(Received for publication, April 18, 1995; and in revised form, May 31, 1995)
From the
We previously reported (Liang, Q., He, J.-S., and Fulco, A.
J.(1995) J. Biol.Chem. 270, 4438-4450) that
Bm3R1, a repressor regulating the expression of P450
P450
Figure 1:
Comparison of the binding affinity of
wild-type and mutant Bm3R1 to Barbie box sequences. The gel mobility
shift assay was carried out as described under ``Experimental
Procedures'' section. The labels are as follows: E,
protein extracts of E. coli cells carrying vector
pKKf223-3 (as a negative control; Bm3R1, protein
extracts of E. coli cells harboring pGS101, a construct for
over-expression of Bm3R1; G39E-Bm3R1, protein extracts of E. coli cells harboring pGS102, a construct for
over-production of G39E-Bm3R1; BB
Since pentobarbital was previously shown to strongly
inhibit the interaction between Bm3R1 and O
Figure 2:
Competitive assays for the binding
affinity of Bm3R1 to Barbie box elements. Each binding reaction (lane)
contained 4 ng of probe and 4 µg of proteins. 60-fold of competitor
was added in each competition assay. Lane designations for E, Bm3R1, BB
Figure 3:
Protection of the BB
Figure 4:
Protection of the BB
Figure 5:
DNA sequence of 5` regulatory region of
the P450
Figure 6:
Protection of the O
Figure 7:
Comparison of the binding affinity of
wild-type and mutant Bm3R1 to the O
Because the interaction between Bm3R1 and
O
Figure 8:
Competitive titration assays for the
interaction between probe C and Bm3R1. A designates a 177-bp
DNA fragment containing the O
Figure 9:
Heterologous CAT constructs for assaying
the effects of Bm3R1 in vivo. Only the portion covering the
promoter and the promoterless CAT gene is shown in the figure for each
construct. Open reading frames are represented by boxes.
Promoters are indicated by arrows. In the A
orientation constructs (designated by an A in the names of the
constructs), CAT was placed downstream from a small P450 coding region.
In the B orientation constructs (designated by a B in the name
of constructs), CAT was placed downstream from the Bm1P1 or Bm3P1
coding regions. Barbie box elements BB
Figure 10:
CAT activity comparison between wild-type
and G39E mutant B. megaterium cells transformed by
heterologous CAT constructs. Each pair of comparisons was assayed for
CAT expression in wild-type and G39E mutant B. megaterium cells grown under identical conditions. The measurement of CAT
activity in soluble protein extracted from B. megaterium cells
grown in the presence (PENT+) or absence (PENT-) of 4 mM pentobarbital is described in
detail under ``Experimental Procedures.'' For A orientation
constructs, cell cultures were induced overnight (15 h) by 4
mM pentobarbital; for B orientation constructs, the
pentobarbital induction time was 2-4 h. The CAT activity value
shown in each plot is a mean (+S.D.) of three sets of data from
three completely separate cell cultures. CAT activity was then measured
3 times in each cell culture.
The
regulatory effects of Bm3R1 in vivo were further evaluated by
estimating the protein levels of cytochromes P450
Figure 11:
Western blot analysis of cytochrome
P450
Three lines of evidence indicate that Bm3R1, a repressor of
the P450 In our initial experiments to
determine whether Bm3R1 could interact with Barbie box sequences, we
used Bm3R1 that had been highly purified from extracts of E. coli cells transformed by pGS101(3) . Although purified Bm3R1
did recognize Barbie box sequences in these experiments, the
interaction was weak and unstable and the interaction complex had very
slow mobility. When crude extracts from the same E. coli cells
transformed by pGS101 were tested in gel shift assays, a very strong
interaction complex between Bm3R1 and Barbie box probes was obtained
(as discussed above). One plausible explanation may be related to the
fact that highly purified Bm3R1 exists primarily in highly aggregated
forms as reported previously(3) . The aggregated Bm3R1 protein
may not bind to or may have greatly reduced affinity for Barbie box
sequences. The soluble protein in crude extracts from
pGS101-transformed E. coli may maintain the Bm3R1 in
nonaggregated or lower aggregated (i.e. dimeric) forms, which
may be the species involved in binding to Barbie box elements. Our
finding that Bm3R1 interacted with the O This alignment makes clear the similarity between the Barbie box
sequences and portions of the palindromic operator sequences. In
particular, the poly(A) motif is highly conserved. The recognition
of several operator sites by one repressor protein is not a unique
finding. One well established model is the regulon of SOS DNA repair
genes in E. coli(17) . A single repressor, LexA, for
SOS DNA repair genes (dinF, uvrA, dinA, dinB, uvrB, sulA, umuC, umuD and recA), scattered over a number of nonadjacent sites
on the E. coli chromosome, can specifically recognize each
operator site and repress the transcription of all of these genes. The
fact that Bm3R1 can recognize all three operator sites indicates that
the P450 CAT
assays with the heterologous CAT constructs of the regulatory regions
of P450 genes (Fig. 10) and the Western immunoblotting analyses (Fig. 11) on the expression of P450 cytochromes in wild-type and
the G39E mutant B. megaterium cells confirm that Bm3R1 is a
repressor of both P450 Based primarily on the results of our experiments showing
that the barbiturate-mediated induction of cytochrome P450s in B.
megaterium involve coordinately regulated and mechanistically
related derepressions, we propose the following model for the
transcriptional regulation of the P450
The
nucleotide sequence(s) reported in this paper has been submitted to the
GenBank®/EMBL Data Bank with accession number(s) X16610 [GenBank]and JO4832.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
in Bacillus megaterium,
could bind to Barbie box sequences in the 5`-flanking regions of
barbiturate-inducible genes. We've now shown that pentobarbital
does not inhibit in vitro binding of Bm3R1 to the P450
and P450
Barbie boxes (BB
and BB
), although
the palindromic operator sequence (O
) of P450
did have a strong competitive
effect on such binding. G39E-Bm3R1, a mutant of Bm3R1, did not bind to
either Barbie box. In the presence of Bm3R1, portions of the regulatory
regions of P450
and P450
were protected from DNase I
digestion. These included 11 of the 15 base pairs of BB
plus 7 base pairs 3` to BB
, BB
plus 16
base pairs 3` to BB
, and, in the 5`-flanking region of P450
, segments covering most of two
palindromic sequences (O
and O
) of 24 and 52
base pairs. These DNase I-protected regions (including O
)
showed considerable sequence identity, especially in a conserved
poly(A) motif. Barbiturates did not inhibit binding of Bm3R1 to
O
O
in vitro while G39E-Bm3R1
did not bind. The regulatory effects of Bm3R1 on P450
and P450
were also
evaluated in vivo using heterologous chloramphenicol
acetyltransferase constructs and Western blotting. In the G39E mutant
strain, both P450
and P450
were constitutively expressed,
and the regulatory proteins Bm1P1 and Bm3P1, although still
pentobarbital-inducible, had significantly higher basal levels of
synthesis. In toto, our results show that Bm3R1 represses both P450
and P450
expression and that it may effect
this by coordinate binding to operator and Barbie box sequences to
produce looping of the P450
and P450
regulatory regions through
protein-protein interaction.
and P450
are
barbiturate-inducible P450 cytochromes from Bacillus megaterium that were discovered, characterized, cloned, and sequenced in our
laboratory(1) . Induction of the P450
gene involves the barbiturate-mediated and coordinately
regulated release of repression caused by the binding of a protein,
Bm3R1, to a 20-bp (
)palindromic operator sequence located in
the 5`-flanking regions of the P450 structural
genes(2, 3) . More recently, it has been shown that
certain peroxisome proliferators are even more active than barbiturates
as inducers of the P450
gene and
apparently act by a similar mechanism(4) . Confirmation that
Bm3R1, which contains a helix-turn-helix DNA-binding motif, is the
critical factor in the barbiturate-mediated regulation of the
expression of P450
was obtained by the
characterization of the defect in a B. megaterium mutant that
constitutively expressed P450
(2) .
Complementation of this constitutive mutant by a DNA fragment
containing the wild-type bm3R1 gene caused repression of
P450
synthesis in the mutant. Sequence analysis of the bm3R1 gene and its upstream region from this mutant identified
a single base change in the codon for residue 39 that resulted in a Gly
to Glu substitution in the
-turn region of the DNA-binding motif.
Unlike wild-type Bm3R1, the mutant protein did not bind to a 20-bp
palindromic operator site in the regulatory region of P450
(2, 3) nor to
other regulatory DNA sequences such as Barbie box elements in the
5`-flanking regions of the P450
and P450
genes that have also been
identified as binding sites for
Bm3R1(5, 6, 7) . Barbie box elements are
homologous 15-17-bp DNA sequences that are found in essentially
all eukaryotic and prokaryotic genes whose 5`-flanking regions are
known and that encode barbiturate-inducible
proteins(3, 6) . Indeed, in a recent publication (8) it has been reported that a Barbie box element is
critically involved in the expression of the phenobarbital-inducible
-acid glycoprotein gene of the rat. Transfection of
rat primary hepatocytes with a CAT construct containing a portion of
the Barbie box regulatory region induced basal expression of
chloramphenicol acetyltransferase activity, which was increased by
phenobarbital and dexamethasone treatment of cells. Induction of
chloramphenicol acetyltransferase activity by phenobarbital was
abolished when rat hepatocytes were transfected by constructs with a
mutation or deletion of the Barbie box sequence. In B.
megaterium, mutation in a 4-bp sequence, AAAG (found in the same
relative position in all Barbie box elements), in the P450
Barbie box led to the
constitutive synthesis of cytochrome P450
; mutation of
the same region in the P450
Barbie box
significantly increased the expression of P450
in response to pentobarbital induction but left the basal
levels unaffected(6) . It was also shown that Bm3R1 could
specifically interact with the Barbie box sequences but that mutated
Barbie boxes showed a decreased binding affinity for Bm3R1 compared
with their wild-type counterparts. Barbie box sequences were also shown
to specifically interact with barbiturate-inducible putative positive
regulatory factors of B. megaterium cells(6) . In the
present paper, we characterize in detail the specific interactions
between Bm3R1 and the Barbie box elements and three operator sites in
the regulatory regions of the P450
and P450
genes.
Materials
Oligonucleotides used in gel mobility
shift assay and polymerase chain reaction procedures were synthesized
by Integrated DNA Technologies Inc. Restriction endonucleases, T4
polynucleotide kinase, T4 DNA ligase, Klenow fragment of DNA
polymerase, and DNase I were purchased from either New England Biolabs
or Life Technologies, Inc. Taq DNA polymerase was a product of
Promega. [-
P]ATP and
[
-
P]dNTP were from DuPont NEN.
[1-
C]Acetyl coenzyme A and dNTP were bought from
Amersham Corp. Poly(dI-dC)
poly(dI-dC) was from Pharmacia Biotech
Inc. Geneclean® kits were ordered from Bio-101. Rabbit polyclonal
antibodies to cytochrome P450
and P450
were prepared in our lab as described previously (9) .
Goat anti-rabbit IgG conjugated to horseradish peroxidase was purchased
from Bio-Rad Laboratories. DNA sequences of cytochrome P450
and P450
genes, available under the GeneBank accession number X16610
and JO4832, respectively, were cloned in our
laboratory(10, 11) . All chemicals used in the
experiments were reagent grade or better.
Bacterial Strains and Recombinant
Plasmids
Escherichia coli DH5 (recA
, F
, endA1,
gyrA96, thi-1, hsdR17, supE44, relA1) was used for plasmid
transformation and preparation. Plasmid vectors for DNA recombinant
manipulation were pTZ19R and pUC19. E. coli JM109 was used as
the host for the overproduction of wild-type Bm3R1 and G39E-Bm3R1.
Plasmid constructs for overproduction of wild-type Bm3R1 (pGS101) and
G39E-Bm3R1 (pGS102) were described in detail
previously(2, 3) . Plasmid pUBcat(12) , an E. coli-B. megaterium shuttle vector containing a promoterless
CAT gene, was utilized in the analysis of Bm3R1 effects on the
transcriptional regulation of the P450
and P450
genes.
Heterologous CAT recombinant plasmids (pcat1A, pcat1B, pcat3A, and
pcat3B) used in this study were constructed as described in detail
previously(6) . B. megaterium ATCC14581, the original
strain from which the P450
and P450
genes were cloned in our
laboratory(11, 12) , was used as the wild-type host
for transcriptional regulation analysis of P450
and P450
genes and
constructs. G39E, a strain of B. megaterium ATCC14581
carrying a point mutation in the bm3R1 gene resulting in a
G39E substitution in Bm3R1 repressor(2) , was also used in
experiments presented here.
Protein Preparations
E. coli cells
harboring pGS101 (or pGS102, pKK223-3) were grown in 500 ml of
culture medium overnight at 37 °C in the presence of 1 mM
isopropyl-1-thio--D-galactopyranoside inducer with
shaking and then harvested by centrifugation. The cells were
resuspended in 20 ml of 25 mM Tris-Cl buffer (pH 8.0)
containing 1 mg/ml lysozyme and 1 mM dithiothreitol and
incubated at 37 °C for 5 min. The cells were then broken by pulsed
sonication for 6 min. The resulting preparation was centrifuged at
40,000
g for 30 min to remove cell debris, and the
supernatant solution was treated with ammonium sulfate (40% of
saturation) to yield a protein precipitate. The precipitated protein
pellet was resuspended in 5 ml of 25 mM Tris-Cl buffer (pH
8.0) and dialyzed twice against 2-liter volumes of 25 mM Tris-Cl buffer (pH 8.0) to remove the ammonium sulfate. The
resulting proteins were used for gel retardation and footprinting
experiments.
Preparation of DNA Fragments
All oligonucleotides
used in the gel retardation experiments reported here were
double-stranded, although single-stranded sequences are shown. The
sequences used here that contained Barbie box elements were prepared as
described previously(6) . These included BB, the P450
Barbie box
(5`-CCATAAAAAGCTGGTGCGTATGCC-3`), and BB
, the P450
Barbie box
(5`-GCATATCAAAAGCTGGTGGAATTT-3`). In each case, the bases
depicted in boldface correspond to the 15-bp Barbie box sequence.
O
, the P450
palindromic
operator sequence (5`-CGAATGAACGTTCATTCCG-3`) described previously (2) was used in some of the experiments reported here as were
the previously described (7) plasmid constructs pBM1-385,
pBM1-385A, and pBM1-385B. Plasmid pBM1-385A was
digested with HindIII and labeled with
[
-
P]dNTP using the Klenow fragment of DNA
polymerase. After cutting with EcoRI, an end-labeled DNA
fragment of 177 bp (corresponding to bp -44 to -221 of the
5`-flanking region of the P450
structural gene) containing the P450
operator sites was obtained from low melting point agarose
gel electrophoresis using the Geneclean kit. A similar strategy was
used in obtaining a one-end-labeled DNA fragment of 208 bp (-222
to -429) containing BB
and an one-end-labeled DNA
fragment of 385 bp (-44 to -429) containing both BB
and operator sites. All of these three radioactive labeled DNA
fragments were used in gel mobility shift assays. The 385-bp DNA
fragment, by labeling at different ends, was also used in the DNase I
footprinting assays on BB
and operator sites, respectively.
The construct pBM3-9 containing a 447-bp fragment that
incorporated the P450
promoter region
including the P450
Barbie box
(BB
) (6) was digested with SalI and then
labeled with [
-
P]dNTP using the Klenow
fragment of DNA polymerase. This 447-bp DNA fragment was then excised
by HindIII digestion to render it labeled at one end and
purified by low melt agarose gel electrophoresis and the Geneclean kit.
The resulting DNA fragment was used for the DNase I footprinting study
of BB
.
Western Blotting Procedures
SDS-polyacrylamide gel
electrophoresis was carried out by a procedure based on that of Laemmli (13) . Immunoblotting for the detection of cytochrome P450 and P450
was performed as described by Harlow and Lane (14) with some modifications. The proteins in the SDS-PAGE gel
were transferred onto a nylon membrane using a Bio-Rad Trans-blot cell.
After being dried in a refrigerator overnight, the membrane was blocked
by incubation for 1 h in 5% nonfat dried milk in 1
TPBS (137
mM NaCl, 2.7 mM KCl, 4.3 mM
Na
HPO
-7H
O, 1.4 mM KH
PO
, 0.3% Tween 20) at room temperature
on a shaker. A preparation of 5% nonfat dried milk in 1
TPBS
and 1
TPBS was used alternatively in rinsing and washing the
membrane after binding the primary antibodies and the
peroxidase-conjugated secondary antibodies. Polypeptides corresponding
to P450
and P450
were detected with hydrogen
peroxide and 4-chloro-1-naphthol.
Other Methods
Gel mobility shift assays were
performed as described previously (6) except that some reaction
mixtures contained barbiturate inducers. Pentobarbital and methohexital
were dissolved in 10 mM potassium carbonate and added in a
mixture with proteins and probe to initiate the binding reaction. DNase
I footprinting analyses were performed as described previously (6) except that different binding buffers were used. The buffer
used in the experiments reported here contained 30 mM Tris-Cl
(pH 8.0), 100 mM KCl, 0.2 M dithiothreitol, 0.1
mM EDTA, 100 µg/ml bovine serum albumin, 1 µg of
poly(dI-dC)poly(dI-dC), and 10% glycerol. Chemical
(Maxam-Gilbert) sequencing reactions (G+A) were carried out by
standard procedures(15) . The procedures for CAT assays were
described in detail previously(6) . Techniques for
transformation of B. megaterium protoplasts by plasmids and
subsequent regeneration were based on Imanaka's
procedure(16) .
Comparison of Binding Affinity of Wild-type Bm3R1 and
G39E-Bm3R1 to Barbie Box Sequences
In a previous
report(6) , we found that Bm3R1, a repressor of the P450 gene(2) , specifically
interacted with Barbie box elements in the 5`-flanking regions of P450
structural genes of B. megaterium. In a side-by-side
comparison using gel mobility shift assays with wild-type Bm3R1 and
G39E
Bm3R1 overproduced in E. coli cells, we now found
that the G39E
Bm3R1 failed to interact with the Barbie box
sequences (Fig. 1). With wild-type Bm3R1, a protein-Barbie box
complex was detected in the assay, and the intensity of this complex
(band) was enhanced with increasing levels of Bm3R1 protein (see lanes2-5 for BB
and lanes12-15 for BB
).
However, when G39E
Bm3R1 was substituted for Bm3R1 at the same
concentrations, no protein-Barbie box complex could be detected (see lanes7-10 for BB
and lanes17-20 for BB
). This
indicates that G39E-Bm3R1 not only lost binding affinity for the
operator (O
) site of the P450
gene (2, 3) but also for the Barbie box
elements.
, the
Barbie box sequence of the P450
gene; BB
, the Barbie box sequence of the P450
gene. Each binding reaction
(lane) contained 4 ng of
P-labeled Barbie box sequence as
probe and 4 µg of protein. The complex formed by Bm3R1 and the
Barbie box probe is indicated by a boldfacearrow; a
weak band formed by E. coli protein and the Barbie box is
marked by a smallarrow.
in
vitro(3) , we set up a similar gel retardation experiment
to see if pentobarbital had inhibitory effects on the interaction
between Bm3R1 and Barbie box sequences. The results (data not shown)
were negative; pentobarbital had no inhibitory effects on the binding
of Bm3R1 to either BB
and BB
.
Competition Assays Probing the Interaction between Bm3R1
and Barbie Box Sequences
Since Bm3R1 could bind to BB and BB
as well as to O
, we carried out
competition experiments by gel retardation to determine the relative
binding affinities of the three sites for Bm3R1. Three conclusions
could be drawn from the results of these experiments as shown in Fig. 2. First, it is apparent that the three sequences are
strong competitors of each other for binding to Bm3R1 (in Fig. 2, compare lanes4, 6, and 7 with lane3 and lanes11, 13, and 14 with lane10). Second,
the Barbie box sequences have higher affinities than O
for the binding to Bm3R1 (compare, in Fig. 2, lane4 with 7 for BB
/O
and lane11 with 14 for
BB
/O
). Third, it appears that Bm3R1 has a
slightly higher affinity for BB
than for BB
(compare lane6with 4 or lane 11 with 13 in Fig. 2).
and BB
are the same as described in the
legend for Fig. 1as are the conditions used to carry out the
assays. Other lane designations include BB
m, the mutant Barbie box
sequence of P450
; BB
m, the mutant Barbie box
sequence of P450
; and O
, the operator sequence of P450
. The complex formed by Bm3R1 and
the Barbie box probe is indicated by a boldfacearrow; a weak band formed by E. coli protein and
the Barbie box probe is marked by a smallarrow.
Footprinting Analysis of the Bm3R1-Barbie Box
Interactions
To determine which DNA sequences of the regulatory
regions interacted with the repressor Bm3R1, as detected by the gel
retardation experiments presented above, one-endlabeled DNA fragments
containing Barbie boxes were used as probes for DNase I footprinting
assays with crude extracts of transformed E. coli cells in
which Bm3R1 was overproduced by the construct pGS101(3) . The
results of footprinting assays are shown in Fig. 3and Fig. 4. In a 447-bp fragment of the P450 regulatory region containing the BB
sequence, an in vitro footprint appeared in response to increasing protein
concentration (Fig. 3A) that covered a 18-bp region
(-219 to -237). The protected region included 11 of the 15
bp of BB
and 7 additional bp 3` to this Barbie box sequence (Fig. 3B) and is almost identical to the region (bp
-219 to -239) protected by positive factors from B.
megaterium(6) . It is thus clear from these results that
both repressor Bm3R1 and as yet unidentified positive factors compete
for binding at the Barbie box site in the 5`-flanking region of the P450
gene. In the 385-bp fragment
containing the BB
sequence from the P450
5`-flanking region, a region
protected from DNase I digestion was detected over a 31-bp span
(-285 to -316), which included the entire Barbie box and 16
bp 3` to BB
(Fig. 4). Analysis of this protected
region reveals an 8-bp tandem repeat separated by 14 bp (Fig. 5). The first half of the repeat overlaps with one-half of
the 6 bp inverted repeat, which bound to putative positive factors of B. megaterium(6) and resides within the Barbie box.
The overlap of these two different repeats provides additional evidence
in support of our previous observation (6) that Bm3R1 and
positive factors compete for binding to the Barbie box site, presumably
in a manner affecting the transcriptional regulation of P450
.
region of P450
from DNase I by Bm3R1. In panelA, the protected region is indicated on the rightmargin by a solidbracket. Numbers on the leftmargin identify the
positions of bases relative to the translational start site of the P450
gene. A 447-bp SalI-HindIII fragment containing BB
excised from pBM3-9 was end-labeled with
P at
the SalI site, incubated with a protein extract of E. coli cells in which Bm3R1 was overproduced by the construct pGS101, and
then subjected to DNase I digestion as described under
``Experimental Procedures.'' E indicates the lane
containing the protein extract of E. coli cells carrying
vector pKK223-3 (the vector for construct pGS101). Bm3R1 indicates lanes containing the protein extract of E. coli cells in which Bm3R1 was overproduced. In panelB, the DNA sequence covering the protected region and the
BB
element is shown. BB
is identified by boldface. The protected sequence is indicated in the same
manner as in panelA.
region of P450
from DNase I digestion by Bm3R1. A 385-bp EcoRI-HindIII fragment containing BB
from
pBM1-385 was end-labeled with
P at the EcoRI site, incubated with a protein extract of E. coli cells in which Bm3R1 was overproduced by the construct pGS101, and
then subjected to DNase I digestion. E indicates the lane
containing the protein extract of E. coli cells carrying
vector pKK223-3 (the vector for construct pGS101). Bm3R1 indicates lanes containing the protein extract of E. coli cells in which Bm3R1 was overproduced. The protected region is
indicated on the rightmargin by a solidbracket. Numbers on the leftmargin identify the positions of bases relative to the translational
start site of the P450
gene.
gene showing the locations of three
protected footprints on BB
, O
, and O
sites in the presence of Bm3R1. The three footprinted regions are underlined. Three pairs of inverted repeats are indicated by
pairs of arrows numbered 1, 2, and 3; a pair
of tandem repeats is indicated by a pair of tandemarrows, each marked with a. The 15 bp of the
BB
sequence are in boldface as are the bases of
the -10 and -35 regions. The O
and O
sequences are in lower case. The P450
promoter is labeled -35 and -10; the Bm1P1 promoter is labeled -10
and -35. Each transcription start site is indicated by an vertical arrow; the the bases of translation start codons for
are marked with asterisks.
Binding of Bm3R1 to Operator Sites of the P450
In the
footprinting analysis of the 385-bp fragment from the P450Promoter
regulatory region, two sites in
addition to the BB
region appeared to be protected from
DNase I digestion by Bm3R1. To precisely determine the location of
these two footprints on the P450
regulatory region, the 385-bp DNA fragment was excised from
pBM1-385,
P-labeled at its HindIII end, and
then used as probe in footprinting experiments (Fig. 6). The
first protected region covered a 21-bp sequence (-68 to
-188), while the second spanned 44 bp (-112 to -155).
DNA sequence analysis of these footprints indicated that the first
protected site was located within an imperfect 24-bp palindromic
sequence (Fig. 5) and the second protected site within an
imperfect 53-bp palindromic sequence (two 24-bp segments forming a
perfect inverted repeat interrupted by a 5-base segment, TAATT, as
shown in Fig. 5). The 24-bp palindromic sequence site overlaps
two promoter regions oriented in opposite directions. The first
promoter, which drives the transcription of the P450
gene, and the second, which
directs the expression of Bm1P1 overlap in their -35
regions. The 53-bp palindromic sequence covers the -10 region of
the P450
promoter and the initiation
site of the P450
transcript. Based on
their structural positions on the regulatory region and their ability
to interact with repressor Bm3R1, the 24-bp palindromic sequence is
designated as operator site one (O
), and the 53-bp
palindromic sequence is designated as operator site two
(O
). Bm3R1 may thus suppress the expression of both the P450
and Bm1P1 genes by
binding to O
and O
as well as to
BB
.
and
O
regions of the P450
gene from
DNase I digestion by Bm3R1. A 385-bp EcoRI-HindIII
fragment containing the O
and O
sites from
pBM1-385 was end-labeled with
P at the HindIII site, incubated with a protein extract of E. coli cells in which Bm3R1 was overproduced by the construct pGS101, and
then subjected to DNase I digestion. E indicates the lane
containing a protein extract of E. coli cells carrying vector
pKK223-3 (the vector for construct pGS101). Bm3R1 indicates lanes containing protein extract of E. coli cells in which Bm3R1 was overproduced. The protected regions are
indicated on the rightmargin by solidbrackets. Numbers on the leftmargin identify the positions of bases relative to the
translational start site of the P450
gene. Sequencing analysis on the footprints are shown in Fig. 5.
The Interaction between Bm3R1 and
O
Once it was established that Bm3R1 could
interact with OO
as Revealed by Gel Mobility
Shift Assays
and O
, we next asked whether
the G39E
Bm3R1-mutated protein retained this attribute.
G39E
Bm3R1, overproduced in E. coli by construct pGS102 (2) , was used in a gel shift assay with a 177-bp DNA fragment
containing both O
and O
utilized as a probe;
the results with G39E
Bm3R1 were directly compared with those
using wild-type Bm3R1 obtained by over-expression by pGS101 in E.
coli. (Fig. 7). A Bm3R1
DNA interaction complex
appeared with increasing levels of Bm3R1 protein (Fig. 7, see lanes2-5); the intensity of the
complex (band) decreased as the level of the unlabeled DNA fragment of
177 bp (as specific competitor) was increased in the reaction (Fig. 7, seelanes6-8). On the other hand, G39E
Bm3R1
appeared not to interact with O
and O
, and no
an equivalent band could be detected in the assay (Fig. 7, see lanes12-15). This observation is
similar to our previous finding (2, 3) that Bm3R1
could specifically interact with operator site (O
) of the P450
gene while G39E
Bm3R1 lost
such interaction.
O
sequences. compet. A designates a 177-bp DNA fragment
containing the O
O
sites; E,
protein extracts of E. coli cells carrying vector
pKK223-3 (as a negative control); Bm3R1, protein
extracts of E. coli cells harboring pGS101, a construct for
the over-expression of Bm3R1; G39E-Bm3R1, protein extracts of E. coli cells harboring pGS102, a construct for the
over-production of the mutated repressor, G39E
Bm3R1. Each binding
reaction (lane) contained 5 ng of
P-labeled fragment A.
The complex formed by Bm3R1 and the probe is indicated by a boldface arrow; a weak band formed by E. coli protein
and the probe is marked by a smallarrow.
was strongly inhibited by barbiturates (pentobarbital
and methohexital) in vitro(3) , a similar experiment
was carried out to determine whether barbiturates inhibited the
interaction between Bm3R1 and O
O
. The
results (data not shown) revealed that neither pentobarbital nor
methohexital had inhibitory effects on the interaction between Bm3R1
and O
O
in vitro. This implies
that the detailed induction mechanism mediated by barbiturates for the P450
gene may differ in certain
respects from that of the P450
gene.
Competitive Assays for Binding of Bm3R1 to the
O
Based on the observation that Bm3R1 could interact
with OO
and BB
Sites of the P450
Regulatory
Region
and O
as well as with BB
on
the P450
regulatory region, we carried out gel shift
assays designed to determine whether BB
and
O
O
enhanced or competed with each other
for binding to Bm3R1. Three different DNA fragments of the 5`
regulatory region of the P450
gene
were used both as probes and competitors in the experiment (see
``Preparation of DNA Fragments'' under ``Experimental
Procedures'' for details). The first was a 177-bp DNA fragment
(designated A) containing the O
O
sites;
the second was a 208-bp fragment (designated B) containing the BB
site; the third, a 385-bp fragment (designated C), consisted of A
+ B and thus contained all three sites. These results (data not
shown) indicate that DNA fragment C (containing both O
,
O
, and BB
) had a higher binding affinity for
Bm3R1 than did an equimolar mixture of DNA fragment A (containing
O
and O
only) and B (containing BB
only). We next performed a competitive titration assay on the
interaction between DNA fragment C (as probe) and Bm3R1 using fragment
C or a combination of fragment A and B as competitors. As Fig. 8shows, fragment C strongly titrated its own
probe
Bm3R1 complex (in Fig. 8, compare lanes4-8 with lane3).
Conversely, the combination of fragment A and B had a relatively weak
competitive effect on the interaction of probe C with Bm3R1 (in Fig. 8, compare lanes10-14 with lanes9 and 4-8).
The fact that a 20-fold excess of fragment C competes more effectively
for Bm3R1 in the presence of probe C than a 150-fold excess of the A
+ B combination implies that when BB
and
O
O
are separated on different DNA
fragments, they compete with each other for binding to Bm3R1, but when
they are located on the same DNA fragment they mutually enhance binding
to Bm3R1. One can thus infer that the complex formed by the binding of
Bm3R1 to O
O
and BB
coordinately in the same DNA fragment is much more stable than
that produced by Bm3R1 binding to O
O
or
to BB
alone. The higher stability of the complex could
perhaps be due to the protein-protein interaction between Bm3R1
molecules bound to the operator sites and BB
element. Such
protein-protein interaction may lead to bending or rolling of the
regulatory region of the P450
gene
into a DNA loop with consequent repression of the expression of the P450
and Bm3P1 genes.
O
sites; B designates a 208-bp fragment containing the BB
element; and C designates a 385-bp fragment containing
the O
O
and BB
sequences. Lanes1 and 17 contained probe C only. Each
binding reaction (lanes2-16)
contained 5 ng of
P-labeled C probe and 6 µg of
protein extract of E. coli cells in which Bm3R1 was
overproduced by pGS101. Added competitors were unlabeled. The bracket designated A+B contained equimolar
amounts of fragments A and B. The competitors were added to probe C in
a range of 20-150-fold by weight in the side by side titration
assays. The locations of specific Bm3R1-probe C interaction complexes
are indicated by an arrow.
Regulatory Effects of Bm3R1 in Vivo
Once we had
determined that Bm3R1 specifically interacted, in vitro, with
operator sites and Barbie box elements on the regulatory regions of the
P450 genes in B. megaterium, we next utilized a CAT assay
system to evaluate the regulatory effects of Bm3R1 in vivo.
The regulatory region (0.5 kb) of the P450 gene was cloned into pUBcat in both orientations using the
unique HindIII site upstream from the promoterless CAT gene.
The resulting two constructs were designated pcat1A and pcat1B (see Fig. 9). The same strategy was used in cloning the regulatory
region (1.6 kb) of P450
into pUBcat,
and the resulting two constructs were designated pcat3A and pcat3B (Fig. 9). These four heterologous CAT constructs were used to
transform B. megaterium ATCC14581 (as wild-type source) and
its G39E mutant strain. The G39E mutant strain was derived from
ATCC14581 and identified as carrying a G39E
Bm3R1 gene in which a
glycine codon was mutated to a glutamate codon at residue position
39(2) . G39E
Bm3R1 protein encoded by this mutated gene
failed to interact in vitro with Barbie box elements and
operator sites as described above. For cell cultures carrying A
orientation constructs (pcat1A and pcat3A), growth in the presence of
pentobarbital was carried out overnight (about 15 h); for B orientation
constructs (pcat1B and pcat3B), cell cultures were induced by
pentobarbital for 2-4 h as described previously(6) . Each
pair of wild-type and mutant B. megaterium strains carrying
the same construct were assayed for CAT activity under identical
conditions (Fig. 10). For wild-type B. megaterium cells
transformed by construct pcat1A, a considerable level of CAT activity
was produced in the absence of the barbiturate, although CAT activity
was increased 2.3-fold when these cells were grown in the presence of 4
mM pentobarbital (Fig. 10A). On the other
hand, G39E mutant cells carrying pcat1A expressed CAT at a high
constitutive level and showed essentially no response to pentobarbital.
The constitutive level of CAT in the mutant cells was, in fact, higher
than the induced level in the wild-type cells, an indication that
G39E
Bm3R1 failed to suppress the expression of the P450
gene. For wild-type cells
transformed by pcat1B, pentobarbital induced a 4-fold increase in the
level of CAT activity (Fig. 10B). For the G39E mutant,
although the cells carrying pcat1B had a pentobarbital-induced level of
CAT activity 3 times the basal level, the basal level of CAT activity
was still as high as the induced level of CAT in the wild-type cells.
This implies that Bm1P1 is also negatively regulated by Bm3R1
and that the G39E mutation in Bm3R1 leads to high basal expression of Bm1P1. The extremely high induction level of Bm1P1 in G39E
mutant cells could result from a barbiturate-responsive activator that
competes with wild-type Bm3R1 for binding to
O
O
sites. This activator is probably
Bm1P2 which was recently found to inhibit the binding of Bm3R1 to
BB
and which competed with Bm3R1 for binding to
O
O
(7) . For pcat3A, no
significant difference was observed in CAT activity between the
wild-type and the mutant B. megaterium transformant cells (Fig. 10C), presumably because it contained a 1.6-kb
DNA fragment constituting the 5` regulatory region of the P450
gene. This fragment included a
wild-type bm3R1 gene, which complemented the defects of the
mutant copy of the bm3R1 gene in the G39E mutant cell genome.
However, the same 1.6-kb DNA fragment is contained in pcat3B, but the
CAT activities produced (Fig. 10D) were similar to those from
pcat1B in wild-type and mutant B. megaterium cells (see Fig. 10B). That is, the basal level of CAT activity in
the transformant G39E cells was as high as the induction level in the
transformant wild-type cells and pentobarbital induction resulted in a
6-fold increase of CAT activity in the transformant G39E cells. It may
be that the complementation of the wild-type bm3R1 gene from
pcat3B is incomplete with respect to the transcriptional regulation of
the Bm3P1 gene in G39E cells and that G39E
Bm3R1 gives
rise to a high level of leakage of Bm3P1 expression.
and BB
and operator sites O
, O
, and O
are also indicated in the figure.
and
P450
in B. megaterium cells by Western blotting
analysis. Fig. 11, A and B, shows these
results for cytochromes P450
and P450
,
respectively. In wild-type B. megaterium cells, the basal
levels of P450
and P450
were very low but
detectable (Fig. 11, A and B, see lane1), while the pentobarbital-induced levels were high (Fig. 11, A and B, see lane2). In the G39E mutant cells, however, both P450
and P450
were highly constitutively expressed, although their
pentobarbital-induced levels were slightly higher than their basal
levels (Fig. 11, A and B, see lanes 3 and 4). This indicates that the mutation in Bm3R1 (G39
mutated to E39) results in derepression of both P450
and P450
. It is thus clear that Bm3R1 acts
as a repressor for the P450
and P450
genes in B. megaterium.
and P450
expressed in wild-type and
G39E mutant B. megaterium cells. The experiments were carried
out as described under ``Experimental Procedures.''
Cytochrome P450s were detected from protein extracts of B.
megaterium cells grown in the absence (PENT-) or
presence (PENT+) of 4 mM pentobarbital. In panelA, each lane contained 50 µg of soluble
protein extracts; in panelB, each lane contained 100
µg of soluble protein extracts. The locations of cytochrome
P450
and P450
are indicated on the rightmargin by arrows.
gene(2) , binds to
Barbie box elements on the regulatory region of the P450 genes of B. megaterium. First of all, gel retardation assays revealed a
strong, specific DNA-protein interaction complex that formed in the
reaction between protein extracts of E. coli cells carrying
pGS101 (a construct for the overproduction of Bm3R1 protein) and a
probe containing a Barbie box sequence ( Fig. 1and Fig. 2). This complex was not formed in the reaction with
extracts of E. coli harboring the control vector. Second,
G39E-Bm3R1, previously been shown to lack binding affinity for
O
(2, 3) , also failed to interact with
Barbie box elements (Fig. 1). Finally, footprinting experiments
revealed that the BB
and BB
regions were
protected by Bm3R1 against DNase I digestion ( Fig. 3and Fig. 4). This finding is in good agreement with our previous
reports in which removal (5) or mutation (6) of
BB
resulted in the highly constitutive expression of P450
, while the mutation of BB
(6) caused a significant increase of P450
expression. The demonstration of
Bm3R1 interaction with Barbie box elements thus provides convincing
evidence in support of our previous suggestion (6) that
repressor and positive factor binding sites overlap in the Barbie box
sequence and that Bm3R1 and positive factors compete with each other
for binding to the Barbie box region with consequent effects on the
regulation of P450 transcription.
and O
sites of the P450
gene as
demonstrated by DNase I protection experiments ( Fig. 5and Fig. 6) and gel shift assays ( Fig. 7and Fig. 8)
was unexpected. Since Bm3R1 was initially shown to bind to the operator
site (O
) of the P450
gene(2, 3) , we searched for sequence
similarities between O
and the two operator sites of the
P450
regulatory region. Our analysis revealed that all
three operator sites are palindromic sequences. Also, the DNA sequences
of the footprints of these sites are AT-rich and have poly(A) or
poly(T) motifs. For O
, the footprinting sequence is
ttTTTCCTTGATAACCAAGTAAAaa (A+T = 70%); for O
,
CTATTAGTACATTTTTATACTAATTGTATAAAAATGTACTAATA (A+T = 84%);
and for O
, TAGCGGAATGAACGTTCATTCCGTTTTt (A+T
= 62%). A sequence-homologous alignment for the foot-printing
sequences of operator sites and the Barbie box sequences is shown
following with half of each palindromic operator sequence underlined:
O
, TAAccaagTAAAAA; O
,
TAAttgtATAAAAAtGTAcTaAtaGTatA; O
, AAAAAcGgAaTGAacGTtcA;
BB
, ATAAAAAGCTGGTGc; BB
, ATcAAAAGCTGGTGg.
and P450
genes are coordinately regulated
in the same regulon. Results in our previous reports also implied that P450
and P450
are coordinately regulated. Thus, BB
and
BB
were recognized by the same positive factors (6) and Bm3R1 interactions with BB
or BB
were inhibited by Bm1P1 and Bm1P2 proteins(7) .
and P450
genes in vivo. The
functions of Bm3R1 were also characterized by DNA-protein interaction
assays in vitro. Bm3R1 can specifically bind to the operator
sites and Barbie box elements located on the 5` regulatory regions of
P450 genes. Since Barbie box elements are found in essentially all of
the 5`-flanking regions of the eukaryotic genes that encode
barbiturate-inducible proteins(6) , it should be interesting to
see whether barbiturate-inducible P450 genes in eukaryotes also have
the Barbie box cognate binding factor analogous to Bm3R1. In rat,
Barbie box sequences in CYP2B1/B2 were reported (18) to interact with proteins from liver cell nuclear extracts
from untreated rats to form a Barbie box-protein interaction complex.
Recently, Dr. R. A. Lubet and his co-workers (
)at the
National Cancer Institute also made a similar observation in the rat CYP2B1 system. They found that a protein in rat liver nuclear
extracts from animals untreated by barbiturates could bind to a 30-bp
DNA fragment from the 5`-flanking region of the CYP2B1 gene.
This protein disappeared, however, and was replaced by a smaller Barbie
box-binding protein in liver nuclear extracts from
phenobarbital-treated animals. As shown by Padmanaban and his
co-workers(18, 19) , this fragment contained a Barbie
box sequence and functioned as a positive cis-acting element
in regulating the transcription of the CYP2B1/B2 genes and in
mediating the inductive effects of phenobarbital. Together, these data
imply that, in the absence of barbiturate inducers, a protein binds to
Barbie box elements that represses the expression of CYP2B1/B2. In the presence of barbiturates, this repressor
protein appears to be displaced from the Barbie box by a positive
factor with the concomitant activation of CYP2B1/B2 transcription. Very
recently, Padmanaban et al.(20) reported that the
positive factor binding to the Barbie box in the presence of
barbiturates was derived by phosphorylation of the Barbie box-binding
repressor. Although these reports generally agree with our previous
suggestion (6) that significant aspects of the mechanism of
barbiturate-mediated induction of P450s (and related enzymes) are
conserved between prokaryotes and eukaryotes and that the cis-acting Barbie box sequences and its cognate protein
factors are important conserved components, the regulation of
barbiturate-mediated induction of P450s in eukaryotes may be much more
complex. In addition to the Barbie box elements, several other cis-acting elements also have been reported. Hahn et al.(21) reported a phenobarbital-responsive enhancer domain
between positions 5.9-1.1 kb of the chicken CYP2H1 gene.
Jaiswal et al.(22) found a functional glucocorticoid
response element located approximately 1.3 kb upstream of the
transcription initiation site of the CYP2B2 gene. Fernandez,
Shervington, and their co-workers (23, 24) identified
a DNA sequence located between positions 199 and 183 of the CYP2B2 promoter that interacted with nuclear proteins and was involved in
basal and phenobarbital-induced transcription of the CYP2B2 gene.
and P450
genes. In the
absence of inducers, Bm3R1 exists in a conformation that binds to
operator and Barbie box sites and in so doing enhances DNA loop
formation in the 5`-flanking region of the P450 structure gene by
protein-protein interaction between the Bm3R1 molecules. As a
consequence, the P450 gene promoters are not accessible to positive
factors, and RNA polymerase and transcription of the P450 genes is
inhibited. In the presence of barbiturates, Bm3R1 assumes another
allosteric form that can still bind to O
, O
,
and Barbie box elements, but not to O
(3) .
However, under the influence of Bm1P1 and Bm1P2(7) , the
Bm3R1-inducer complexes dissociate from O
, O
,
and Barbie box sequences and DNA looping is reversed. The positive
factors and RNA polymerase can now access the promoters of the P450
genes to greatly accelerate the rate of synthesis of the P450 mRNAs and
cause the observed induction of the P450 genes by barbiturates.
We appreciate helpful discussions with Dr. Harvey
Herschman and Dr. Kevin McEntee. We especially thank Jian-Sen He from
this laboratory for providing the pBM1-385 constructs and Lisha
Chen and Keynes Tong, also from this laboratory, for their excellent
technical assistance in several of the experiments reported here.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.