©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Role of Barbie Box Sequences as cis-Acting Elements Involved in the Barbiturate-mediated Induction of Cytochromes P450 and P450 in Bacillus megaterium(*)

(Received for publication, July 15, 1994; and in revised form, October 11, 1994)

Qianwa Liang Jian-Sen He Armand J. Fulco (§)

From the Department of Biological Chemistry and the Laboratory of Structural Biology and Molecular Medicine, School of Medicine, University of California, Los Angeles, California 90024-1737

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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 (^1)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.




EXPERIMENTAL PROCEDURES

Materials

Restriction endonucleases, T(4) DNA ligase, T(4) polynucleotide kinase, and DNase I were purchased from either New England Biolabs or Life Technologies, Inc. Taq DNA polymerase was a product of Promega. Oligonucleotides used in gel retardation and PCR were synthesized by Oligos etc., Inc. [1-^14C]Acetyl-coenzyme A, dNTP, and [-P]ATP were obtained from Amersham Corp. Poly(dI-dC)bullet(dI-dC) and Nest-Deletion kits were from Pharmacia LKB Biotechnology. Sequenase kit and Escherichia coli vector pTZ19R were from U. S. Biochemical Corp. GeneClean kits were ordered from BIO 101 Inc. The genes encoding cytochromes P450 and P450BM, including 5`-flanking regulatory regions, were cloned and sequenced in our laboratory(3, 13) ; these sequences are available under GeneBank accession numbers X16610 and J04832, respectively. All chemicals used in the experiments were reagent grade.

Site-directed Mutagenesis of Barbie Boxes by PCR

The 5`-regulatory regions of the P450 gene (0.5 kb) and the P450 gene (1.6 kb) were each cloned into plasmid vector pTZ19R. Oligonucleotide primers (designated BB, BB, BB, and BB) used for the introduction of mutations into the Barbie boxes of the P450 and P450 genes by PCR are described in Fig. 1. Each pair of mutant oligonucleotides contained a Barbie box sequence with 2 bp changes in the most conserved sequence (AAAG in the wild type) as shown in Table 1. PCR was set up as reported previously (19) except that 3 mM MgCl(2) was substituted for 1.5 mM MgCl(2) in the reaction. For introducing mutations in the Barbie box of P450, the reaction was carried out in an automatic thermal cycler for 4 cycles of 1 min at 94 °C, 1 min at 50 °C, and 0.5 min at 72 °C and then for 20 cycles of 1 min at 92 °C, 1 min at 58 °C, and 0.5 min at 72 °C. The 72 °C incubation of the last cycle was extended for an extra 5 min before the reaction mixture was cooled to room temperature. The DNA region (250 bp) between the 5` end of the P450 regulatory region and the P450 wild type Barbie box (BB(1)) was mutated and amplified by PCR with primer BB and the universal primer for the vector pTZ19R. The DNA region (370 bp) between BB(1) and the 3` end of the P450 regulatory region was mutated and amplified by PCR with primer BB and the reverse primer from the vector. After treatment with T(4) DNA polymerase in the presence of dNTP, these two mutated DNA fragments (250 and 370 bp) were purified by low melt agarose electrophoresis and GeneClean kit. The whole length of mutated P450 regulatory region (0.5 kb) was produced by placing the two mutated DNA fragments (overlapping at the BB(1) sequence) in the PCR cycler for 10 cycles of 1 min at 92 °C, 1 min at 55 °C, and 1 min at 72 °C and then for 10 cycles of PCR amplification with the universal and reverse primers. The mutated P450 regulatory region was digested with HindIII and cloned into pTZ19R. The same strategy, employing PCR was used for site-directed mutagenesis of the wild type Barbie box (BB(3)) of the P450 regulatory region. The mutated P450 regulatory region (1.6 kb) was also cloned into pTZ19R. Both the P450 and P450 mutated regulatory regions were further analyzed by DNA sequencing to confirm the desired mutations in the Barbie boxes.


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(1) by PCR; BB and BB were used for BB(3) mutation. All of the Barbie box sequences shown (BB(1), BB, BB(3), 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-beta-D-thiogalactoside inducer; BB(1) (lanes 1-5) and BB (lanes 6-8) designate the wild type and mutant Barbie box sequences, respectively, of the P450 gene; BB(3) (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)bullet(dI-dC) and single stranded oligonucleotides (for details, see ``Experimental Procedures''). BB(1) and BB(3) 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(1) and BB(3) 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(1), BB(3)) 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(1), BB, BB(3), 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(3) 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.



Bacterial Strains and Plasmid Constructs

E. coli DH5alpha (recA, F, endA1, gyrA96, thi-1, hsdR17, supE44, relA1) was used for plasmid transformation and preparation. A plasmid vector, pTZ19R, was used for recombinant DNA manipulation. Plasmid pUB(16) , an E. coli-B. megaterium shuttle vector containing a promoterless CAT gene, was utilized for analysis of Barbie box effects on the transcriptional regulation of the P450 and P450 genes. B. megaterium ATCC 14581, the original strain from which the P450 and P450 genes were cloned in our laboratory(3, 13) , was used as the wild type source for analysis of transcriptional regulation of the P450 and P450 genes and derived constructs. A B. megaterium ATCC 14581 mutant (designated G39E) constitutive for the expression of cytochrome P450 and previously characterized as resulting from a point mutation in the bm3R1 gene (17) was also used in some of the experiments presented here. E. coli JM109 was used as a host for overexpression of wild type and mutant Bm3R1 protein(17, 18) .

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.



Protein Preparations

Cell free extracts of B. megaterium cells were prepared as described previously (14) with some modification. B. megaterium cells were grown at 35 °C in 500 ml of BM medium, and harvested in early stationary phase. The cells were resuspended in 20 ml of 25 mM Tris-Cl (pH 7.4) buffer containing 1 mg/ml lysozyme, and incubated at 37 °C for 10 min. Then, the cells were broken by pulsed sonication. The resulting preparation was centrifuged at 40,000 times g for 30 min to remove debris. The same procedure was used to prepare protein extract from E. coli cells carrying pGS101 (for overproduction of Bm3R1) or pGS102 (for overproduction of mutant Bm3R1)(17) . However, the preparations from E. coli cells were treated with (NH(4))(2)SO(4) (40% of saturation) and the precipitate was collected(18) . The precipitated proteins, containing Bm3R1 or mutant Bm3R1, were resuspended in 5 ml of 25 mM Tris-Cl, and dialyzed against 2 times 2 liters of 25 mM Tris-Cl buffer to remove the salt.

CAT Activity Assay

The chloramphenicol acetyltransferase (CAT) assay was based on Sleigh's procedure (20) with minor modifications. The reaction was carried out in a total volume of 100 µl containing 20 µl of 8 mM chloramphenicol, 10 µl of 40,000 times g cell-free extract (with a protein concentration of 0.2-1.0 mg/ml), 50 µl of 0.15 M Tris-Cl buffer (pH 7.8), and 20 µl of acetyl-CoA (5 µCi/ml [1-^14C]acetyl-CoA plus 0.5 mM unlabeled acetyl-CoA). After incubating the mixture for 30 min to 1 h at 37 °C (the time of incubation was dependent on the CAT activity of the cell extract), the reaction mixture was heated at 65 °C for 10 min to inactivate deacetylases. The samples were then transferred to an ice bath and 100 µl of distilled H(2)O and 200 µl of ice-cold ethyl acetate were added. After the mixture was vortexed vigorously for 20 s and centrifuged at 14,000 times g for 5 min at room temperature, 100 µl of upper organic phase was transferred to a counting vail, mixed with 5 ml of scintillation fluid, and the radioactivity measured by liquid scintillation counting.

Unidirectional Deletions

A series of 5` to 3` deletion derivatives of the P450 regulatory region were generated as reported previously(4) . Five deletion derivatives around the BB(1) region, harboring 345-, 329-, 318-, 308-, and 294-bp portions of the P450 regulatory region (517 bp in length; Fig. 9A), were selected and cloned into the pUB shuttle vector. For the P450 regulatory region (a 1.6-kb DNA segment inserted into vector pTZ19R), a unidirectional deletion from the 5` end was performed according to the manufacturer's (Pharmacia LKB Biotechnology) protocol included with the Nest-Deletion kit with the 3` overhanging end of a KpnI site serving as the protected end to exonuclease III action and the 5` overhanging end of a SalI site as the deletion end. A deletion derivative (pBM3-9) carrying a 447-bp portion of the P450 regulatory region (containing BB(3)) was selected for footprinting analysis.


Figure 9: Deletion analysis of the Barbie box region of the P450 regulatory region. In panel A, the whole 500-bp sequence of the P450regulatory region and the end points of five deletion derivatives in proximity to the BB(1) 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(1) 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.



Gel Mobility Shift Assay

The gel mobility shift assay was a modification of a standard procedure (21) for the detection of sequence-specific DNA-binding proteins. Each pair of Barbie box oligonucleotides (Fig. 1) was annealed in a buffer containing 20 mM Tris-Cl (pH 8.0), 1 mM EDTA, and 100 mM NaCl by incubating the mixture at 70 °C for 10 min and then cooling slowly to room temperature. After radioactive labeling with [-P]ATP and T4 polynucleotide kinase, the double-stranded DNA probe was purified on a small filtration column. The binding reaction was set up in a total volume of 30 µl by mixing 4 ng of DNA probe and various amounts of cell extract (0.5-20 µg of protein) in a binding buffer consisting of 12 mM HEPES-NaOH (pH 7.9), 4 mM Tris-Cl (pH 7.9), 60 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 4% glycerol, 1 µg of double stranded poly(dI-dC)bullet(dI-dC), and 10 ng of the single stranded oligonucleotides, 5`-GGAATTCGTAATGAGATAAGCAGTTCGC-3`. After incubation at room temperature for 30 min, the reaction mixture was loaded onto a 6% polyacrylamide gel for electrophoresis in 1 times Tris glycine buffer. The polyacrylamide gel electrophoresis gel was dried under vacuum and subjected to autoradiography.

DNase I Footprinting Analysis

The P450 regulatory region portion of the 385-bp segment containing BB(1) was obtained by PCR amplification with the introduction of an EcoRI site at its 5` end and a HindIII site at its 3` end. The DNA fragment was digested at the 5` end with EcoRI, filled in with [alpha-P]dNTP using the Klenow fragment of DNA polymerase, and purified by gel filtration. For footprinting analysis on the P450 regulatory region, pBM3-9 was digested with SalI and then labeled with [alpha-P]dNTP using the Klenow fragment of DNA polymerase. The P450 DNA fragment was excised by HindIII digestion to render it one end-labeled and then purified by low melt agarose gel electrophoresis and by use of the GeneClean kit. The footprinting reaction was set up in a total volume of 30 µl which contained 1 µl of probe (about 5 ng DNA), 20 µl of cell extract (0.25-2 mg/ml of proteins), 10% glycerol, 1 µg of poly(dI-dC)bullet(dI-dC), 12 mM Tris-Cl (pH 7.9), 12 mM HEPES-NaOH (pH 7.9), 60 mM KCl, 1 mM EDTA, and 1 mM dithiothreitol. After a 30-min incubation at room temperature the samples were placed into an ice bath for 10 min. Following a 2-min incubation of the samples at 25 °C, 10 mM MgCl(2) and 2 ng of DNase I were added to digest unprotected regions of DNA for 40 s at 25 °C before the addition of 100 µl of stop solution (1 M ammonium acetate, 200 µg/ml tRNA, 10 mM EDTA, and 1% sodium dodecyl sulfate). The samples were extracted with phenol/chloroform and precipitated with isopropyl alcohol. After vacuum drying, each sample was resuspended in 8 µl of loading buffer (50% deionized formamide, 10 mM EDTA, 0.02% bromphenol blue, and 0.02% xylene cyanol) and heated at 75 °C for 2 min before 4-µl samples were loaded onto the gel. The chemical (Maxam-Gilbert) sequencing reaction (G + A) was carried out exactly as described in a standard procedure (22) .


RESULTS

Analysis of Barbie Boxes in the Regulatory Regions of the Cytochrome P450 and P450 Genes by Site-directed Mutagenesis

The introduction of site-specific mutations in Barbie box sequences by PCR is described under ``Experimental Procedures.'' The resulting mutant Barbie boxes and their wild type counterparts are shown in Fig. 1. Because both P450 and P450 Barbie boxes (BB(1) and BB(3)) are located in coding regions, the mutations in BB(1) and BB(3) were designed to produce no change or minimum change in the corresponding polypeptide sequences. In BB(1), the highly conserved sequence AAAG was mutated to GAAC with the two corresponding amino acid residues Leu and Phe changed to similar residues, Val and Leu in the predicted polypeptide sequence. Although the function of the polypeptide encoded by this open reading frame has not yet been fully characterized, the 2-amino acid residue change resulting from the mutations in BB(1) did not result in a significant change in the protein secondary structure as predicted by computer analysis (MacVector Protein Analysis Toolbox). For BB(3), the change from AAAG to GAAA involved silent mutations since no amino acid residue was changed in the Bm3R1 protein, a repressor for the P450 gene(17) .

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 P450and 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(1)) 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(1) 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(1) region. For constructs of pcat3A (wild type BB(3)) 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(3) 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(1) 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(3) 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.



Studies on the Binding Affinity of Bm3R1 to Wild Type and Mutant Barbie Boxes

The results of the mutation of Barbie box sequences, as presented above, strongly suggested the involvement of a repressor that could bind to the Barbie box region. Based on previous results from our laboratory(4, 17, 18) , a likely candidate was Bm3R1, a repressor of the P450 gene(17) . As shown in Fig. 4, in gel mobility shift assays Bm3R1 could indeed interact specifically with Barbie box sequences. The specificity of Bm3R1-binding to Barbie box sequences was supported by two lines of evidence. First of all, a strong band was detected in the assay with protein extracts of E. coli cells carrying pGS101, a construct for overproduction of Bm3R1(17) , but was not detected with extracts of E. coli cells harboring pKK223-3, the original plasmid vector of pGS101 (compare lane 3 with lane 2 for BB(1) and lane 11 with lane 10 for BB(3), both in Fig. 4). Second, the strong band disappeared after the addition of 60-fold unlabeled Barbie box sequence (in Fig. 4compare lane 3 with 4 for BB(1) and lane 11 with 12 for BB(3)). In competition assays, we noted that Bm3R1 still recognized the mutated Barbie boxes (in Fig. 4see lane 6 for BB; lane 14 for BB). However, the mutant Barbie boxes had lower affinities than their wild type counterparts for binding Bm3R1 (in Fig. 4compare lane 5 with 4 or lane 7 with 8 for BB/BB(1); lane 13 with 12 or lane 15 with 16 for BB/BB(3)). The decreased binding affinity of Bm3R1 to the mutated Barbie boxes provides an additional explanation for our observation that the mutation in BB(1) resulted in the constitutive expression of P450 and an increase of the expression of Bm1P1 and that the mutation in BB3 increased the expression of P450 and Bm3P1 in response to barbiturate induction.

Binding of B. megaterium Proteins to Barbie Boxes

In a previous report from our laboratory (4) it was shown that Barbie boxes from the regulatory regions of both the B. megaterium P450 genes and those of the rat CYP2B1 and CYP2B2 genes interacted with a partially purified protein from B. megaterium cells grown in the absence of barbiturates. In the gel retardation experiments presented here, we detected two different Barbie box DNA-protein interaction complexes (designated fast mobility complex, F, and slow mobility complex, S) with whole cell extracts of B. megaterium (Fig. 5). When using whole cell extracts of B. megaterium grown in the absence of barbiturates, the F band was weak and the S band was very weak for BB(1) (Fig. 5, lanes 2-4), but both F and S bands were relatively strong for BB(3) (Fig. 5, lanes 12-14). When using whole cell extracts of B. megaterium grown in the presence of 4 mM pentobarbital, the intensity of these 2 bands were enhanced (5-10-fold) for either BB(1) or BB(3) (see lanes 7-9 and 17-19, Fig. 5). The intensity of these 2 bands increased with increasing amounts of whole cell extracts (from 5 to 20 µg of protein) and disappeared (or almost disappeared) with addition of 60-fold unlabeled Barbie box DNA as a competitor in the reaction (see lanes 5 and 15 for BB(1), lanes 10 and 20 for BB(3), Fig. 5). One possible interpretation of these data was that these DNA-protein complexes were formed by the specific binding of positive regulatory factors to Barbie box sequences. Both of these complexes had lower mobility (i.e. higher apparent molecular weight) on the gel than the complex reported previously from protein partially purified from extracts of B. megaterium grown in the absence of barbiturates(4) . In our present experiments using whole cell extracts rather than specific fractions from extracts fractionated on a DEAE-cellulose column, we could not detect an equivalent band. The several possible explanations for this result are considered in detail under ``Discussion.''

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(1), 11 and 12 for BB(3) 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.

Competition Studies of the Binding of Positive Factors to Wild Type and Mutant Barbie Boxes

As noted above, Barbie box mutations resulted in the highly constitutive expression of P450, higher inducible expression of P450, and an increase in positive Barbie box-binding factors. We next carried out experiments to determine whether mutations in Barbie box sequences altered the interaction between a Barbie box and positive factors from B. megaterium cells. Fig. 7shows the results of gel retardation experiments performed with wild type and mutant BB(1) and BB(3) and with the 20-bp palindromic sequence of the operator site of the P450 gene as competitors. At least 3 conclusions can be derived from these results. First, based on the competition experiments, the mutant Barbie box (BB and BB) sequences have higher binding affinities for the positive factors than do their wild type counterparts (compare lane 14 with 13 and lane 19 with 20 as shown in Fig. 7A for BB(1)/BB and lane 9 with 10 and lane 19 with 20 in Fig. 7B for BB(3)/BB). Second, wild type BB(3) has higher affinity than wild type BB(1) for these positive factors (compare lane 15 with 13 in Fig. 7A; lane 4 with 6 and 13 with 15 in Fig. 7B). Third, the P450 operator is a strong competitor of both BB(1) and BB(3) in forming the slow mobility complex but not for the formation of the fast mobility complex (compare lane 16 with 12 in Fig. 7A; lane 7 with 3 and 16 with 12 in Fig. 7B). This last observation implies that there is at least one positive regulatory factor which can bind, in B. megaterium cells, to both the P450 operator site and the Barbie box element.

Footprinting Analysis

To determine which portions of the regulatory regions interacted with the positive factors that were detected in gel retardation experiments by their ability to bind to Barbie box probes, DNA fragments containing Barbie boxes were used as probes for footprinting studies with whole cell extracts of B. megaterium cells grown in the presence of 4 mM pentobarbital. The result of footprinting analysis with the BB(1) region of P450 are shown in Fig. 8. In the 385-bp fragment from P450, 5`-flanking region, two in vitro footprints were detected (Fig. 8A). The first covers an 11-bp region (from -304 to -314) located inside the BB(1) sequence. Inspection of this protected sequence reveals that it contains half of a small inverted repeat (underlined in Fig. 8B). The other half of the inverted repeat is covered by the second footprint (from -319 to -324). When the 447-bp fragment of the P450 regulatory region containing the BB(3) sequence was used in footprinting experiments, we failed to observe clear protection of sequences within the BB(3) region although in some experiments (data not shown), a weakly protected sequence, spanning 20 bp (-219 to -238) and containing 13 of the 15 bp of BB(3), appeared at the higher protein concentrations.


Figure 8: Protection of the BB(1) 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(1) 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(1) element is shown. The pair of arrows indicate a 6-bp perfect inverted repeat. The 15-bp BB(1) is identified by bold type. The protected sequences are indicated in the same manner as in panel A.



Deletion Analysis of the Barbie Box Region of the P450 Regulatory Region

Five deletion derivatives in or near the Barbie box of the P450 regulatory region were cloned into shuttle vector pUB upstream of the promoterless CAT gene. B. megaterium ATCC 14581 transformed by the resulting constructs were evaluated by CAT assay for deletion effects. The structures of these 5 deletion derivatives are shown in Fig. 9A and the levels of CAT expression in B. megaterium cells harboring these pUB constructs are summarized in Fig. 9B. CAT expression from extracts of cells transformed by the intact P450 regulatory region (514 bp, construct 1) and grown in the absence of barbiturates was taken as the basal level of expression. In cells grown in the presence of 4 mM pentobarbital, CAT expression increased about 4.5-fold. Removal of 158 bp from the 5` end of the P450 regulatory region (construct 2) did not significantly change either basal or induced CAT expression. However, deletion to position -329 in the P450 regulatory region (construct 3), reduced the induced level of CAT activity to half that of construct 1. Deleting 11 bp further (construct 4) led to severe inhibition of cell growth by 4 mM pentobarbital. When pentobarbital in the growth medium was reduced to 3 mM, cells harboring construct 4 grew well and CAT expression levels were similar to those of construct 3. Surprisingly, when deletion into the Barbie box sequence was carried out (construct 5), the remaining portion of the P450 regulatory sequence failed to respond to pentobarbital; the induction level of CAT activity was essentially the same as the basal level. These results suggest that there is a positive regulatory factor binding site located in a region between -345 and -307 (probably the region from -329 to -307) and that the 5`-half of the Barbie box is part of the positive factor binding site. This is congruent with our results from the footprinting analysis of BB(1) that indicates that the inverted repeat is a binding site for positive factors. The first 4 deletion constructs had no effect on the basal level of CAT expression but when deletion went beyond the Barbie box sequence (construct 6), CAT was constitutively expressed at a very high level. The basal and induced levels of CAT activity did not differ significantly from each other and were about double the induced level of CAT activity from construct 1 (intact regulatory region). This result is consistent with our previously reported finding that removal of the whole BB(1) region results in the complete derepression of CAT synthesis(4) . It thus seems likely that a repressor binding site is located within the sequence spanning bp -308 and -295, a region that includes the 3`-half of the Barbie box. However, based on the mutation analysis of BB(1), in which the mutation of AAAG to GAAC led to the constitutive expression of P450 (see Fig. 3A), the repressor binding site must actually include the whole Barbie box and span a region between bp -316 and -295. That is, the repressor binding site and positive factor binding site overlap in the Barbie box sequence. This observation implies that positive factors and repressor compete with each other for binding to the Barbie box region, a site putatively critically involved in the regulation of P450 transcription in B. megaterium.


DISCUSSION

In gel retardation experiments with either BB(1) or BB(3) (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(1) and BB(3). 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(1) and BB(3), 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(1) and BB(3) 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(1) and BB(3) (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 (^2)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(1) on the regulatory region of P450 (Fig. 9). The removal of the binding site (including the 5`-half of BB(1)) 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(1) and BB(3) 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(3) (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(3) 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(3) 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(1). 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. (^3)Mutation in BB(1) (AAAG to GAAC) leads to high levels of synthesis of the putative positive factors (for example, Bm1P1 synthesis is stimulated 10-fold when BB(1) is mutated; Fig. 3B), which presumably leads to increased competition with or exclusion of Bm3R1 binding at the BB(1) site. In support of this hypothesis, we have recently found that Bm1P1 protein inhibits the interaction between Bm3R1 and BB(1)(^4)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.


FOOTNOTES

*
The work was supported by National Institutes of Health Research Grant GM23913 and by the Director of the Office of Energy Research, Office of Health and Environmental Research, Contract DE-FC03-ER06015. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: University of California, Laboratory of Structural Biology and Molecular Medicine, 900 Veteran Ave., Los Angeles, CA 90024-1786. Tel.: 310-825-8750; Fax: 310-825-9433; fulco{at}lbes.medsch.ucla.edu.

(^1)
The abbreviations used are: bp, base pair(s); kb, kilobase pair(s); CAT, chloramphenicol acetyltransferase; PCR, polymerase chain reaction.

(^2)
Dr. Ronald Lubet, personal communication.

(^3)
L.-P. Wen, J. S. He, Q. Liang, and A. J. Fulco, unpublished experiments.

(^4)
K. Tong, J.-S. He, and A. J. Fulco, unpublished experiments.


ACKNOWLEDGEMENTS

We thank Keynes Tong from this laboratory for his excellent technical assistance in several of the experiments reported here.


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