(Received for publication, December 9, 1996, and in revised form, March 10, 1997)
From the Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709
Induction of drug- and carcinogen-metabolizing cytochrome P450s by xenobiotic chemicals is a common cellular defense mechanism, usually leading to increased detoxification of xenobiotics but sometimes, paradoxically, to formation of more toxic and carcinogenic metabolites. Phenobarbital (PB) is an archetypal representative for chemicals including industrial solvents, pesticides, plant products, and clinically used drugs that induce several genes within CYP subfamilies 2B, 2A, 2C, and 3A in rodents and humans. Although the transcription of these CYP genes is activated by PB, the associated molecular mechanisms have not yet been elucidated. Here we have analyzed, in detail, enhancer activity of a far upstream region of mouse Cyp2b10 gene and report a 132-base pair PB-responsive enhancer module (PBREM) with a 33-base pair core element containing binding sites for nuclear factor I- and nuclear receptor-like factors. Mutations of these binding sites abolish the ability of PBREM to respond to inducers in mouse primary hepatocytes.
The cytochrome P450s (CYPs)1 comprise a superfamily of heme-thiolate proteins (1) with diverse functions from the synthesis and degradation of steroid hormones and fatty acid derivatives to metabolism of xenobiotic chemicals such as drugs, industrial chemicals, environmental pollutants, and carcinogens (2-5). Induction of P450s by xenobiotic chemicals is a common phenomenon, conserved throughout the vertebrate kingdom, insects, and bacteria. Xenobiotic inducers can be sorted into distinct classes based on the subsets of P450s induced (6, 7); for instance, polycyclic aromatic hydrocarbons and peroxisome proliferators are known to activate CYP1A and CYP4A genes through ligand-dependent aryl hydrocarbon and peroxisome proliferator-activated receptors, respectively (7, 8). For many other CYP genes and the majority of xenobiotics, however, the molecular basis of induction is virtually unknown (1, 7, 9). Phenobarbital (PB) represents a large number of structurally unrelated chemicals that induce the same set of CYP genes including members within subfamilies 2A, 2B, 2C, and 3A, with CYP2B forms being activated most effectively (for review, see Ref. 10). PB also induces various transferases and enzymes of heme metabolism and affects processes of cell growth and cell-cell communication (10). The signaling pathways through which PB acts to regulate the induction process of CYP genes are mostly unknown due to the loss of PB response in hepatoma cells and the lack of other reliable in vitro assays. Also, the findings on PB-responsive DNA regulatory elements of these CYP genes have been quite inconsistent (11-16). We have addressed the above problems by developing a primary hepatocyte culture in which both endogenous and transfected Cyp2b10 genes remain transcriptionally inducible by PB-like chemicals (17). Using this culture system, we now identify and dissect a PB-responsive enhancer module PBREM from the mouse Cyp2b10 gene and show that binding sites for both nuclear factor I- and nuclear receptor-like factors are involved in induction.
The 177-bp Cyp2b10 DNA fragment
(2426/
2250 bp) from plasmid BglPst4 (17) was amplified using
primers 2B10-S (5
cagggatccTGTCTGGATCAGGACACA) and 2B10-AS (5
cagggatccTCAGTGCCAGATCAACCA). Cyp2b9 gene DNA fragment
(
898/
765 bp) (18) was amplified from BamHI-digested CD-1
mouse genomic DNA (2 µg) with primers 2B9-S (5
cttggatccATTAAAATCTGGTTACCAAGGAGGAAGAAAAGA) and 2B9-AS (5
cttggatccCCAGCTTTGCAGGAGCAAAATCCTGGTGTCATT). The amplified DNAs were
digested with BamHI and ligated into BamHI site
of pBLCAT2 (tkCAT) plasmid (19). Various deletions of
Cyp2b10 177-bp fragment (depicted in Fig. 3) were done using
appropriate 20-24-mer primers harboring a BamHI site at
5
-ends for cloning as above. Internal deletions of elements pB
and pC
were generated using primers 2B10-S, 2B10-AS, and 20-mer primers with a
5
-end EcoRI site annealing to elements pB, pB
, pC, and pD.
In wild-type Cyp2b10 fragment, this resulted in a 3-bp
mutation (
2336 GTACTT to GaAtTc)
without any change in the spacing between elements pB
and pC. All
mutations from the wild-type sequence shown hereafter are indicated by
underlined, lowercase characters. To expedite the mutation of pC, the
spacer region between pC and pD was first changed to an XbaI
site (
2298 GCCTGA to tCtaGA), and pC was then mutated by changing six nucleotides from Cyp2b10 sequence to
those in corresponding Cyp2b9 position by primer MUT-pC
(
799
ctgtctagaAAGTcctTGaTGGCACTGTGtCAAGaTCAGGAAA). Mutagenesis of the minimal enhancer construct pB
-C (
2364/
2297) were done using primers B
-mut1 (
2364
ctgggatccAAACATGGTacagTCGGGCACA), B
-mut2 (
2364
ctgggatccAAACATGGTGATTTCGGtactAGAATCTGT), pC-NFIm (
2297
ctgggatccGCAAGTTGATGcatagACTGTGCCAA), and pC-NRm (
2297 ctgggatccGCAAGTTGATGGTGGCACTGTGCCAAccagAAGAAAGTAC).
Plasmids
4300CAT,
1404CAT, and
566CAT were described previously
(17). Plasmid
2397CAT was constructed by amplifying the
Cyp2b10 region between
2397 and
1404 bp using
proof-reading Pfu DNA polymerase and primers containing
HindIII sites. The amplified DNA was inserted into
HindIII-digested
4300CAT plasmid. Plasmid
1850CAT was
generated from
4300CAT by partial PvuII digestion and
self-ligation. Appropriate recombinant plasmid DNAs produced in
Escherichia coli TG-1 cells were purified twice on CsCl
gradients and verified by DNA sequencing over the amplified regions.
The quality and supercoiling of plasmid DNAs were checked by agarose
gel electrophoresis.
Preparation, Transfection, and Culture of Mouse Primary Hepatocytes
Two-month-old C57BL/6 males were purchased from
Jackson Laboratory (Bar Harbor, MA). About 25 × 106
mouse hepatocytes were electroporated with 30 µg each of individual enhancer/tkCAT reporter plasmids including 10 µg of pSVgal control plasmid (Promega) to normalize results between different plasmid DNAs
as described previously (17). Equal aliquots from a transfected cell
pool were dispensed into four 60-mm dishes to assure identical transfection efficiencies among treatments (17), unattached cells were
removed after 30 min, and dishes (about 3 × 106
cells) were incubated with or without inducers for 24 h in
Williams' E-based medium (17) with supplemental 30 mM
pyruvate. Cell extracts (20) were assayed for protein (21) and
-galactosidase (22), heat-treated for 20 min and assayed for CAT
activity (17), and quantitated by radioisotope imager (Molecular
Dynamics). Generally, cell aliquots from the same batch of isolation
gave very similar transfection efficiencies as determined from pilot
experiments with pSVCAT and pSV
gal control plasmids, as also found
by other investigators using electroporation (23). For mRNA
analyses, cells were lysed using Trizol reagent (Life Technologies,
Inc.) after 8 h of treatment, and total RNA samples (10 µg) were
subjected to Northern blot analysis with 32P-labeled 360-bp
CYP2B10 and 180-bp mouse albumin cDNA probes (17).
Crude nuclear extracts (24) from control and
PB-treated mouse livers were enriched through heparin-agarose columns
to remove endogenous DNase activity (25). DNase I protection assays
were performed with 32P-end-labeled 177-bp
Cyp2b10 DNA fragments or pB-C variants (5 × 104 cpm/lane), enriched nuclear extracts (up to 10 µg)
and DNase I (0.5 units) adjusted to equal total protein with bovine
serum albumin, incubated at room temperature for 20 min, and
processed as before (17). Gel shift assays were performed
using 3 µg of crude nuclear extract in 10 µl of 10 mM Hepes, pH 7.6, 0.5 mM dithiothreitol, 15%
glycerol, 2 µg poly(dI-dC), 0.05% Nonidet P-40, 50 mM
NaCl, and about 30,000 cpm of 32P-end-labeled
oligonucleotide probe. The free and protein-bound probes were separated
on 5% acrylamide gels in 0.5 × Tris-Borate-EDTA buffer prior to
autoradiography. The top strands of the probes used were: PBRE,
5
TTAGCAAGAGGGAAGGTCAGAGAAC; PBRE NRm, 5
TTAGCAAGAGGGAAccTCAGAGAAC; pC wt, 5
TACTTTCCTGACCTTGGCACAGTGCCACCATCAACTTG; pC NFIm, 5
TACTTTCCTGACCTTGGCACAGTctatgCATCAACTTG; pC NRm, 5
TACTTTCCTGAggTTGGCACAGTGCCACCATCAACTTG; and pC dm, 5
TACTTTCCTGAggTTGGCACAGTctatgCATCAACTTG.
Our previous reporter gene assays using various deletion
constructs of the Cyp2b10 gene indicated that induction of
CAT activity was 2-3-fold when using either 4300CAT or
1404CAT
plasmids. The inducibility was lost and the basal CAT activity
increased more than 10-fold when sequences downstream of
775 bp were
included (17). These results were confirmed by the experiments depicted in Fig. 1.
4300CAT gave 3.1-fold induction, close to
previously observed values (17). Notably, the CAT activity from
2397CAT plasmid was induced even further, by 7.6-fold in three
different experiments with mouse primary hepatocytes. Removal of the
2397/
1850-bp or the
1850/
1404-bp DNA fragments attenuated the
induction to 2.8- and 2.3-fold, respectively. The
566CAT plasmid
produced high basal, non-inducible CAT activity as found before (17). These findings indicate that Cyp2b10 gene may contain two
regions involved in PB induction, the previously described
1404/
971-bp region (17) and the stronger
2397/
1850-bp
region.
Trottier et al. (11) showed that a 163-bp
Sau3AI-Sau3AI fragment within the 2.3-kb region
of the rat CYP2B2 gene conferred a 3.5-6.6-fold PB
inducibility to a tkCAT reporter in rat hepatocytes, but no further
analysis of this enhancer or its associated factors was reported (11).
Since we found that the
2397/
1850-bp Cyp2b10 DNA
fragment mediated PB induction and contains sequences overlapping with
the rat 163-bp fragment, we cloned the DNA homologous to the rat 163-bp
sequence from the mouse Cyp2b10 gene and found that its
identity to CYP2B2 163-bp fragment is 91%, which is higher than the overall 83% identity in 5
-flanking region (17). The corresponding fragment from non-inducible mouse Cyp2b9 gene
exhibited a lower 70% identity (Fig. 2A). We
then performed DNase I protection assays with the 177-bp
Cyp2b10 DNA and identified three weakly protected (pA, pB, and
pB
) and three strongly protected (pC, pD, and pE) nuclear protein
binding regions (Fig. 2B; also see Fig. 2A,
bracketed areas). None of these six regions displayed any
noticeable differences in binding patterns between control and
PB-treated mouse nuclear extracts in gel shift assays (Fig. 2C) or in footprint assays (not shown). These findings
suggest that pre-existing DNA-binding factors are being modified in
response to PB or that if distinct DNA-binding species activated by PB really exist, they are not detectable by the DNA and oligonucleotide probes used here.
We then examined whether this Cyp2b10 DNA had any
PB-inducible enhancer activity in mouse primary hepatocytes. Fig.
3A, top panel, shows that the
endogenous CYP2B10 mRNA was strongly increased by PB (lane
2) and TCPOBOP (lane 4) but not by 3,3-DCP (lane 3), which is an inactive TCPOBOP derivative (17, 26). The mouse
albumin mRNA, used as a control, did not respond to the inducers.
The same pattern of induction by PB and TCPOBOP (
11-fold) was
conferred to the tk promoter by the insertion of 177-bp
Cyp2b10 DNA (Fig. 3A, middle panel),
whereas the tk promoter alone was not activated by any of the compounds
(Fig. 3A, bottom panel). It is notable that the
extent of induction was at least as high as with the
2397CAT
construct in Fig. 1. Furthermore, the dose responses of the 177-bp
Cyp2b10 DNA-driven CAT activity paralleled that of
endogenous CYP2B10 mRNA (Fig. 3B). The maximal levels of
CYP2B10 mRNA were achieved with 50 nM TCPOBOP and 0.3 mM PB, which induced CAT activity 6.0- and 8.6-fold,
respectively. These results indicate that the mouse 177-bp DNA sequence
acts as a PB-responsive enhancer with the same chemical specificity and dose-responsiveness as the endogenous Cyp2b10 gene. These
data strongly suggest that the 177-bp DNA element mediates PB induction in vivo.
Since
there were at least six DNA regions capable of nuclear protein binding
within the 177-bp DNA sequence, we next determined their functional
role for PB inducibility. Primary hepatocytes were transfected with
various DNA deletions linked to tkCAT reporter plasmid (Fig.
4). The deletion of pA had only slight effects on the
basal activity or inducibility (compare lanes 1 and
2, and 5 and 7). Depending on the
presence of other elements, the deletion of pE tended to decrease the
inducibility by elevating the basal activity about 2-fold at most
(compare lanes 1 and 4, and 2 and 6). Due to high basal activity of construct 2364/
2250
(lane 3), deletion of pE in this case actually increased the
-fold inducibility (compare lanes 3 and 8). The
simultaneous removal of pA and pE decreased the inducibility only by
25% (compare lanes 1 and 7). These results
suggest that the elements pA and pE do not have a major role in the
function of the 177-bp Cyp2b10 enhancer. The removal of pB
attenuated the induction response by 40-60%, mostly due to increases
in the basal CAT activity (e.g. compare lanes 2 and 3, and 7 and 9). The
deletion of pD also attenuated the induction response by 25-50% due
to decreases in induced CAT activity but without affecting the basal
levels (compare lanes 4 and 5, 6 and
7, and 8 and 9). Finally, the
construct
2364/
2297 reproducibly displayed about 3-fold induction
indicating that regions pB
plus pC harbored the
inducer-dependent DNA segment (lane 9).
However, neither pB nor pC alone could confer any significant
inducibility to the tk promoter (Fig. 4, lanes 10 and
11), suggesting that sequences within both regions are
required for induction response. When multimerized DNA fragments were
inserted in front of the tk promoter, CAT activity was induced about
2-fold with pC but less with pB
(lanes 12 and
13). Although the extent of induction was too low to draw a
definite conclusion, pC appeared to have a more central role than pB
in the observed enhancer activity of the pB
-C construct. Consistent
with this notion, the most dramatic loss of inducibility (from 6.8- to
1.4-fold) was observed when pC was deleted from the 177-bp
Cyp2b10 DNA (lanes 14 and 16). The
importance of pB
for enhancer activity was also confirmed by the fact
that deletion of pB
attenuated the induction to 2.1-fold (lane
15). In addition to these results, we found that the 177-bp
Cyp2b10 DNA conferred PB inducibility also to SV40 and
proximal Cyp2b10 (
64CAT) (17) promoters, regardless of its
orientation or distance from promoter (data not shown), indicating that
the 177-bp Cyp2b10 DNA is a functional enhancer. In summary,
our data suggest that pC and pB
have a major role in determining the
inducibility while pB and pD also contribute to the full enhancer
activity by modulating the basal and PB-induced activity levels,
respectively. Because of this multifactorial nature, we designate the
132-bp Cyp2b10 fragment (
2397/
2265 bp) as the
Phenobarbital Responsive
Enhancer Module (PBREM).
The mouse Cyp2b9 gene encodes the female-specific steroid
16-hydroxylase (18), which is related to Cyp2b10 but not
induced by PB (27). The DNA sequence most similar to PBREM was
amplified from the Cyp2b9 gene, linked into tkCAT plasmid,
and transfected into mouse hepatocytes. As compared with the
Cyp2b10 PBREM, the Cyp2b9-derived DNA sequence
could not confer any inducibility to the tk promoter-driven CAT
activity, indicating that Cyp2b10 PBREM displays the
predicted genetic specificity of induction (compare PBREM and
Cyp2b9 in Fig. 5, A and
B). Additionally, the Cyp2b9-driven CAT activity
was not increased more than 1.4-fold by TCPOBOP, 3,3
-DCP, or PB (data
not shown). Considering these findings and the key role of pC in PBREM
function, the sequence of pC was mutated by converting six nucleotides
to those in the Cyp2b9 gene while keeping the protected
elements pB, pB
, and pD in the Cyp2b10 gene intact. For
easier cloning, this was done by first changing the spacer region
between pC and pD into an XbaI site, which did not affect
the inducibility (PBREM-Xba in Fig. 5, A and B).
The subsequent replacement of six nucleotides in Cyp2b10 pC
element by corresponding nucleotides in Cyp2b9 gene resulted
in the loss of induction of CAT activity (PBREM-mut pC in Fig. 5,
A and B), underscoring the importance of pC as
the core inducible element within PBREM. The nucleotide sequence of the
33-bp pC fragment contained a perfect nuclear factor I (NFI) binding
site (TGGN7CCA) and a putative nuclear receptor (NR)
binding motif (AGGTCA). Intriguingly, both binding sites are mutated in the noninducible Cyp2b9 gene, the 5
NFI motif TGG to
TGa, and the NR motif AGGTCA to AGaTCA (Fig.
2A).
Functional Analysis of NFI and NR Sites within Element pC
We
mutated the NFI and NR binding sites within the minimal inducible
fragment pB-C (
2364/
2297) and examined how these mutations affected binding of nuclear proteins and inducibility of CAT reporter activity. Fig. 6, A and B indicate
that nucleotides including NFI and NR motifs between
2326 and
2300
on top strand (upper panel) and between
2333 and
2304 on
bottom strand (lower panel) were protected from DNase I
digestion in the wild-type pB
-C DNA. Binding to NFI site was
dramatically decreased in pC NFI mutant (
2312 GCCAC to
ctatg) so that now only NR motif and seven adjoining nucleotides were protected. In addition, a DNase I hypersensitive site
appeared at
2310/
2313, just downstream from the NR site (Fig. 6,
A and B, solid arrows). Mutation of
the NR site (AGGTCA to AccagA) did not affect NFI binding
but somewhat reduced the extent of protection at 5
end of pC at
2332,
2329, and
2326 (Fig. 6, A and B).
Thus, the patterns of nuclear protein binding to pC were altered by NFI
and NR mutations, whereas a mutation at pB
had no effect on protection
of pC.
The altered binding patterns were also confirmed in gel shift assays.
Previously, we identified a 25-bp DNA element (1219/
1195) highly
similar to a portion of the 163-bp CYP2B2 fragment identified by
Trottier et al. (11, 17). This 25-bp probe, here termed PBRE, formed two complexes with liver nuclear extracts (Fig.
6C, lane 1), which could be competed by 50-fold
excess of either PBRE or pC oligonucleotides (lanes 2 and
4) but not by oligonucleotides containing mutations at NR
motifs (lanes 3 and 5). In line with the DNase I
protection assay, mutation of NFI motif considerably reduced binding to
pC, and the major remaining complex now comigrated with the top PBRE
complex (compare lanes 6 and 8). When both NFI and NR motifs were mutated from the pC probe, this major complex disappeared (compare lanes 6 and 7). In
competition experiments, 50-fold excess of PBRE competed for the
formation of faster-migrating complexes of pC which were also abolished
by mutation of NR motif in pC (lanes 9-11). Finally, the
binding to pC NFI mutant harboring an intact NR site could be competed
relatively efficiently by PBRE (lanes 12-14). These results
indicate that pC and PBRE appear to bind similar factors, that pC can
bind both NFI-like and NR-like factors, and the binding of
NR-like factors are dependent on the integrity of the AGGTCA
motif.
The same mutated DNA fragments used for DNase I protection were used in
CAT reporter gene assays (Fig. 7). In five independent transfections, the mutation of NFI binding site reduced the basal CAT
activity to 63% and abolished the induction (pC NFIm). The mutation of
the NR site increased the basal activity to 221%, but the inducibility
was again lost (pC NRm). Mutations of a putative D-binding protein site
within pB reduced the basal activity but had smaller effects on the
induction response (pB
mut1, pB
mut2), confirming the principal role of
pC as the core enhancer element. These results are consistent with pC
being occupied by at least two factors, NFI- and NR-like proteins.
Although both sites are needed to confer inducibility to the minimal
pB
-C construct, they appear functionally different, with NFI-like
protein acting as an activator and NR-like protein as a repressor,
respectively.
The barbiturate-regulated induction mechanism of bacterial
CYP102 gene is well characterized, with a 17-bp so called
Barbie box sequence as the cis-acting element (14, 28). The
nature of PB-dependent regulatory elements of mammalian
CYP genes, on the other hand, has been very controversial.
Some studies indicated that Barbie box-like sequences are present in
PB-inducible mammalian gene promoters (at 136/
127 bp in rat
1-acid glycoprotein gene and at
89/
73 bp in
CYP2B2 gene) and that they bind nuclear proteins in a
PB-dependent manner (13, 14). Similar results were reported with CYP2B2 sequences (
98/
68 bp) overlapping the Barbie
box (29). Another group found that the CYP2B2 Barbie box was
not protected, and two elements at
199/
183 bp and at
72/
31 bp formed nuclear protein complexes that were more abundant after PB
administration (15). However, these studies have relied mainly on
in vitro protein binding or in vitro
transcription experiments. Only one report showed that mutation of the
Barbie box eliminated the 1.6-fold induction by PB of rat
1-acid glycoprotein promoter-driven reporter gene in
primary hepatocytes (13).
Several groups have documented that nuclear proteins do not bind to
Barbie box-like sequences in 1-acid glycoprotein (30, 31) or in mouse (17) and rat CYP2B genes (15, 32, 33). Consistent with the absence of any significant protein binding, we
found, using DNA transfection assays in primary hepatocytes, that
Barbie box-like sequences did not have a major role in basal or
PB-induced Cyp2b10 gene transcription (17). Later on, it was
shown that mutation of a Barbie box-like sequence in the proximal CYP2B2 promoter did not affect PB-inducibility of reporter
gene DNA by in situ injection into rat liver (34). In
contrast to somewhat variable results reported on the proximal regions
of CYP2B genes (13, 15, 16), recent studies on transgenic
mouse lines carrying either 19 or 0.8 kbp of CYP2B2
5
-flanking sequences (12) and on transient transfections with
Cyp2b10 (17) or CYP2B2 genes (11, 34) suggest
that PB responsiveness resides in the distal part of CYP2B
genes. Transient transfections carried out with the Cyp2b10
gene in the present study and functional studies utilizing different
methodologies with the rat CYP2B2 gene (11, 34) converge
well together, indicating that a PB-responsive element is located
around
2.3 kbp region of CYP2B genes. Notably, this region
does not contain any Barbie box-like sequences.
We have now identified, for the first time, the core PB-inducible
element that appears to be a classical enhancer and provided some
evidence for associated factors. Interestingly, we did not detect any
differences in protein binding to PBREM between the control and
PB-treated liver nuclear extracts. This implies that PB might act by
modifying pre-existing DNA-binding factors, which is consistent with
the insensitivity of CYP2B10 mRNA induction to
inhibition of protein synthesis by cycloheximide in this hepatocyte system (17). Alternatively, PB might modify the function, affinity, and/or amount of factors associated with the pre-existing DNA-binding proteins. More importantly, we found that the core inducible element is
capable of binding NFI- and NR-like factors, both necessary for
induction response. We found that NFI and C/EBP could bind to
32P-labeled pC element in gel shift assays since antibodies
raised against these transcription factors were able to supershift some of the pC complexes (data not shown). Our previous studies indicated that another PB-responsive element was located in the
1.4/
1.0-kbp region of Cyp2b10 gene although it could not confer
PB-inducibility to heterologous promoters in primary hepatocytes (17).
We found, however, that this element contained a 25-bp sequence (PBRE), which was very similar to a portion in the rat 163-bp CYP2B2
gene fragment and contained an AGGTCA motif. We therefore proposed that
a NR-like protein may have a role in PB induction of CYP2B genes (17). The PBRE also bears similarity to sequences within the
present PBREM. It can be aligned with pB (at
2386/
2362 on top
strand) but also with pC (at
2333/
2309 on bottom strand). According
to our gel shift assays, factors binding to PBRE appear be related to
those occupying the NR site within pC, the DNA region most important
for PBREM function.
The PB signal, activating the Cyp2b10 gene, may primarily target the NR-binding (repressor) factor rather than the NFI-like (activator) protein for several reasons. First, NFI isoforms are ubiquitous and present in many tissues and cell lines (35), whereas the Cyp2b10 gene induction is liver-specific (17). Second, NFI binding sites are present, e.g. in mouse albumin (36), rat tyrosine aminotransferase (37), rat CYP2A2 (38), and mouse Cyp2d9 (39) gene promoters, and none of these genes is PB-inducible (17, 40, 38, 27). Third, in our unpublished experiments,2 the PBREM-driven CAT activity was found to be quite high and non-inducible in several continuous cell lines. It may be speculated that the NR-like repressor protein, critical for induction, is missing or inactive in these cell lines. However, as our transfection assays indicate, both NFI-like and NR-like factors are important for the function of PBREM and identification of both factors in further studies is required to elucidate their respective roles in PB-induced gene expression.
While the sequence and function of PBREM are well conserved between the
rat and mouse CYP2B genes, we did not find identical or
highly similar sequences in the reported sequences of PB-inducible genes within families CYP2A, CYP2C, and
CYP3A. This may be related to the fact that CYP2B
forms are also the most efficiently induced by PB (7, 10). Presuming a
key role for a NR-like protein in PB induction, it is possible that
divergence of DNA elements such as PBREM in CYP2B genes has
resulted in substitution of the NFI site by other CCAAT-like sequences
such as C/EBP, CP2, or CP1 sites (35) or by other transcription factor
sites, leading to different organization and perhaps to different
efficiency of these DNA elements in other PB-responsive CYP
genes. In this view, we found elements composed of NR- and CCAAT-like
binding sites at 2.7/
2.6 and
1.0/
0.9 kbp in CYP2A1
gene (38); at
2.25/
2.1 kbp, close to a DNase I hypersensitive site
in CYP2C1 gene (41); at
2.2 kbp in CYP2C8 gene
(42), and at
0.65 and
0.15 kbp in CYP3A genes (43, 44).
The functional role of these elements, reminiscent of PBREM in these
PB-inducible genes, remains to be tested. As a pleiotropic inducer, PB
may alter gene functions through different pathways and regulatory
mechanisms. In this respect, and given the sequence divergence
discussed above, it is noteworthy that the extent and kinetics of
induction among the PB-responsive genes have been reported to differ
(45, 46).
In conclusion, we have characterized and dissected a multifactorial
PB-responsive enhancer at 2.3 kbp in mouse Cyp2b10 gene in
primary hepatocytes. In transient transfection assays, the enhancer
responds to inducers with identical chemical specificity as the
endogenous Cyp2b10 gene. The inducibility of the enhancer is
lost by introduction of naturally occurring mutations from the
non-inducible Cyp2b9 gene. The function of the enhancer is dependent on the integrity of both nuclear factor I- and nuclear receptor-like sites within a 33-bp core element. Further analysis of
PBREM and its binding proteins will provide a central framework that
may apply to many other PB-inducible genes.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U67059[GenBank].
We thank Dr. Cary Weinberger and Dr. Gordon Ibeanu for comments on the manuscript and Mr. Rick Moore for help with liver perfusions and DNA sequencing.