (HNF-4) (15). The application of the PEPCK GRU model to PB regulation of CYP2B2 does not imply that any particular recognition site is shared between the two response units, but merely that PB induction of CYP2B2 requires interactions among multiple regulatory proteins and cis-acting elements constituting a PBRU.
The essential element of the PBRU model then is that more than one
sequence element within the 163-bp Sau3AI fragment is
required to confer PB responsiveness. From our results we conclude that a central core of the fragment, between coordinates 2257 and
2207,
is inactive alone but can confer PB responsiveness when combined either
with upstream or downstream accessory elements within the fragment. In
support of this hypothesis, one such accessory site, designated AF1,
has been localized between coordinates
2188 and
2172. AF1
presumably represents an accessory factor binding site. The protein(s)
responsible for the F0/F0' footprint may also be responsible for AF1
activity, since these footprints overlap with the AF1 sequence.
The level of PB responsiveness of construct 2257/
2172 (Fig.
1B) is essentially the same as that of construct
2257/
2155 (Fig. 1A), and both are about 2-fold lower
than the wild-type
2317/
2155 construct. This indicates that
sequences between
2172 and
2155 are not essential for maximal PB
responsiveness and, moreover, suggests that a second accessory element
may be present between
2317 and
2257. Since full PB responsiveness
is retained in 5' deletion constructs up to
2264, the putative second
accessory element may lie between
2264 and
2257. However, it is
also possible that the reduced responsiveness of the
2257/
2155
construct (and of the
2257/
2172 construct) is a consequence of
partial inactivation of essential sequences at the 5' end of the
central core. Experiments are currently underway to resolve these
issues. In any case, removing one (or the other) of the putative
CYP2B2 PBRU accessory sites reduces but does not abolish PB
responsiveness. Similarly, eliminating any one of the three
PEPCK GRU accessory sites reduces but does not abolish
GRU-mediated glucocorticoid responsiveness (17).
Our results indicate that the HX·NF1 complex is required for maximal PB responsiveness (Fig. 6). NF1 proteins are abundant, ubiquitous transcription factors (37), different isoforms of which are products of a multigene family (38). The NF1 consensus binding sites are composed of two motifs, TGG and GCCAA, separated by a 6- or 7-bp spacer, and the protein protects a 25-30-bp region surrounding this sequence from DNase I digestion (25). An NF1 protein is clearly responsible for part of the F1/F1' footprint and the role of NF1 is positive, because mutations which reduce (e.g. NF1 m1) or abolish (e.g. NF1dm2) NF1 binding reduce but do not abolish PB responsiveness (Fig. 6). This result is similar to that of Honkakoski and Negishi (12) with a different NF1 mutation that reduced but did not eliminate PB responsiveness conferred by a subfragment of the mouse Cyp2b10 homolog of the CYP2B2 163-bp Sau3AI fragment. In our hands, mutations of the nuclear receptor half-site element, as in HXm, or of both that element and NF1, as in HXm-NF1dm2, also reduce PB responsiveness, that is they have similar effects to those of mutational inactivation of NF1 alone (Fig. 6). In the mouse Cyp2b10 system, Honkakoski and Negishi (12) reported a more dramatic effect of such mutations in reducing PB responsiveness. Hence, the HX·NF1 complex is clearly essential for conferring maximal PB responsiveness.
The protein binding to the hexamer half-site creating the HX portion of the F1/F1' footprint is unknown, but there are several possibilities. Inspection of the sequence surrounding the HX·NF1 complex reveals a recognition site for the orphan nuclear receptor, fetoprotein transcription factor (22, 39), and MatInspector finds a match to recognition sites for three other orphan receptors, including chicken ovalbumin upstream promoter transcription factor and HNF-4. Preliminary experiments have revealed binding of both HNF-4 (40) and fetoprotein transcription factor2 to the double-stranded HX oligo. Which of these or other proteins interacts functionally with the HX·NF1 complex to elicit maximal PB responsiveness remains to be determined.
The nature of the proteins responsible for the F0/F0' and F2/F2' footprints is currently unknown. They are of particular interest because F2/F2' overlaps the central core identified as essential for PB responsiveness whereas F0/F0' overlaps the accessory AF1 site.
The protein responsible for the F3-F4/F3' footprints is presumably a member of the nuclear receptor superfamily, because of the overlap with ER-7 site and because the F3-F4 footprint is competed by the HX oligo containing the AGGTCA hexamer half-site. The ER-7 region is not required for PB responsiveness, so it is clearly not an essential site, although it may be an accessory site. Similar conclusions were reached by Honkakoski and Negishi (12) with regard to the corresponding Cyp2b10 pB sequence.
Our observation that mutation of a putative GRE leads to a major reduction in PB responsiveness is particularly intriguing, as it raises the possibility that a glucocorticoid receptor-like molecule may be involved in conferring PB responsiveness. The putative PBRU GRE matches the GRE consensus at only 7 of 12 positions. Hence, like the GREs forming part of the PEPCK GRU (14), it is not a typical GRE.
Much previous effort has been concentrated on promoter-proximal sequences, particularly the Barbie box (29, 30) and their possible role in conferring PB responsiveness on the rat CYP2B1 and CYP2B2 genes. However, mutation of the Barbie box sequence in the context of the CYP2B2 promoter and 5'-flank does not affect PB responsiveness either in rat hepatocytes transfected by injection in situ (11) or in transfected primary rat hepatocytes (Fig. 6). Hence, while promoter-proximal sequences are doubtless required for basal transcriptional activity (9, 36), there is at present no convincing evidence for their involvement in conferring PB responsiveness.
The nucleotide sequences of the coding regions of CYP2B1 and CYP2B2 are about 97% identical (41, 42), and this extends over at least 1 kb of their 3'-flanking regions (43) and over some 2.3 kb of their 5'-flanking regions (13, 44). This, plus the fact that the responses of liver CYP2B1 and CYP2B2 to PB treatment are very similar (although the basal level of CYP2B2 is somewhat higher than that of CYP2B1 (45, 46)), explains why CYP2B1 and CYP2B2 are sometimes treated as though they were a single gene (47, 48). Nevertheless, there are striking differences in the tissue-specific expression of CYP2B1 and CYP2B2, most notably in lung where CYP2B2 is not expressed, whereas CYP2B1 is expressed constitutively but is not PB-inducible (46). Because the 163-bp Sau3AI fragment is similarly positioned and, except for a single nucleotide difference, identical in sequence in CYP2B1 and CYP2B2 (13, 44) (see also Fig. 2), we will have to look elsewhere to explain the differences between CYP2B1 and CYP2B2 expression in the lung. It seems likely that the CYP2B2 PBRU and CYP2B1 PBRU are both inactive in the lung and that CYP2B1 expression is turned on because of the constitutive presence of some other transcription factor, the activity of which depends on one of the subtle sequence differences between the CYP2B1 and CYP2B2 5'-flanking regions.
The molecular details as to how the presence of PB leads to activation of transcription via the PBRU remain to be determined. However, a recent report of results obtained by in vivo footprinting suggests that PB may modify the interaction of transcription factors with the PBRU in chromatin (48). Although this provides further support for the role of the PBRU in transcriptional activation, further characterization of the proteins that bind to it will be necessary to elucidate the mechanism.
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ACKNOWLEDGEMENTS |
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We thank Normand Marceau and members of his laboratory for advice and access to facilities for isolating rat hepatocytes; Carl Séguin for carefully reading the manuscript; Simon Labbé for advice on DNase I footprinting; Anne Belzil for technical assistance; Pierre Paquin and Guy Langlois for photographic work; and Jacques Côté, Josée Aubin, and members of the Luc Bélanger laboratory for advice.
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FOOTNOTES |
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* This work was supported by a grant from the Medical Research Council.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.
Both authors contributed equally to this work.
§ To whom correspondence should be addressed: Centre de recherche, L'Hôtel-Dieu de Québec, 11 côte du Palais, Québec, Canada G1R 2J6. Tel.: 418-691-5548; Fax: 418-691-5439; E-mail: Alan.Anderson{at}bio.ulaval.ca.
1
The abbreviations used are: CYP, cytochrome
P450; PB, phenobarbital; bp, base pair(s); NF1, nuclear factor 1; PBRU,
phenobarbital response unit; GRU, glucocorticoid response unit; PEPCK,
phosphoenolpyruvate carboxykinase; oligo, oligodeoxyribonucleotide;
CAT, chloramphenicol acetyltransferase; GRE, glucocorticoid response
element; HNF-4, hepatocyte nuclear factor-4; dATPS, deoxyadenosine
5'-(
-thio)triphosphate.
2 C. Stoltz, L. Galarneau, L. Bélanger, and A. Anderson, unpublished results.
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REFERENCES |
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