(Received for publication, July 15, 1994; and in revised form, October 11, 1994)
From the
In a previous publication (He, J.-S., and Fulco, A. J.(1991) J. Biol. Chem. 266, 7864-7869), we reported that a
15-17-base pair DNA sequence (designated a Barbie box element) in
the 5`-regulatory regions of cytochrome P450and P450
genes from Bacillus
megaterium was recognized by a barbiturate-regulated protein. It
is now recognized that essentially all eukaryotic and prokaryotic genes
whose 5`-flanking regions are known and that encode
barbiturate-inducible proteins contain the Barbie box element. A 4-base
pair sequence (AAAG) is found in the same relative position in all
Barbie box elements. In B. megaterium, mutation of the Barbie
box located in the P450
gene leads to
the constitutive synthesis of cytochrome P450
and a
10-fold increase of expression of Bm1P1, a small gene located
upstream of the P450
gene, that
encodes a putative regulatory protein. Mutation of the P450
Barbie box significantly
increased the expression of both P450
and Bm3P1 (another small gene located upstream of
the P450
gene that encodes a second
putative regulatory protein) in response to pentobarbital induction but
left the basal levels unaffected. In gel mobility shift assays, Bm3R1,
a repressor of the P450
gene, was
found to specifically interact with the Barbie box sequences of the B. megaterium P450 genes. Mutated Barbie boxes showed a
decreased binding affinity for Bm3R1 compared to their wild type
(unmutated) counterparts. Barbie box sequences were also shown to
specifically interact with putative positive regulatory factors of B. megaterium cells. These putative positive factors were
induced by pentobarbital and were also present at high levels during
late stationary phase of B. megaterium cell cultures grown in
the absence of barbiturates. The mutated Barbie box sequences had
greater binding affinity for these positive factors than did unmutated
Barbie box sequences. DNase I footprinting analysis of the 5`-flanking
region of the P450
gene revealed that
these positive factors protected a segment of DNA covering a portion of
the Barbie box sequence and a small flanking region. Similar
footprinting experiments with the 5`-flanking region of the P450
gene failed, however, to
unambiguously reveal protected sequences in the Barbie box region. The
evidence suggests that the positive factors and Bm3R1 compete with each
other for binding to the Barbie box region, especially in the
5`-flanking region of the P450
gene,
and for putative roles in the regulation of transcription from the B. megaterium P450 genes. This inference was further supported
by evidence derived from a nested-deletion analysis in and around the
Barbie box of the P450
regulatory
region that showed that a repressor binding site and a positive factor
binding site overlap at the Barbie box sequence. In toto,
these experimental results indicate that, in B. megaterium at
least, the Barbie box sequences are important cis-acting
elements for coordinately regulating the barbiturate-mediated
expression of the P450
and P450
genes and the genes encoding
their positive regulatory factors.
Cytochromes P450 and P450
are two
barbiturate-inducible P450 monooxygenases from Bacillus megaterium that were discovered, cloned, sequenced, and characterized in our
laboratory(1) . P450
(molecular mass 47.5
kilodaltons) shows sequence similarity to P450
and to
several other bacterial P450s and is moderately
barbiturate-inducible(2, 3, 4) ;
P450
(molecular mass 117.7 kilodaltons) is a
catalytically self-sufficient fatty acid monooxygenase exhibiting
significant structural homology to the 2-component microsomal
P450-reductase systems of
eukaryotes(5, 6, 7, 8, 9, 10, 11, 12, 13) and can be induced several hundredfold by
barbiturates(14, 15) . We subsequently cloned the
genes encoding these two P450s, including the apparently complete
regulatory regions, studied them with respect to the regulation of
their expression, and adduced experimental evidence that
barbiturate-mediated induction of P450
and P450
of B. megaterium may be mechanistically related to the
analogous induction of cytochromes P4502B1 and P4502B2 of the
rat(1, 4, 10, 16, 17, 18) .
Analyses of the 5`-regulatory sequences of the genes encoding these
four P450 cytochromes revealed a string of 17 bp (
)in the
5`-flanking region of each that shared a high degree of sequence
identity. Labeled oligonucleotide probes of each of these sequences
were tested in gel retardation assays with protein obtained from B.
megaterium grown either in the presence or absence of barbiturates
or with protein from nuclear extracts from livers of rats left
untreated or injected with phenobarbital. Each of the four 17-mers
bound strongly to a single protein in partially fractionated extracts
from bacteria grown in the absence of barbiturates but this binding was
dramatically reduced when extracts from pentobarbital- or
phenobarbital-grown cells were used. Conversely, the probes complexed
weakly to one protein band from nuclear extracts from untreated rats
but much more strongly with protein from phenobarbital-treated rats.
Similar effects could be obtained by prolonged incubation with
phenobarbital of either soluble protein from the bacteria grown in the
absence of barbiturates or nuclear extract protein from untreated rats.
The potential importance of this sequence, designated a ``Barbie
box'' (18) is highlighted by the observation that
essentially all of the eukaryotic genes whose 5`-flanking regions are
known and which encode barbiturate-inducible proteins contain 15 bp
sequences (the last 15 bp of the 17 bp sequences) that are highly
homologous to the 15-bp Barbie box sequence of the P450
gene and to a consensus sequence
that strongly binds the barbiturate-regulated proteins of rat and B. megaterium. These sequences are shown in Table 1. In
particular, there is an identical 4-bp sequence (AAAG) that is found in
the same relative location within the 15-bp Barbie box sequence of each
gene that seems to be critical for DNA-protein interaction. One
somewhat baffling finding reported in our published results (4) was that the barbiturate-regulated rat protein that bound
to Barbie box sequences appeared to be a positive regulator (i.e. was induced or transformed into a DNA-binding protein by
barbiturates) while the barbiturate-negatively regulated bacterial
protein appeared to be a repressor. Our results reported here offer a
possible explanation to this puzzle and establish the critical nature
of the Barbie box. We have now detected several proteins from B.
megaterium in cells grown in the presence of barbiturates which,
like the putative repressor previously identified, bind to the Barbie
box. These protein factors may be analogous to the rat positive
regulatory factor and may function by binding to the Barbie box and
displacing the repressor. As the results we report here indicate, this
putative repressor is probably identical to Bm3R1, a helix-turn-helix
DNA-binding protein shown previously to bind, in the absence of
barbiturates, to a 20-bp palindromic operator sequence near the
promoter region of the P450
gene(17) . In the presence of inducer-barbiturates,
however, Bm3R1 is inhibited from binding to its operator and induction
ensues(18) . We also present evidence in the present report
that the induction of P450
by barbiturates may also
involve competition between a positive DNA-binding protein and the
Bm3R1 repressor.
Figure 1:
Wild type and mutant
Barbie box sequences of P450 and
P450
. The 15 bp of each Barbie box sequence are
shown in bold and the target nucleotides in site-directed
mutagenesis are underlined. DNA sequences are written in
triplets according to their positions in the open reading frames. The
codons involved in mutations are indicated with their corresponding
amino acid residues. Oligonucleotide BB
and BB
were used as primers for site-directed mutagenesis of BB
by PCR; BB
and BB
were used for
BB
mutation. All of the Barbie box sequences shown
(BB
, BB
, BB
, and BB
)
were used in DNA-protein interaction assays (see gel retardation
experiments in Fig. 4, Fig. 5, Fig. 6, and Fig. 7).
Figure 4:
Comparison assays of the binding affinity
of Bm3R1 to wild type and mutant Barbie boxes. The gel mobility shift
assay was carried out as described under ``Experimental
Procedures.''. C (lanes 2 and 10),
soluble protein extracts of E. coli cells carrying
pKK223-3 (as a negative control); Bm3R1 (lanes 3-8 and 11-16), soluble protein in extracts of E.
coli cells harboring pGS101, a construct for overexpression of
Bm3R1, grown in the presence of 1 mM isopropyl--D-thiogalactoside inducer; BB
(lanes 1-5) and BB
(lanes
6-8) designate the wild type and mutant Barbie box
sequences, respectively, of the P450
gene; BB
(lanes 11-13) and
BB
(lanes 14-16) refer to the wild type and
mutant Barbie box sequences, respectively, of the P450
gene. Each binding reaction
(lane) contained 4 ng of
P-labeled Barbie box sequence and
4 µg of protein. Sixty-fold by weight of competitor was added in
each competition assay. The complex formed by Bm3R1 and the Barbie box
probe is indicated by a bold arrow; a weak band formed by E. coli protein and the Barbie box probe is marked by a small arrow.
Figure 5:
Barbie box DNA-protein interaction assays
using whole cell extracts. Various dilutions of crude protein extracts
from B. megaterium grown in the presence (BM+)
or absence (BM-) of 4 mM pentobarbital were
incubated with 4 ng of double-stranded Barbie box sequences (shown in Fig. 1) end-labeled with P in the presence of
double-stranded poly(dI-dC)
(dI-dC) and single stranded
oligonucleotides (for details, see ``Experimental
Procedures''). BB
and BB
refer to the wild
type Barbie box sequences of the P450
and P450
genes,
respectively. For each competition assay (lane), 60-fold of specific
competitor was used. Two DNA-protein complexes (bands) were detected
and are designated as slow mobility complex (S) and fast
mobility complex (F).
Figure 6:
Barbie box DNA-protein binding comparisons
between protein extracts from G39E mutant cells and those from wild
type cells of B. megaterium. Each lane (reaction) contained 15
µg of protein and 4 ng of probe. The conditions were the same as
described in the legend for Fig. 5. G39E-
indicates lanes containing protein extracts from G39E mutant cells
grown in the absence of barbiturates; G39E+ indicates
lanes containing protein extracts from G39E mutant cells grown in the
presence of 4 mM pentobarbital; BM- indicates
lanes containing protein extracts from wild type cells grown in absence
of barbiturates; BM+ indicates lanes containing protein
extracts from B. megaterium cells grown in the presence of 4
mM pentobarbital; BM20- indicates lanes
containing protein extracts from a B. megaterium cell culture
grown to very late stationary phase in the absence of barbiturates; BM20+ indicates lanes containing protein extracts from a B. megaterium cell culture grown to very late stationary phase
in the presence of 4 mM pentobarbital; BB and BB
designate the wild type Barbie box
sequences of P450
and P450
genes respectively. The slow and
fast complexes are marked S and F,
respectively.
Figure 7:
Competition assay for wild type
(BB, BB
) and mutant (BB
,
BB
) Barbie boxes. Each binding reaction (lane) contained
20 µg of protein and 4 ng of probe. The conditions were the same as
described in the legend for Fig. 5. Unlabeled BB
,
BB
, BB
, BB
, and the P450
operator sequence
(O
, 20-mer) were used as competitors in the reactions. Panel A shows the results of competition assays for BB1 and
BB
; panel B, competition assays for BB
and BB
. BM- indicates lanes
containing protein extracts from B. megaterium cells grown in
absence of barbiturates; BM+ indicates lanes containing
protein extracts from B. megaterium cells grown in the
presence of 4 mM pentobarbital. The positions of the slow and
fast complexes are indicated in the figure by S and F, respectively.
After confirming, by sequence
analysis, that we had obtained the desired mutations in the Barbie box
sequences, the mutated and wild type P450 and P450
regulatory regions, respectively, were
cloned into pUB
in both orientations using the unique HindIII site on the vector. Eight heterologous CAT constructs
were produced and designated pcat1A, pcat1mA, pcat1B, pcat1mB, pcat3A,
pcat3mA, pcat3B and pcat3 mB (Fig. 2).
Figure 2: Heterologous CAT constructs for assaying the effects of mutation on Barbie boxes. 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 A orientation constructs (designated by an ``A'' in the names of constructs), CAT was placed downstream from a small P450 coding region. In B orientation constructs (designated by a ``B'' in the name of constructs), CAT was placed downstream from the Bm1P1 or Bm3P1 coding regions. These eight different constructs were used to transform B. megaterium cells.
Figure 9:
Deletion analysis of the Barbie box region
of the P450 regulatory region. In panel A, the whole 500-bp sequence of the P450
regulatory region and the end
points of five deletion derivatives in proximity to the BB
region are shown. These five deletion derivatives were checked by
DNA sequencing analysis to confirm their end points. The numbering in
the sequence is from the translational start site of P450
. The BB
sequence is double underlined. Also indicated are the -35 and
-10 sequences (underlined) and the transcriptional start
point of the P450
gene. The five
deletion derivatives and the P450
regulatory region were cloned into vector pUB
.
Each insert is indicated by a number (which is also the
construct number shown in panel B) at its 5` end (a
superscript base). These six heterologous CAT constructs were used to
transform B. megaterium cells. In panel B, the levels
of CAT expression directed by these six constructs are indicated. B. megaterium transformed by these CAT constructs was grown in
the absence or presence of 4 mM pentobarbital and then assayed
for CAT activity (see ``Experimental Procedures'' for
detail). Each value in the plot is the average of four separate assays. PENT- indicates values for cells grown in the absence of
pentobarbital; PENT+ indicates values for cells grown in
the presence of 4 mM pentobarbital. Cells carrying constructs
4 or 6 were induced by 3 mM pentobarbital in the growth medium
since cells grown in 4 mM pentobarbital was severely
growth-inhibited.
As described under
``Experimental Procedures,'' wild type and mutated regulatory
sequences of both P450 and P450
were cloned into the E. coli-B.
megaterium shuttle vector, pUB
, each in both
orientations. The resulting eight different heterologous CAT constructs
are illustrated as in Fig. 2. In the ``A
orientation,'' the promoterless CAT gene was placed just
downstream from the 5` coding regions (5 amino acid residues for the P450
constructs; 27 amino acid residues for the P450
constructs) of P450 genes. Since there are
three stop codons upstream from the AUG start codon of the CAT gene, no
translational read-through into the CAT coding region is possible and
the effects of a mutation in a Barbie box would be directly reported by
the promoterless CAT gene. The 5`-regulatory regions of the P450
and P450
genes
each contain a small open reading frame in the opposite orientation to
and upstream from the P450 coding region. In the 5`-regulatory region
of P450
, the two opposite promoters overlap (Fig. 2); in P450
, the two opposite
promoters are adjacent. Based on the structural relationships and
orientations of these promoters, it was conceivable that mutations in
the Barbie box sequences could affect transcription of the two small
coding regions. To test this hypothesis, we placed the CAT reporter
gene at the 5` ends of the regulatory sequences of the P450
and P450
genes (the
``B orientation'' constructs in Fig. 2). All eight CAT
constructs were used to transform B. megaterium ATCC 14581.
Each pair of constructs containing the wild type or mutant Barbie boxes
were analyzed for CAT expression in the resulting transformed cells
under identical conditions (Fig. 3). Under our normal overnight
growth conditions (15-16 h of culture growth in the presence or
absence of 4 mM pentobarbital), B. megaterium cells
harboring pcat1A (wild type BB
) produced a significant
level of CAT activity in the absence of barbiturate and this activity
increased about 2.5-fold when the cells were grown in the presence of 4
mM pentobarbital (Fig. 3A). We were surprised
to find, however, that the cells carrying pcat1 mA (mutant Barbie box
BB
) expressed CAT at a high constitutive level and showed
no response to growth in the presence of pentobarbital. Thus, it seemed
likely that the 2-bp mutation in BB
resulted in the
elimination of or a significant decrease in repressor binding, so that
the repressor was unable compete with positive factors for the BB
region. For constructs of pcat3A (wild type BB
) and
pcat3 mA (mutant BB
), the basal levels of CAT expression
in the transformants were essentially the same. However, pcat3 mA
produced a higher induced level of CAT activity than pcat3A (Fig. 3C). The former showed a 9-fold increase in CAT
activity in response to pentobarbital induction, whereas the latter
increased 5-fold. This suggests that the 2-bp mutation in BB
may have enhanced its binding affinity for barbiturate-responsive
factors that increased the level of CAT expression by pcat3mA. For
constructs in the B orientation, no significant induction of CAT
activity was observed with overnight culture growth in the presence of
4 mM pentobarbital although pcat1 mB produced 10-fold more CAT
activity than pcat1B in transformed cells grown in either the presence
or absence of 4 mM pentobarbital (data not shown). Since it
seemed possible that the small open reading frame (designated Bm1P1) in
the regulatory region of the P450
gene as well
as the open reading frame designated Bm3P1 in the 5`-flanking region of
the P450
gene might encode trans-acting factors involved in the transcriptional
regulation of the P450 genes, we decided to investigate CAT expression
by these constructs in more detail. Since the synthesis of P450
proteins in B. megaterium is increased rapidly in response to
barbiturate induction(1, 14) , the genes encoding
regulatory factors might turn on only during the early period of
induction. In order to ascertain the optimal timing for assaying
barbiturate-mediated induction in the ``B'' orientation
constructs, we carried out the following experiment. After the addition
of 4 mM pentobarbital to a 2-liter early log phase culture
(0.5 A
OD) of B. megaterium cells
carrying pcat1A or pcat3A, a 50-ml sample from each culture was
collected every 2 h and assayed for CAT activity. The same procedure
was carried out with cultures growing in the absence of pentobarbital.
After 2 h, the induction level of CAT activity increased 2-fold
relative to the basal level in B. megaterium cells harboring
pcat1A. For cells containing pcat3A, the induction reached 4-fold at 4
h. Thus 2- and 4-h incubation times with pentobarbital were utilized
for CAT activity assays on the B orientation constructs of P450
and P450
,
respectively. The results, shown in Fig. 3, indicate that both Bm1P1 and Bm3P1 were positive in response to
pentobarbital induction. The induction level of CAT activity was 4.5
times the basal level in cells transformed by pcat1B, the construct was
designed to test the promoter associated with Bm1P1 (Fig. 3B). For cells transformed by the construct
containing the mutant Barbie box (pcat1mB), both the basal and induced
levels of CAT activity were about 10-fold higher than for cells
transformed by pcat1B (Fig. 3B). For cells containing
pcat3B, the induction level of CAT activity was twice the basal level
after 4 h of pentobarbital induction (Fig. 3D). For
cells transformed by pcat3mB, the basal level of CAT activity was the
same as for the pcat3B cells but the induction level of CAT activity
was 4 times higher than the basal level or about twice the induction
level of pcat3B transformed cells. The increase of CAT expression
levels in cells containing pcat1mB and pcat3mB suggest that both Bm1P1 and Bm3P1 are under the negative regulation of
a repressor (presumably Bm3R1) bound to Barbie box elements. The
positive response of Bm1P1 and Bm3P1 to pentobarbital
induction implies that these two small open reading frames could indeed
encode two positive regulatory proteins involved in
barbiturate-mediated induction of P450 transcription in B.
megaterium. The results of this group of experiments utilizing CAT
expression as a reporter can be summarized as follows. The mutation of
BB
leads to constitutive expression of P450
and a 10-fold increase of expression of Bm1P1 (Fig. 3, A and B,
respectively), while mutation of BB
significantly increases
the expression of P450
and Bm3P1 in
response to pentobarbital induction but leaves the basal levels
unaffected. These results indicate that the Barbie boxes are important cis-acting elements for coordinately regulating the
barbiturate-mediated expression of the P450s and their positive
regulatory factors in B. megaterium.
Figure 3: CAT activity comparisons between constructs carrying mutant and wild type Barbie boxes. Each pair of constructs was assayed for CAT expression in B. megaterium under identical conditions. The measurement of CAT activity in soluble protein extracted from B. megaterium cells grown in the presence or absence 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 in each of which CAT activity was measured three times. PENT-, cells grown in the absence of pentobarbital; PENT+, cells grown in the presence of 4 mM pentobarbital.
Although Bm3R1 could specifically bind to Barbie box sequences (Fig. 4), G39E-Bm3R1, a mutant Bm3R1 protein isolated from the
G39E strain of B. megaterium ATCC 14581 constitutive for the
expression of P450(17) and tested in
an equivalent experiment (data not shown) lacked such binding activity.
It should be remembered, in this regard, that the G39E mutant of Bm3R1,
unlike its wild type counterpart, also lacks binding affinity for the
20-bp palindromic operator sequence involved in the regulation of
expression of both the bm3R1 and P450
genes(17) . To determine if Bm3R1 participates in the
formation of the two DNA-protein complexes reported here (Fig. 5), we set up a parallel gel retardation experiment with
whole cell extracts of the G39E mutant strain of B.
megaterium. As Fig. 6shows, the 2 bands clearly appeared
when gel shift assays were carried out with whole cell extracts of the
G39E mutant cells grown in absence of barbiturate (see lanes 3, 4,
13, and 14 in Fig. 6) and were enhanced when
extracts of G39E mutant cells grown in the presence of 4 mM pentobarbital were used (see Fig. 6, lanes 5, 6,
15, and 16). These results thus imply that Bm3R1 was not
a component of the two Barbie box DNA-protein complexes. Surprisingly,
when cell cultures were grown into very late stationary phase (i.e. growth for about 20 h after the addition of pentobarbital), the
intensity of these 2 bands was reversed on gels run with extracts of
cells grown in either the absence or presence of 4 mM pentobarbital (see lanes 1 and 2 for
BB
, 11 and 12 for BB
in Fig. 6). That is, cell extracts from cultures grown to very late
stationary in the absence of barbiturates contained a high level of
Barbie box-binding factors but these factors were almost undetectable
in very late stationary phase cultures grown in the presence of 4
mM pentobarbital. One partial explanation could be a
significant increase in a putative endogenous inducer for the positive
regulatory factors during very late stationary phase in cultures grown
in the absence of barbiturates. Our original observation relevant to
this point was that the highest specific monooxygenase activity of
P450
, in cell-free preparations from cells grown in the
absence of exogenous inducer, was obtained from cultures harvested
during the stationary phase of growth; cultures harvested during log
growth yielded little if any hydroxylase activity(14) . It is
possible that in cultures grown to late stationary phase in the
presence of pentobarbital (which presumably mimics an endogenous
inducer), the extremely high P450 levels already present may result in
feedback inhibition of the expression of the genes encoding the
positive factors.
Figure 8:
Protection of the BB region of P450
from DNase I by positive factors
in crude extracts of B. megaterium cells grown in the presence
of 4 mM pentobarbital (BM
). In panel
A, the protected regions are indicated on the right margin by solid brackets. Numbers on the left margin identify
the positions of bases relative to the translational start site of the P450
gene. A 385-bp EcoRI-HindIII fragment containing BB
produced by PCR was end-labeled at one end with
P at
the EcoRI site, incubated with BM
protein,
and then subjected to DNase I digestion as described under
``Experimental Procedures.'' In each reaction mixture, 5 ng
of the
P-labeled DNA fragment was used. Lane 1 contained no BM
protein; lanes 2-5 contained 40, 20, 10, and 5 µg of BM
protein,
respectively. In panel B, the DNA sequence covering the
protected regions and the BB
element is shown. The pair of arrows indicate a 6-bp perfect inverted repeat. The 15-bp
BB
is identified by bold type. The protected
sequences are indicated in the same manner as in panel
A.
In gel retardation experiments with either BB or
BB
(the wild type Barbie box sequences in the regulatory
regions of the P450
and P450
genes, respectively), two Barbie box DNA-protein complexes were
detected with extracts from B. megaterium ATCC 14581 grown in
the presence or absence of pentobarbital. However, pentobarbital
increased protein binding to BB
and BB
. Our
finding is similar to that in a report describing an inducible P450
system from Streptomyces griseolus(23) . Proteins from S. griseolus ATCC 11796 grown in the presence or absence of
the cytochrome P450
inducers, sulfonylurea or
phenobarbital, interacted with a 280-bp 5`-flanking fragment of the P450
gene containing what we now define as a
Barbie box sequence. Two shifted complexes were formed but DNA binding
was 3-5-fold higher when cells were grown in the presence of the
inducers. The Barbie box sequence was protected in both complexes when
they were subjected to DNase I digestion. Barbie box sequences that
were recognized by protein from nuclear extracts have also been
reported for barbiturate-inducible eukaryotic P450s. In the rat, Barbie
box sequences in CYP2B1/B2 specifically interacted with
proteins from liver cell nuclear extracts from untreated rats (forming
one complex) and from phenobarbital-treated rats (two
complexes)(24) . Phenobarbital appeared to increase synthesis
of protein factor(s) that bound to a 223-bp 5`-flanking fragment of the CYP2B1/B2 genes that contained Barbie box
sequences(25) . We also detected Barbie box-binding protein in
nuclear extracts obtained from rats treated with
phenobarbital(4) . In B. megaterium, the two complexes
may be formed from different Barbie box-binding factors since the P450
operator sequence could compete with the
Barbie box sequences in the slower mobility complex but not with the
Barbie box in the faster complex. Even though the band formed from the
faster mobility complex with protein from uninduced cultures was quite
strong for both BB
and BB
, it was enhanced
under inducing conditions. Although the nature of the protein factor(s)
involved in the formation of these two complexes remains to be
determined, we think that the evidence supports the conclusion that
they contain proteins that are involved in the regulation of expression
of the B. megaterium P450 genes.
We reported previously (4) that a factor in a partially purified protein fraction from
cell extracts of B. megaterium cells grown in the absence of
barbiturates could bind to the BB and BB
sequences. In light of our finding that Bm3R1, partially purified
from extracts of E. coli cells harboring a plasmid for the
overexpression of Bm3R1, can specifically interact with BB
and BB
(Fig. 4), it seemed likely that this
factor was Bm3R1, the repressor for P450
.
Still, the fact that a Bm3R1-Barbie box binding band was not detected
in gel retardation assays with whole cell extracts of B. megaterium grown in the absence of barbiturates (Fig. 5Fig. 6Fig. 7) was an unexpected finding.
There are several possible explanations for this result. The most
likely hypothesis, we think, is that the stationary phase cultures
(pentobarbital minus) from which the extracts were prepared contained
levels of Bm3R1 too low to compete effectively with the levels of the
positive Barbie box-binding factors. Our original experiments (4) demonstrating that a factor (presumably Bm3R1), present in
extracts from B. megaterium cells grown in the absence of
barbiturates, could bind to Barbie box sequences, were carried out not
with total cell extracts but with DEAE-cellulose fractions in which the
repressor was enriched and the ``positive'' binding factors
removed. It is also conceivable that the predominant form of Bm3R1 in
extracts from stationary phase B. megaterium differs in some
way (phosphorylation, polymerization) from Bm3R1 obtained from
overexpression in E. coli(17, 18) , a
hypothesis currently being tested in our laboratory. Nevertheless, it
is apparent from the sum of the experimental evidence presented here
that, in B. megaterium at least, both positive and negative
factors apparently compete for binding at Barbie box sites as putative
regulators of P450 transcription. A similar observation has been made
in the rat CYP2B1 system by Dr. Ronald Lubet (
)and
his co-workers at the National Cancer Institute. They detected, by gel
retardation assays, a protein in rat liver nuclear extracts from
animals untreated by barbiturates that bound to a 30-bp DNA fragment
from the 5`-flanking region of the CYP2B1 gene. This fragment
contained a Barbie box sequence (see Table 1) and has been
reported to function as a positive cis-acting element in
regulating the transcription of the CYP2B1/B2 genes and in
mediating the induction of these genes by
phenobarbital(24, 25) . According to Dr. Lubet, this
protein disappeared in gel mobility shift assays and was replaced by a
smaller Barbie box-binding protein when a nuclear extract of liver from
phenobarbital-treated animals was used. Our footprinting analysis on
the 5`-flanking region of P450
indicates that
the Barbie box region of this gene is part of a binding site for
putative positive factor(s), a hypothesis supported by the results of
unidirectional deletions around BB
on the regulatory region
of P450
(Fig. 9). The removal of the
binding site (including the 5`-half of BB
) for the putative
positive factor(s) eliminates the barbiturate-inducibility of
P450
. It should also be noted that in the S. griseolus system (23) mentioned above, site-specific
DNA binding activity involved two similar 8-bp inverted repeat
sequences in the P450
regulatory region with half of the
inverted repeat being located in a Barbie box sequence (see Table 1).
In B. megaterium, mutations in BB and BB
showed significant effects on the
transcription of the reporter gene CAT. The mutated Barbie boxes
designated BB
and BB
(Fig. 1),
displayed a lower affinity for the Bm3R1 repressor and a higher binding
affinity for the positive protein factors than did their wild type
counterparts ( Fig. 4and Fig. 7). These data are
congruent with our finding that mutation in BB
(AAAG to
GAAA) caused an increase in the pentobarbital-induced level of CAT
expression. Under noninducing conditions, Bm3R1 is bound to the
operator site (17) and, we think, to the BB
site of P450
as well. In the presence of barbiturate,
the binding of Bm3R1 to the operator site is inhibited (18) and
Bm3R1 also fails to compete effectively with positive factors for
binding to the mutated BB
site ( Fig. 4and Fig. 7) with a resultant increase in inducible CAT activity. For
the P450
gene, the synthesis of both cytochrome
P450
and its positive regulatory factor(s) may be
negatively regulated by the binding of Bm3R1 to BB
. In this
regard, we have found that the Bm3R1-defective (G39E) mutant of B.
megaterium is not only constitutive for the synthesis of
cytochrome P450
but for cytochrome P450
synthesis as well. (
)Mutation in BB
(AAAG
to GAAC) leads to high levels of synthesis of the putative positive
factors (for example, Bm1P1 synthesis is stimulated 10-fold when
BB
is mutated; Fig. 3B), which presumably
leads to increased competition with or exclusion of Bm3R1 binding at
the BB
site. In support of this hypothesis, we have
recently found that Bm1P1 protein inhibits the interaction between
Bm3R1 and BB
(
)and thus gives rise to highly
constitutive expression of the reporter gene, CAT.
The data from gel
retardation, footprinting, and deletion assays and site-directed
mutagenesis together establish that Barbie box sequences are important cis-acting elements in regulating the basal level of
expression and the barbiturate-mediated induction of P450s in B.
megaterium. Obviously, this cis-acting element does not
function alone but presumably acts synergistically with other cis-acting elements (such as the operator site of P450) on the 5`-regulatory regions of the B. megaterium P450s in conjunction with trans-acting
factors to mediate the increase of P450 synthesis in response to
barbiturates. The possibility that other cis-acting elements
in addition to those identified in B. megaterium are involved
in eukaryotes in the regulation of barbiturate-mediated induction of
P450s and related enzymes is supported by findings from several
laboratories. Thus Hahn et al.(26) identified a
phenobarbital-responsive enhancer domain between position -5.9 kb
and -1.1 kb of the chicken CYP2H1 gene and Jaiswal et al.(27) found a functional glucocorticoid response
element located approximately 1.3 kb upstream of the transcription
initiation site of the barbiturate-inducible CYP2B2 gene. More
recently it has been reported (28) that sequences within
-800 bp to -20 kb of the 5`-flanking region of the rat CYP2B2 gene confer critical regulatory information necessary
for phenobarbital induction and tissue-specific expression in
vivo. However, the results from the -800-bp CYP2B2 transgene in this report may be open to alternate interpretations.
The 800-bp regulatory region (containing a Barbie box sequence) in the
transgene designated ``[minus]800 bp CYP2B2 transgene'' may not be able to protect the transgene from the
influence of its 5`-flanking genomic DNA sequences (such as enhancer or
other promoter sequences). If the transgene integrated downstream of a
promoter or an enhancer, the expression of the transgene may not
reflect the nature of the 800-bp CYP2B2 promoter, even
assuming that it carries all of the phenobarbital-responsive cis-acting elements.
Thus, although the regulatory systems involved in barbiturate-mediated enzyme induction in eukaryotes may generally be more complex than the system operating in B. megaterium, the preponderance of evidence, from our laboratory and from others, implies that significant aspects of the mechanism of barbiturate-mediated induction of P450 monooxygenases and related enzymes in prokaryotes and eukaryotes have been conserved in the course of evolution and that the cis-acting Barbie box sequences and probably their binding proteins are important conserved components of this mechanism.