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Volume 272, Number 47, Issue of November 21, 1997
pp. 29423-29425
(Received for publication, September 19, 1997)
From the Department of Molecular and Integrative Physiology and the
College of Medicine, University of Illinois at Urbana-Champaign,
Urbana, Illinois 61801
ABSTRACT Phenobarbital is a classical inducer of the drug
metabolizing cytochrome P450 genes, but the molecular mechanism of
induction has not been elucidated. Functional analyses have identified
a phenobarbital-responsive unit in the rat CYP2B1/2 and
mouse Cyp2b10 genes about INTRODUCTION Phenobarbital
(PB)1 has pleiotropic effects
in the liver, affecting the mRNA levels of as many as 50 genes (1)
as well as cellular morphology. The regulation of the microsomal drug
metabolizing system by PB has been known for about 40 years (2), but
the mechanism of transcriptional activation of the genes for
cytochromes P450, the key enzymes in this system, is not understood.
The rat CYP2B1 and CYP2B2 genes have nearly
identical sequences in the proximal promoter and PBRU regions and are
induced by PB by at least 200- and 40-fold, respectively (3). A
PB-responsive enhancer region has been identified at about EXPERIMENTAL PROCEDURES Rats were injected intraperitoneally
with either 100 mg/kg PB or isotonic saline and were sacrificed 6 h later. The livers were removed, and nuclear extracts were prepared as
described (7). For in vitro DNase I footprinting analysis,
end-labeled DNA fragments containing the PBRU were prepared by PCR
using the labeled primers S3 or A3 (see Fig. 3) for the sense or
antisense strand, respectively. Binding conditions were as described
previously for gel shift assays (8). 40,000 cpm of the probes were
incubated at 0 °C for 6 min in the absence or the presence of 30 µg of protein of rat liver nuclear extract and 2.5-12.5 or 150 ng of
DNase I for the naked DNA or DNA incubated with proteins, respectively. Dideoxynucleotide DNA sequencing ladders were used as markers.
[View Larger Version of this Image (38K GIF file)]
Rats were treated as above, nuclei
were isolated from the liver and incubated with 90-120 µg/ml DNase I
for 10 min at 0 °C, and genomic DNA was isolated as described (9).
Purified genomic DNA was incubated with 0.5-2.5 × 10 RESULTS Incubation
in vitro of the CYP2B1/2 PBRU region with liver nuclear
extracts from control and PB-treated rats resulted in DNase I
protection patterns that were very similar (Fig.
1) as was also observed with the mouse
Cyp2b10 gene (6). Strong protection was detected for both
the antisense and sense strands from about
[View Larger Version of this Image (59K GIF file)]
In contrast
to the in vitro footprints, binding of proteins to the PBRU
in native chromatin was dramatically altered by 6 h after PB
treatment. In liver nuclei from control rats, strong protection of both
the antisense and sense strands was observed from about
[View Larger Version of this Image (79K GIF file)]
DISCUSSION Protein binding to a region centered on the NF-1 site is
consistent with functional analysis of the region. In
CYP2B1/2, a core sequence of 37 bp centered on the NF-1 site
was necessary but not sufficient for PB induction.2
Sequence from either side of the NF-1 region was able to confer PB
inducibility extending to either Because purified NF-1 protects a region of about 25 bp (11), the
protection of 24-29 bp in the control native chromatin samples may be
primarily the result of NF-1 binding. The similarity of this binding
with that in vitro and the observation that the affinity of
NF-1 for DNA in nucleosomal structures was decreased (12) suggests that
the DNA in the chromatin at the NF-1 site is in an open conformation.
This conclusion is also supported by the detection of DNase I
hypersensitivity in PBRU chromatin of both control and PB-treated
animals (13). The protection of 60 bp in the PB-treated samples, which
differs from the in vitro binding, indicates that PB
treatment alters the chromatin structure, which then influences the
binding. The extended protection after PB treatment compared with both
control native chromatin and in vitro footprints suggests
that PB-induced changes in the chromatin structure facilitate
additional binding of proteins to the PBRU either directly or by
binding to NF-1 to form a larger complex.
Although evidence has been presented for PB-responsive elements in the
proximal promoter regions of CYP2B1/2 (14, 15), most recent
data favor a PB-responsive enhancer at about Two basic models have been proposed for ligand-mediated transcriptional
activation. In first model, proteins are not bound to the regulatory
region until a ligand-induced change in the chromatin structure enables
the binding of positive regulatory factors (17, 18). Interestingly, in
the two cited studies, NF-1 is a key protein that binds to chromatin in
response to ligand treatment. In the second model, regulatory proteins
are bound in the uninduced state but are either inactive or suppressed
by negative coregulators. The ligand activates the factors directly or
indirectly by altering chromatin conformation, so that binding of
positive coregulators is favored over that of negative coregulators (19). The results presented in this paper are most consistent with the
second of these mechanisms, because strong binding of protein, possibly
NF-1, to the PBRU in chromatin is observed in the untreated animal, in
which liver expression of CYP2B1 is undetectable and that of
CYP2B2 is very low (20). The observation that binding of
proteins to the PBRU in vitro is not affected by PB
treatment is also consistent with the second mechanism, in which
activity or conformation of proteins already bound to the DNA is
affected. The alteration of the protected region in native chromatin by PB treatment suggests that either the conformation of the binding proteins is changed or additional proteins are recruited to the regulatory protein complex and that the change is dependent on the
chromatin structure. The most straightforward interpretation of these
results is that NF-1 is bound in the untreated animal in an inactive
state. PB treatment results in a change in chromatin structure, which
results in recruitment of coregulators and/or additional DNA binding
regulatory factors producing a transcriptionally active regulatory
complex. This process may be accompanied by further modifications of
chromatin such as histone acetylation, which could result from
recruitment of activator coregulators or their exchange for suppressor
coregulators, which have histone acetylase and deacetylase activities,
respectively (21-23). Characterization of the complex of proteins that
bind to the PBRU will be required to establish the mechanism.
FOOTNOTES ACKNOWLEDGEMENTS We thank Siqing Liu for the preparation of
nuclear extracts for the in vitro footprints and Ilia
Rivera-Rivera and Yue Wang for carrying out the procedures with animals
and technical assistance. PCR primer oligonucleotides were selected
with the assistance of the Genetic Engineering Facility of the
Biotechnology Center of the University of Illinois at
Urbana-Champaign.
REFERENCES
COMMUNICATION:
Phenobarbital Alters Protein Binding to the CYP2B1/2
Phenobarbital-responsive Unit in Native Chromatin*
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
2.3 kilobase pairs from the
transcriptional start site, but little or no changes in protein binding
to this region were observed in vitro. To examine the role
of chromatin structure, protein binding to the phenobarbital-responsive
unit assessed by in vitro DNase I footprinting was compared
with that assessed by DNase I in vivo footprints in native
chromatin. A region centered on a putative nuclear factor-1 site was
the major protected region in in vitro footprints, and
there were no detectable differences in binding between extracts from
control and phenobarbital-treated animals. In contrast, phenobarbital
treatment dramatically altered the protection pattern in native
chromatin. In control samples a core region of about 25 base pairs (bp)
centered on the nuclear factor-1 site was protected. However, after
phenobarbital treatment, the protection of this core region was
increased, and more dramatically the region of protection was extended
20 bp to either side so that a total of about 60 bp were protected.
These results provide the first evidence that phenobarbital treatment
alters the composition or architecture of proteins binding to the
phenobarbital-responsive unit region and indicate that chromatin
structure is important in this process. Because proteins are bound to
the region in the untreated animal, the mechanism of induction involves
the activation of proteins bound to the region and possibly recruitment
of additional regulatory proteins rather than conversion of a closed
chromatin structure to an open one that can bind regulatory
factors.
2.3
kilobase pairs in the rat CYP2B1/2 (4, 5), and an analogous
region is present in the mouse (6) Cyp2b10 genes. Multiple
regulatory elements contribute to the PB effect so that this enhancer
has been termed the PB response unit (PBRU) or module (6). Despite the
functional data implicating the PBRU in the PB response, analysis of
protein binding by either gel shift assays in rat2 (4) and
mouse genes (6) or by in vitro footprinting in the mouse
gene (6) has revealed little or no change in protein binding to this
region after PB treatment. Here we show that a 25-bp core region of the
PBRU DNA is protected from DNase I in native chromatin in rat liver
nuclei from control animals and that PB treatment increases the
protection from DNase I digestion in the core region and substantially
extends the strongly protected region to 60 bp. These results indicate
that PB treatment alters the composition or structure of the protein
complex binding to the PBRU in native chromatin.
Fig. 3.
Sequence of the CYP2B1/2 PBRU.
Arrows underlining the sequence indicate the sequence and
positions of the primers used for ligation-mediated PCR.
Open, hatched, and solid bars indicate regions protected from DNase I cleavage by protein binding for the
in vitro, control native chromatin, and PB-treated native chromatin footprints, respectively. Bars above and below the
sequence are for the sense and antisense strands, respectively.
Asterisks indicate hypersensitive sites in the in
vitro footprints, and arrowheads indicate
hypersensitive sites for the chromatin footprints.
5 units DNase I/µg DNA at 37°C for 5 min. 2-3 µg
of the genomic DNA was subjected to ligation-mediated PCR as described
(10). Annealing temperatures for the primers S1/A1, S2/A2, and S3/A3 were 56, 60, and 67 °C, respectively. A G-ladder as a marker was generated by ligation-mediated PCR of purified genomic DNA that had
been incubated with 0.5% dimethylsulfate at room temperature for 2 min
followed by piperidine treatment as described (10). Reproducible
results were obtained with five to seven ligation-mediated PCR
reactions for each strand from two sets of control and PB-treated rats.
2180 to 2205, which
includes an NF-1 motif that has been shown to bind to proteins that
react with antiserum to NF-1.2 A second
region of protection was observed from about 2221 to 2234. Several
hypersensitive sites were detected in the regions from 2144 to
2173, from 2213 to 2217, and at 2237 flanking the protected
regions. Similar footprints in the control and PB-treated samples
suggest that PB does not change the concentration or affinity of
proteins binding to the PBRU region, although binding of different proteins to the same site cannot be excluded.
Fig. 1.
Binding of liver nuclear proteins from
control (C) and PB-treated (PB) rats to the
antisense and sense strands of the PBRU region detected by in
vitro footprinting. DNA was incubated with 2.5 (left
lanes) or 12.5 ng (right lanes) DNase I, and DNA with
nuclear extract added was incubated with 150 ng of DNase I. The
protected regions are indicated by brackets, and
arrows denote hypersensitive sites. The indicated nucleotide
positions were determined by sequencing ladders run in parallel (not
shown).
2180 to
2210, and hypersensitive sites were observed at 2162/2170 and
2225/2227 (Fig. 2). The protected
region, centered on the NF-1 site, was very similar to the protection observed in the in vitro footprints, and the hypersensitive
sites were within regions of hypersensitivity observed in
vitro, although the protected region from 2221 to 2234
observed in the in vitro experiment was not present (Fig.
3). In the footprints from nuclei of
PB-treated rats, the level of protection within the control footprint
region was increased, and more dramatically the footprints for both the
antisense and sense strands were extended about 20 bp to either side of
the control footprints (Fig. 2). The total protected region was 60 bp
from 2163 to 2222 (Figs. 2 and 3). The extended protection was
particularly evident for the region from about 2163 to 2182.
Interestingly, this region exhibited DNase I hypersensitivity in both
control and PB-treated in vitro footprints, which is
suggestive of protein interaction. Reproducible results were obtained
in samples from two sets of rats analyzed independently as shown.
Fig. 2.
Binding of protein to native chromatin in
nuclei from control and PB-treated rats detected by in vivo
footprinting. DNase I sensitivity of the sense strand and the
antisense strand for two independent experiments for control
(C) and PB-treated (PB) animals is shown. For the
sense strand, a repeat analysis of the second set of animals is shown
in which more similar amounts of radioactivity were analyzed for the
control and PB samples so that the differences are illustrated better.
DNase I sensitivity of isolated DNA (DNA) is also shown. DNA
was amplified and labeled by ligation-mediated PCR as described under
"Experimental Procedures." Brackets with
dashed and solid lines mark the protected regions for the control and PB-treated samples, respectively, and
arrows indicate hypersensitive sites. The indicated
nucleotide positions were determined from sequencing ladders and from
G-ladders generated from genomic DNA by ligation-mediated PCR (not
shown).
2258 on the 5
side or to 2170 on
the 3 side. Likewise, in the mouse Cyp2b10 gene, a
corresponding 32-bp core sequence was defined by transcriptional
analysis, and an additional flanking sequence from either side of the
core was required to confer maximal PB induction (6). The region from 2170 to 2258, defined functionally, largely overlaps the protected region in chromatin in the PB-treated samples from 2161 to 2223. In
agreement with the functional studies, protein is bound to the
CYP2B1/2 core region in both the control and PB-treated
samples in native chromatin, whereas the binding extends further into the flanking regions in the PB-treated samples.
2.3 kilobase pairs as
the primary mediator of the response to PB. The enhancer region from
the rat CYP2B1/2 genes or the homologous region from the
mouse Cyp2b10 gene has been shown to mediate the response in
transfected primary hepatocytes or after transfection of rat liver
in situ (4-6), and sequences upstream of 800 bp are
required for PB responsiveness in transgenic mice (16). The present
studies, which show for the first time that PB treatment dramatically
alters the pattern of protein binding to chromatin in the PBRU region,
provide additional evidence that this distal enhancer plays a major
role in PB induction.
To whom correspondence should be addressed: Dept. of Molecular and
Integrative Physiology, University of Illinois at Urbana-Champaign, 524 Burrill Hall, 407 S. Goodwin Ave., Urbana, IL 61801. Tel.: 217-333-1146; Fax: 217-333-1133; E-mail: byronkem{at}uiuc.edu.
1
The abbreviations used are: PB, phenobarbital;
PBRU, phenobarbital-responsive unit; NF-1, nuclear factor-1; PCR,
polymerase chain reaction; bp, base pair(s).
2
S. Liu, Y. Park, I. Rivera-Rivera, H. Li, and B. Kemper, submitted for publication.
Volume 272, Number 47,
Issue of November 21, 1997
pp. 29423-29425
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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