©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transcriptional Regulation of the Genes Encoding Cytochromes P450 and P450 in Bacillus megaterium by the Binding of Bm3R1 Repressor to Barbie Box Elements and Operator Sites (*)

(Received for publication, April 18, 1995; and in revised form, May 31, 1995)

Qianwa Liang 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

We previously reported (Liang, Q., He, J.-S., and Fulco, A. J.(1995) J. Biol.Chem. 270, 4438-4450) that Bm3R1, a repressor regulating the expression of P450 in Bacillus megaterium, could bind to Barbie box sequences in the 5`-flanking regions of barbiturate-inducible genes. We've now shown that pentobarbital does not inhibit in vitro binding of Bm3R1 to the P450 and P450 Barbie boxes (BB(3) and BB(1)), although the palindromic operator sequence (O) of P450did have a strong competitive effect on such binding. G39E-Bm3R1, a mutant of Bm3R1, did not bind to either Barbie box. In the presence of Bm3R1, portions of the regulatory regions of P450 and P450 were protected from DNase I digestion. These included 11 of the 15 base pairs of BB(3) plus 7 base pairs 3` to BB(3), BB(1) plus 16 base pairs 3` to BB(1), and, in the 5`-flanking region of P450, segments covering most of two palindromic sequences (O and O) of 24 and 52 base pairs. These DNase I-protected regions (including O) showed considerable sequence identity, especially in a conserved poly(A) motif. Barbiturates did not inhibit binding of Bm3R1 to O(I)bulletOin vitro while G39E-Bm3R1 did not bind. The regulatory effects of Bm3R1 on P450 and P450 were also evaluated in vivo using heterologous chloramphenicol acetyltransferase constructs and Western blotting. In the G39E mutant strain, both P450 and P450 were constitutively expressed, and the regulatory proteins Bm1P1 and Bm3P1, although still pentobarbital-inducible, had significantly higher basal levels of synthesis. In toto, our results show that Bm3R1 represses both P450 and P450expression and that it may effect this by coordinate binding to operator and Barbie box sequences to produce looping of the P450 and P450regulatory regions through protein-protein interaction.


INTRODUCTION

P450 and P450 are barbiturate-inducible P450 cytochromes from Bacillus megaterium that were discovered, characterized, cloned, and sequenced in our laboratory(1) . Induction of the P450 gene involves the barbiturate-mediated and coordinately regulated release of repression caused by the binding of a protein, Bm3R1, to a 20-bp (^1)palindromic operator sequence located in the 5`-flanking regions of the P450 structural genes(2, 3) . More recently, it has been shown that certain peroxisome proliferators are even more active than barbiturates as inducers of the P450 gene and apparently act by a similar mechanism(4) . Confirmation that Bm3R1, which contains a helix-turn-helix DNA-binding motif, is the critical factor in the barbiturate-mediated regulation of the expression of P450 was obtained by the characterization of the defect in a B. megaterium mutant that constitutively expressed P450(2) . Complementation of this constitutive mutant by a DNA fragment containing the wild-type bm3R1 gene caused repression of P450 synthesis in the mutant. Sequence analysis of the bm3R1 gene and its upstream region from this mutant identified a single base change in the codon for residue 39 that resulted in a Gly to Glu substitution in the beta-turn region of the DNA-binding motif. Unlike wild-type Bm3R1, the mutant protein did not bind to a 20-bp palindromic operator site in the regulatory region of P450(2, 3) nor to other regulatory DNA sequences such as Barbie box elements in the 5`-flanking regions of the P450and P450 genes that have also been identified as binding sites for Bm3R1(5, 6, 7) . Barbie box elements are homologous 15-17-bp DNA sequences that are found in essentially all eukaryotic and prokaryotic genes whose 5`-flanking regions are known and that encode barbiturate-inducible proteins(3, 6) . Indeed, in a recent publication (8) it has been reported that a Barbie box element is critically involved in the expression of the phenobarbital-inducible alpha(1)-acid glycoprotein gene of the rat. Transfection of rat primary hepatocytes with a CAT construct containing a portion of the Barbie box regulatory region induced basal expression of chloramphenicol acetyltransferase activity, which was increased by phenobarbital and dexamethasone treatment of cells. Induction of chloramphenicol acetyltransferase activity by phenobarbital was abolished when rat hepatocytes were transfected by constructs with a mutation or deletion of the Barbie box sequence. In B. megaterium, mutation in a 4-bp sequence, AAAG (found in the same relative position in all Barbie box elements), in the P450 Barbie box led to the constitutive synthesis of cytochrome P450; mutation of the same region in the P450 Barbie box significantly increased the expression of P450 in response to pentobarbital induction but left the basal levels unaffected(6) . It was also shown that Bm3R1 could specifically interact with the Barbie box sequences but that mutated Barbie boxes showed a decreased binding affinity for Bm3R1 compared with their wild-type counterparts. Barbie box sequences were also shown to specifically interact with barbiturate-inducible putative positive regulatory factors of B. megaterium cells(6) . In the present paper, we characterize in detail the specific interactions between Bm3R1 and the Barbie box elements and three operator sites in the regulatory regions of the P450 and P450 genes.


EXPERIMENTAL PROCEDURES

Materials

Oligonucleotides used in gel mobility shift assay and polymerase chain reaction procedures were synthesized by Integrated DNA Technologies Inc. Restriction endonucleases, T4 polynucleotide kinase, T4 DNA ligase, Klenow fragment of DNA polymerase, and DNase I were purchased from either New England Biolabs or Life Technologies, Inc. Taq DNA polymerase was a product of Promega. [-P]ATP and [alpha-P]dNTP were from DuPont NEN. [1-^14C]Acetyl coenzyme A and dNTP were bought from Amersham Corp. Poly(dI-dC)bulletpoly(dI-dC) was from Pharmacia Biotech Inc. Geneclean® kits were ordered from Bio-101. Rabbit polyclonal antibodies to cytochrome P450 and P450 were prepared in our lab as described previously (9) . Goat anti-rabbit IgG conjugated to horseradish peroxidase was purchased from Bio-Rad Laboratories. DNA sequences of cytochrome P450 and P450 genes, available under the GeneBank accession number X16610 and JO4832, respectively, were cloned in our laboratory(10, 11) . All chemicals used in the experiments were reagent grade or better.

Bacterial Strains and Recombinant Plasmids

Escherichia coli DH5alpha (recA, F, endA1, gyrA96, thi-1, hsdR17, supE44, relA1) was used for plasmid transformation and preparation. Plasmid vectors for DNA recombinant manipulation were pTZ19R and pUC19. E. coli JM109 was used as the host for the overproduction of wild-type Bm3R1 and G39E-Bm3R1. Plasmid constructs for overproduction of wild-type Bm3R1 (pGS101) and G39E-Bm3R1 (pGS102) were described in detail previously(2, 3) . Plasmid pUBcat(12) , an E. coli-B. megaterium shuttle vector containing a promoterless CAT gene, was utilized in the analysis of Bm3R1 effects on the transcriptional regulation of the P450 and P450 genes. Heterologous CAT recombinant plasmids (pcat1A, pcat1B, pcat3A, and pcat3B) used in this study were constructed as described in detail previously(6) . B. megaterium ATCC14581, the original strain from which the P450 and P450 genes were cloned in our laboratory(11, 12) , was used as the wild-type host for transcriptional regulation analysis of P450 and P450genes and constructs. G39E, a strain of B. megaterium ATCC14581 carrying a point mutation in the bm3R1 gene resulting in a G39E substitution in Bm3R1 repressor(2) , was also used in experiments presented here.

Protein Preparations

E. coli cells harboring pGS101 (or pGS102, pKK223-3) were grown in 500 ml of culture medium overnight at 37 °C in the presence of 1 mM isopropyl-1-thio-beta-D-galactopyranoside inducer with shaking and then harvested by centrifugation. The cells were resuspended in 20 ml of 25 mM Tris-Cl buffer (pH 8.0) containing 1 mg/ml lysozyme and 1 mM dithiothreitol and incubated at 37 °C for 5 min. The cells were then broken by pulsed sonication for 6 min. The resulting preparation was centrifuged at 40,000 g for 30 min to remove cell debris, and the supernatant solution was treated with ammonium sulfate (40% of saturation) to yield a protein precipitate. The precipitated protein pellet was resuspended in 5 ml of 25 mM Tris-Cl buffer (pH 8.0) and dialyzed twice against 2-liter volumes of 25 mM Tris-Cl buffer (pH 8.0) to remove the ammonium sulfate. The resulting proteins were used for gel retardation and footprinting experiments.

Preparation of DNA Fragments

All oligonucleotides used in the gel retardation experiments reported here were double-stranded, although single-stranded sequences are shown. The sequences used here that contained Barbie box elements were prepared as described previously(6) . These included BB(1), the P450 Barbie box (5`-CCATAAAAAGCTGGTGCGTATGCC-3`), and BB(3), the P450 Barbie box (5`-GCATATCAAAAGCTGGTGGAATTT-3`). In each case, the bases depicted in boldface correspond to the 15-bp Barbie box sequence. O, the P450 palindromic operator sequence (5`-CGAATGAACGTTCATTCCG-3`) described previously (2) was used in some of the experiments reported here as were the previously described (7) plasmid constructs pBM1-385, pBM1-385A, and pBM1-385B. Plasmid pBM1-385A was digested with HindIII and labeled with [alpha-P]dNTP using the Klenow fragment of DNA polymerase. After cutting with EcoRI, an end-labeled DNA fragment of 177 bp (corresponding to bp -44 to -221 of the 5`-flanking region of the P450 structural gene) containing the P450 operator sites was obtained from low melting point agarose gel electrophoresis using the Geneclean kit. A similar strategy was used in obtaining a one-end-labeled DNA fragment of 208 bp (-222 to -429) containing BB(1) and an one-end-labeled DNA fragment of 385 bp (-44 to -429) containing both BB(1) and operator sites. All of these three radioactive labeled DNA fragments were used in gel mobility shift assays. The 385-bp DNA fragment, by labeling at different ends, was also used in the DNase I footprinting assays on BB(1) and operator sites, respectively. The construct pBM3-9 containing a 447-bp fragment that incorporated the P450 promoter region including the P450 Barbie box (BB(3)) (6) was digested with SalI and then labeled with [alpha-P]dNTP using the Klenow fragment of DNA polymerase. This 447-bp DNA fragment was then excised by HindIII digestion to render it labeled at one end and purified by low melt agarose gel electrophoresis and the Geneclean kit. The resulting DNA fragment was used for the DNase I footprinting study of BB(3).

Western Blotting Procedures

SDS-polyacrylamide gel electrophoresis was carried out by a procedure based on that of Laemmli (13) . Immunoblotting for the detection of cytochrome P450 and P450 was performed as described by Harlow and Lane (14) with some modifications. The proteins in the SDS-PAGE gel were transferred onto a nylon membrane using a Bio-Rad Trans-blot cell. After being dried in a refrigerator overnight, the membrane was blocked by incubation for 1 h in 5% nonfat dried milk in 1 TPBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na(2)HPO(4)-7H(2)O, 1.4 mM KH(2)PO(4), 0.3% Tween 20) at room temperature on a shaker. A preparation of 5% nonfat dried milk in 1 TPBS and 1 TPBS was used alternatively in rinsing and washing the membrane after binding the primary antibodies and the peroxidase-conjugated secondary antibodies. Polypeptides corresponding to P450 and P450 were detected with hydrogen peroxide and 4-chloro-1-naphthol.

Other Methods

Gel mobility shift assays were performed as described previously (6) except that some reaction mixtures contained barbiturate inducers. Pentobarbital and methohexital were dissolved in 10 mM potassium carbonate and added in a mixture with proteins and probe to initiate the binding reaction. DNase I footprinting analyses were performed as described previously (6) except that different binding buffers were used. The buffer used in the experiments reported here contained 30 mM Tris-Cl (pH 8.0), 100 mM KCl, 0.2 M dithiothreitol, 0.1 mM EDTA, 100 µg/ml bovine serum albumin, 1 µg of poly(dI-dC)bulletpoly(dI-dC), and 10% glycerol. Chemical (Maxam-Gilbert) sequencing reactions (G+A) were carried out by standard procedures(15) . The procedures for CAT assays were described in detail previously(6) . Techniques for transformation of B. megaterium protoplasts by plasmids and subsequent regeneration were based on Imanaka's procedure(16) .


RESULTS

Comparison of Binding Affinity of Wild-type Bm3R1 and G39E-Bm3R1 to Barbie Box Sequences

In a previous report(6) , we found that Bm3R1, a repressor of the P450 gene(2) , specifically interacted with Barbie box elements in the 5`-flanking regions of P450 structural genes of B. megaterium. In a side-by-side comparison using gel mobility shift assays with wild-type Bm3R1 and G39EbulletBm3R1 overproduced in E. coli cells, we now found that the G39EbulletBm3R1 failed to interact with the Barbie box sequences (Fig. 1). With wild-type Bm3R1, a protein-Barbie box complex was detected in the assay, and the intensity of this complex (band) was enhanced with increasing levels of Bm3R1 protein (see lanes2-5 for BB(1) and lanes12-15 for BB(3)). However, when G39EbulletBm3R1 was substituted for Bm3R1 at the same concentrations, no protein-Barbie box complex could be detected (see lanes7-10 for BB(1) and lanes17-20 for BB(3)). This indicates that G39E-Bm3R1 not only lost binding affinity for the operator (O) site of the P450 gene (2, 3) but also for the Barbie box elements.


Figure 1: Comparison of the binding affinity of wild-type and mutant Bm3R1 to Barbie box sequences. The gel mobility shift assay was carried out as described under ``Experimental Procedures'' section. The labels are as follows: E, protein extracts of E. coli cells carrying vector pKKf223-3 (as a negative control; Bm3R1, protein extracts of E. coli cells harboring pGS101, a construct for over-expression of Bm3R1; G39E-Bm3R1, protein extracts of E. coli cells harboring pGS102, a construct for over-production of G39E-Bm3R1; BB, the Barbie box sequence of the P450 gene; BB, the Barbie box sequence of the P450 gene. Each binding reaction (lane) contained 4 ng of P-labeled Barbie box sequence as probe and 4 µg of protein. The complex formed by Bm3R1 and the Barbie box probe is indicated by a boldfacearrow; a weak band formed by E. coli protein and the Barbie box is marked by a smallarrow.



Since pentobarbital was previously shown to strongly inhibit the interaction between Bm3R1 and Oin vitro(3) , we set up a similar gel retardation experiment to see if pentobarbital had inhibitory effects on the interaction between Bm3R1 and Barbie box sequences. The results (data not shown) were negative; pentobarbital had no inhibitory effects on the binding of Bm3R1 to either BB(1) and BB(3).

Competition Assays Probing the Interaction between Bm3R1 and Barbie Box Sequences

Since Bm3R1 could bind to BB(1) and BB(3) as well as to O, we carried out competition experiments by gel retardation to determine the relative binding affinities of the three sites for Bm3R1. Three conclusions could be drawn from the results of these experiments as shown in Fig. 2. First, it is apparent that the three sequences are strong competitors of each other for binding to Bm3R1 (in Fig. 2, compare lanes4, 6, and 7 with lane3 and lanes11, 13, and 14 with lane10). Second, the Barbie box sequences have higher affinities than O for the binding to Bm3R1 (compare, in Fig. 2, lane4 with 7 for BB(1)/O and lane11 with 14 for BB(3)/O). Third, it appears that Bm3R1 has a slightly higher affinity for BB(3) than for BB(1) (compare lane6with 4 or lane 11 with 13 in Fig. 2).


Figure 2: Competitive assays for the binding affinity of Bm3R1 to Barbie box elements. Each binding reaction (lane) contained 4 ng of probe and 4 µg of proteins. 60-fold of competitor was added in each competition assay. Lane designations for E, Bm3R1, BB and BB are the same as described in the legend for Fig. 1as are the conditions used to carry out the assays. Other lane designations include BBm, the mutant Barbie box sequence of P450; BBm, the mutant Barbie box sequence of P450; and O, the operator sequence of P450. The complex formed by Bm3R1 and the Barbie box probe is indicated by a boldfacearrow; a weak band formed by E. coli protein and the Barbie box probe is marked by a smallarrow.



Footprinting Analysis of the Bm3R1-Barbie Box Interactions

To determine which DNA sequences of the regulatory regions interacted with the repressor Bm3R1, as detected by the gel retardation experiments presented above, one-endlabeled DNA fragments containing Barbie boxes were used as probes for DNase I footprinting assays with crude extracts of transformed E. coli cells in which Bm3R1 was overproduced by the construct pGS101(3) . The results of footprinting assays are shown in Fig. 3and Fig. 4. In a 447-bp fragment of the P450 regulatory region containing the BB(3) sequence, an in vitro footprint appeared in response to increasing protein concentration (Fig. 3A) that covered a 18-bp region (-219 to -237). The protected region included 11 of the 15 bp of BB(3) and 7 additional bp 3` to this Barbie box sequence (Fig. 3B) and is almost identical to the region (bp -219 to -239) protected by positive factors from B. megaterium(6) . It is thus clear from these results that both repressor Bm3R1 and as yet unidentified positive factors compete for binding at the Barbie box site in the 5`-flanking region of the P450 gene. In the 385-bp fragment containing the BB(1) sequence from the P450 5`-flanking region, a region protected from DNase I digestion was detected over a 31-bp span (-285 to -316), which included the entire Barbie box and 16 bp 3` to BB(1) (Fig. 4). Analysis of this protected region reveals an 8-bp tandem repeat separated by 14 bp (Fig. 5). The first half of the repeat overlaps with one-half of the 6 bp inverted repeat, which bound to putative positive factors of B. megaterium(6) and resides within the Barbie box. The overlap of these two different repeats provides additional evidence in support of our previous observation (6) that Bm3R1 and positive factors compete for binding to the Barbie box site, presumably in a manner affecting the transcriptional regulation of P450.


Figure 3: Protection of the BB(3) region of P450 from DNase I by Bm3R1. In panelA, the protected region is indicated on the rightmargin by a solidbracket. Numbers on the leftmargin identify the positions of bases relative to the translational start site of the P450 gene. A 447-bp SalI-HindIII fragment containing BB(3) excised from pBM3-9 was end-labeled with P at the SalI site, incubated with a protein extract of E. coli cells in which Bm3R1 was overproduced by the construct pGS101, and then subjected to DNase I digestion as described under ``Experimental Procedures.'' E indicates the lane containing the protein extract of E. coli cells carrying vector pKK223-3 (the vector for construct pGS101). Bm3R1 indicates lanes containing the protein extract of E. coli cells in which Bm3R1 was overproduced. In panelB, the DNA sequence covering the protected region and the BB(3) element is shown. BB(3) is identified by boldface. The protected sequence is indicated in the same manner as in panelA.




Figure 4: Protection of the BB(1) region of P450 from DNase I digestion by Bm3R1. A 385-bp EcoRI-HindIII fragment containing BB(1) from pBM1-385 was end-labeled with P at the EcoRI site, incubated with a protein extract of E. coli cells in which Bm3R1 was overproduced by the construct pGS101, and then subjected to DNase I digestion. E indicates the lane containing the protein extract of E. coli cells carrying vector pKK223-3 (the vector for construct pGS101). Bm3R1 indicates lanes containing the protein extract of E. coli cells in which Bm3R1 was overproduced. The protected region is indicated on the rightmargin by a solidbracket. Numbers on the leftmargin identify the positions of bases relative to the translational start site of the P450 gene.




Figure 5: DNA sequence of 5` regulatory region of the P450gene showing the locations of three protected footprints on BB(1), O(I), and O sites in the presence of Bm3R1. The three footprinted regions are underlined. Three pairs of inverted repeats are indicated by pairs of arrows numbered 1, 2, and 3; a pair of tandem repeats is indicated by a pair of tandemarrows, each marked with a. The 15 bp of the BB(1) sequence are in boldface as are the bases of the -10 and -35 regions. The O(I) and O sequences are in lower case. The P450 promoter is labeled -35 and -10; the Bm1P1 promoter is labeled -10 and -35. Each transcription start site is indicated by an vertical arrow; the the bases of translation start codons for are marked with asterisks.



Binding of Bm3R1 to Operator Sites of the P450Promoter

In the footprinting analysis of the 385-bp fragment from the P450 regulatory region, two sites in addition to the BB(1) region appeared to be protected from DNase I digestion by Bm3R1. To precisely determine the location of these two footprints on the P450 regulatory region, the 385-bp DNA fragment was excised from pBM1-385, P-labeled at its HindIII end, and then used as probe in footprinting experiments (Fig. 6). The first protected region covered a 21-bp sequence (-68 to -188), while the second spanned 44 bp (-112 to -155). DNA sequence analysis of these footprints indicated that the first protected site was located within an imperfect 24-bp palindromic sequence (Fig. 5) and the second protected site within an imperfect 53-bp palindromic sequence (two 24-bp segments forming a perfect inverted repeat interrupted by a 5-base segment, TAATT, as shown in Fig. 5). The 24-bp palindromic sequence site overlaps two promoter regions oriented in opposite directions. The first promoter, which drives the transcription of the P450 gene, and the second, which directs the expression of Bm1P1 overlap in their -35 regions. The 53-bp palindromic sequence covers the -10 region of the P450 promoter and the initiation site of the P450 transcript. Based on their structural positions on the regulatory region and their ability to interact with repressor Bm3R1, the 24-bp palindromic sequence is designated as operator site one (O(I)), and the 53-bp palindromic sequence is designated as operator site two (O). Bm3R1 may thus suppress the expression of both the P450 and Bm1P1 genes by binding to O(I) and O as well as to BB(1).


Figure 6: Protection of the O(I) and O regions of the P450 gene from DNase I digestion by Bm3R1. A 385-bp EcoRI-HindIII fragment containing the O(I) and O sites from pBM1-385 was end-labeled with P at the HindIII site, incubated with a protein extract of E. coli cells in which Bm3R1 was overproduced by the construct pGS101, and then subjected to DNase I digestion. E indicates the lane containing a protein extract of E. coli cells carrying vector pKK223-3 (the vector for construct pGS101). Bm3R1 indicates lanes containing protein extract of E. coli cells in which Bm3R1 was overproduced. The protected regions are indicated on the rightmargin by solidbrackets. Numbers on the leftmargin identify the positions of bases relative to the translational start site of the P450 gene. Sequencing analysis on the footprints are shown in Fig. 5.



The Interaction between Bm3R1 and O(I)bulletOas Revealed by Gel Mobility Shift Assays

Once it was established that Bm3R1 could interact with O(I) and O, we next asked whether the G39EbulletBm3R1-mutated protein retained this attribute. G39EbulletBm3R1, overproduced in E. coli by construct pGS102 (2) , was used in a gel shift assay with a 177-bp DNA fragment containing both O(I) and O utilized as a probe; the results with G39EbulletBm3R1 were directly compared with those using wild-type Bm3R1 obtained by over-expression by pGS101 in E. coli. (Fig. 7). A Bm3R1bulletDNA interaction complex appeared with increasing levels of Bm3R1 protein (Fig. 7, see lanes2-5); the intensity of the complex (band) decreased as the level of the unlabeled DNA fragment of 177 bp (as specific competitor) was increased in the reaction (Fig. 7, seelanes6-8). On the other hand, G39EbulletBm3R1 appeared not to interact with O(I) and O, and no an equivalent band could be detected in the assay (Fig. 7, see lanes12-15). This observation is similar to our previous finding (2, 3) that Bm3R1 could specifically interact with operator site (O) of the P450 gene while G39EbulletBm3R1 lost such interaction.


Figure 7: Comparison of the binding affinity of wild-type and mutant Bm3R1 to the O(I)bulletO sequences. compet. A designates a 177-bp DNA fragment containing the O(I)bulletO sites; E, protein extracts of E. coli cells carrying vector pKK223-3 (as a negative control); Bm3R1, protein extracts of E. coli cells harboring pGS101, a construct for the over-expression of Bm3R1; G39E-Bm3R1, protein extracts of E. coli cells harboring pGS102, a construct for the over-production of the mutated repressor, G39EbulletBm3R1. Each binding reaction (lane) contained 5 ng of P-labeled fragment A. The complex formed by Bm3R1 and the probe is indicated by a boldface arrow; a weak band formed by E. coli protein and the probe is marked by a smallarrow.



Because the interaction between Bm3R1 and O was strongly inhibited by barbiturates (pentobarbital and methohexital) in vitro(3) , a similar experiment was carried out to determine whether barbiturates inhibited the interaction between Bm3R1 and O(I)bulletO. The results (data not shown) revealed that neither pentobarbital nor methohexital had inhibitory effects on the interaction between Bm3R1 and O(I)bulletOin vitro. This implies that the detailed induction mechanism mediated by barbiturates for the P450 gene may differ in certain respects from that of the P450 gene.

Competitive Assays for Binding of Bm3R1 to the O(I)bulletO and BB(1) Sites of the P450Regulatory Region

Based on the observation that Bm3R1 could interact with O(I) and O as well as with BB(1) on the P450 regulatory region, we carried out gel shift assays designed to determine whether BB(1) and O(I)bulletO enhanced or competed with each other for binding to Bm3R1. Three different DNA fragments of the 5` regulatory region of the P450 gene were used both as probes and competitors in the experiment (see ``Preparation of DNA Fragments'' under ``Experimental Procedures'' for details). The first was a 177-bp DNA fragment (designated A) containing the O(I)bulletO sites; the second was a 208-bp fragment (designated B) containing the BB(1) site; the third, a 385-bp fragment (designated C), consisted of A + B and thus contained all three sites. These results (data not shown) indicate that DNA fragment C (containing both O(I), O, and BB(1)) had a higher binding affinity for Bm3R1 than did an equimolar mixture of DNA fragment A (containing O(I) and O only) and B (containing BB(1) only). We next performed a competitive titration assay on the interaction between DNA fragment C (as probe) and Bm3R1 using fragment C or a combination of fragment A and B as competitors. As Fig. 8shows, fragment C strongly titrated its own probebulletBm3R1 complex (in Fig. 8, compare lanes4-8 with lane3). Conversely, the combination of fragment A and B had a relatively weak competitive effect on the interaction of probe C with Bm3R1 (in Fig. 8, compare lanes10-14 with lanes9 and 4-8). The fact that a 20-fold excess of fragment C competes more effectively for Bm3R1 in the presence of probe C than a 150-fold excess of the A + B combination implies that when BB(1) and O(I)bulletO are separated on different DNA fragments, they compete with each other for binding to Bm3R1, but when they are located on the same DNA fragment they mutually enhance binding to Bm3R1. One can thus infer that the complex formed by the binding of Bm3R1 to O(I)bulletO and BB(1) coordinately in the same DNA fragment is much more stable than that produced by Bm3R1 binding to O(I)bulletO or to BB(1) alone. The higher stability of the complex could perhaps be due to the protein-protein interaction between Bm3R1 molecules bound to the operator sites and BB(1) element. Such protein-protein interaction may lead to bending or rolling of the regulatory region of the P450 gene into a DNA loop with consequent repression of the expression of the P450 and Bm3P1 genes.


Figure 8: Competitive titration assays for the interaction between probe C and Bm3R1. A designates a 177-bp DNA fragment containing the O(I)bulletO sites; B designates a 208-bp fragment containing the BB(1) element; and C designates a 385-bp fragment containing the O(I)bulletO and BB(1) sequences. Lanes1 and 17 contained probe C only. Each binding reaction (lanes2-16) contained 5 ng of P-labeled C probe and 6 µg of protein extract of E. coli cells in which Bm3R1 was overproduced by pGS101. Added competitors were unlabeled. The bracket designated A+B contained equimolar amounts of fragments A and B. The competitors were added to probe C in a range of 20-150-fold by weight in the side by side titration assays. The locations of specific Bm3R1-probe C interaction complexes are indicated by an arrow.



Regulatory Effects of Bm3R1 in Vivo

Once we had determined that Bm3R1 specifically interacted, in vitro, with operator sites and Barbie box elements on the regulatory regions of the P450 genes in B. megaterium, we next utilized a CAT assay system to evaluate the regulatory effects of Bm3R1 in vivo. The regulatory region (0.5 kb) of the P450 gene was cloned into pUBcat in both orientations using the unique HindIII site upstream from the promoterless CAT gene. The resulting two constructs were designated pcat1A and pcat1B (see Fig. 9). The same strategy was used in cloning the regulatory region (1.6 kb) of P450 into pUBcat, and the resulting two constructs were designated pcat3A and pcat3B (Fig. 9). These four heterologous CAT constructs were used to transform B. megaterium ATCC14581 (as wild-type source) and its G39E mutant strain. The G39E mutant strain was derived from ATCC14581 and identified as carrying a G39EbulletBm3R1 gene in which a glycine codon was mutated to a glutamate codon at residue position 39(2) . G39EbulletBm3R1 protein encoded by this mutated gene failed to interact in vitro with Barbie box elements and operator sites as described above. For cell cultures carrying A orientation constructs (pcat1A and pcat3A), growth in the presence of pentobarbital was carried out overnight (about 15 h); for B orientation constructs (pcat1B and pcat3B), cell cultures were induced by pentobarbital for 2-4 h as described previously(6) . Each pair of wild-type and mutant B. megaterium strains carrying the same construct were assayed for CAT activity under identical conditions (Fig. 10). For wild-type B. megaterium cells transformed by construct pcat1A, a considerable level of CAT activity was produced in the absence of the barbiturate, although CAT activity was increased 2.3-fold when these cells were grown in the presence of 4 mM pentobarbital (Fig. 10A). On the other hand, G39E mutant cells carrying pcat1A expressed CAT at a high constitutive level and showed essentially no response to pentobarbital. The constitutive level of CAT in the mutant cells was, in fact, higher than the induced level in the wild-type cells, an indication that G39EbulletBm3R1 failed to suppress the expression of the P450 gene. For wild-type cells transformed by pcat1B, pentobarbital induced a 4-fold increase in the level of CAT activity (Fig. 10B). For the G39E mutant, although the cells carrying pcat1B had a pentobarbital-induced level of CAT activity 3 times the basal level, the basal level of CAT activity was still as high as the induced level of CAT in the wild-type cells. This implies that Bm1P1 is also negatively regulated by Bm3R1 and that the G39E mutation in Bm3R1 leads to high basal expression of Bm1P1. The extremely high induction level of Bm1P1 in G39E mutant cells could result from a barbiturate-responsive activator that competes with wild-type Bm3R1 for binding to O(I)bulletO sites. This activator is probably Bm1P2 which was recently found to inhibit the binding of Bm3R1 to BB(1) and which competed with Bm3R1 for binding to O(I)bulletO(7) . For pcat3A, no significant difference was observed in CAT activity between the wild-type and the mutant B. megaterium transformant cells (Fig. 10C), presumably because it contained a 1.6-kb DNA fragment constituting the 5` regulatory region of the P450 gene. This fragment included a wild-type bm3R1 gene, which complemented the defects of the mutant copy of the bm3R1 gene in the G39E mutant cell genome. However, the same 1.6-kb DNA fragment is contained in pcat3B, but the CAT activities produced (Fig. 10D) were similar to those from pcat1B in wild-type and mutant B. megaterium cells (see Fig. 10B). That is, the basal level of CAT activity in the transformant G39E cells was as high as the induction level in the transformant wild-type cells and pentobarbital induction resulted in a 6-fold increase of CAT activity in the transformant G39E cells. It may be that the complementation of the wild-type bm3R1 gene from pcat3B is incomplete with respect to the transcriptional regulation of the Bm3P1 gene in G39E cells and that G39EbulletBm3R1 gives rise to a high level of leakage of Bm3P1 expression.


Figure 9: Heterologous CAT constructs for assaying the effects of Bm3R1 in vivo. Only the portion covering the promoter and the promoterless CAT gene is shown in the figure for each construct. Open reading frames are represented by boxes. Promoters are indicated by arrows. In the A orientation constructs (designated by an A in the names of the constructs), CAT was placed downstream from a small P450 coding region. In the B orientation constructs (designated by a B in the name of constructs), CAT was placed downstream from the Bm1P1 or Bm3P1 coding regions. Barbie box elements BB(1) and BB(3) and operator sites O(I), O, and O are also indicated in the figure.




Figure 10: CAT activity comparison between wild-type and G39E mutant B. megaterium cells transformed by heterologous CAT constructs. Each pair of comparisons was assayed for CAT expression in wild-type and G39E mutant B. megaterium cells grown under identical conditions. The measurement of CAT activity in soluble protein extracted from B. megaterium cells grown in the presence (PENT+) or absence (PENT-) of 4 mM pentobarbital is described in detail under ``Experimental Procedures.'' For A orientation constructs, cell cultures were induced overnight (15 h) by 4 mM pentobarbital; for B orientation constructs, the pentobarbital induction time was 2-4 h. The CAT activity value shown in each plot is a mean (+S.D.) of three sets of data from three completely separate cell cultures. CAT activity was then measured 3 times in each cell culture.



The regulatory effects of Bm3R1 in vivo were further evaluated by estimating the protein levels of cytochromes P450 and P450 in B. megaterium cells by Western blotting analysis. Fig. 11, A and B, shows these results for cytochromes P450 and P450, respectively. In wild-type B. megaterium cells, the basal levels of P450 and P450 were very low but detectable (Fig. 11, A and B, see lane1), while the pentobarbital-induced levels were high (Fig. 11, A and B, see lane2). In the G39E mutant cells, however, both P450 and P450 were highly constitutively expressed, although their pentobarbital-induced levels were slightly higher than their basal levels (Fig. 11, A and B, see lanes 3 and 4). This indicates that the mutation in Bm3R1 (G39 mutated to E39) results in derepression of both P450 and P450. It is thus clear that Bm3R1 acts as a repressor for the P450 and P450 genes in B. megaterium.


Figure 11: Western blot analysis of cytochrome P450 and P450 expressed in wild-type and G39E mutant B. megaterium cells. The experiments were carried out as described under ``Experimental Procedures.'' Cytochrome P450s were detected from protein extracts of B. megaterium cells grown in the absence (PENT-) or presence (PENT+) of 4 mM pentobarbital. In panelA, each lane contained 50 µg of soluble protein extracts; in panelB, each lane contained 100 µg of soluble protein extracts. The locations of cytochrome P450 and P450 are indicated on the rightmargin by arrows.




DISCUSSION

Three lines of evidence indicate that Bm3R1, a repressor of the P450 gene(2) , binds to Barbie box elements on the regulatory region of the P450 genes of B. megaterium. First of all, gel retardation assays revealed a strong, specific DNA-protein interaction complex that formed in the reaction between protein extracts of E. coli cells carrying pGS101 (a construct for the overproduction of Bm3R1 protein) and a probe containing a Barbie box sequence ( Fig. 1and Fig. 2). This complex was not formed in the reaction with extracts of E. coli harboring the control vector. Second, G39E-Bm3R1, previously been shown to lack binding affinity for O(2, 3) , also failed to interact with Barbie box elements (Fig. 1). Finally, footprinting experiments revealed that the BB(1) and BB(3) regions were protected by Bm3R1 against DNase I digestion ( Fig. 3and Fig. 4). This finding is in good agreement with our previous reports in which removal (5) or mutation (6) of BB(1) resulted in the highly constitutive expression of P450, while the mutation of BB(3)(6) caused a significant increase of P450 expression. The demonstration of Bm3R1 interaction with Barbie box elements thus provides convincing evidence in support of our previous suggestion (6) that repressor and positive factor binding sites overlap in the Barbie box sequence and that Bm3R1 and positive factors compete with each other for binding to the Barbie box region with consequent effects on the regulation of P450 transcription.

In our initial experiments to determine whether Bm3R1 could interact with Barbie box sequences, we used Bm3R1 that had been highly purified from extracts of E. coli cells transformed by pGS101(3) . Although purified Bm3R1 did recognize Barbie box sequences in these experiments, the interaction was weak and unstable and the interaction complex had very slow mobility. When crude extracts from the same E. coli cells transformed by pGS101 were tested in gel shift assays, a very strong interaction complex between Bm3R1 and Barbie box probes was obtained (as discussed above). One plausible explanation may be related to the fact that highly purified Bm3R1 exists primarily in highly aggregated forms as reported previously(3) . The aggregated Bm3R1 protein may not bind to or may have greatly reduced affinity for Barbie box sequences. The soluble protein in crude extracts from pGS101-transformed E. coli may maintain the Bm3R1 in nonaggregated or lower aggregated (i.e. dimeric) forms, which may be the species involved in binding to Barbie box elements. Our finding that Bm3R1 interacted with the O(I) and O sites of the P450 gene as demonstrated by DNase I protection experiments ( Fig. 5and Fig. 6) and gel shift assays ( Fig. 7and Fig. 8) was unexpected. Since Bm3R1 was initially shown to bind to the operator site (O) of the P450 gene(2, 3) , we searched for sequence similarities between O and the two operator sites of the P450 regulatory region. Our analysis revealed that all three operator sites are palindromic sequences. Also, the DNA sequences of the footprints of these sites are AT-rich and have poly(A) or poly(T) motifs. For O(I), the footprinting sequence is ttTTTCCTTGATAACCAAGTAAAaa (A+T = 70%); for O, CTATTAGTACATTTTTATACTAATTGTATAAAAATGTACTAATA (A+T = 84%); and for O, TAGCGGAATGAACGTTCATTCCGTTTTt (A+T = 62%). A sequence-homologous alignment for the foot-printing sequences of operator sites and the Barbie box sequences is shown following with half of each palindromic operator sequence underlined: O(I), TAAccaagTAAAAA; O, TAAttgtATAAAAAtGTAcTaAtaGTatA; O, AAAAAcGgAaTGAacGTtcA; BB(1), ATAAAAAGCTGGTGc; BB(3), ATcAAAAGCTGGTGg.

This alignment makes clear the similarity between the Barbie box sequences and portions of the palindromic operator sequences. In particular, the poly(A) motif is highly conserved.

The recognition of several operator sites by one repressor protein is not a unique finding. One well established model is the regulon of SOS DNA repair genes in E. coli(17) . A single repressor, LexA, for SOS DNA repair genes (dinF, uvrA, dinA, dinB, uvrB, sulA, umuC, umuD and recA), scattered over a number of nonadjacent sites on the E. coli chromosome, can specifically recognize each operator site and repress the transcription of all of these genes. The fact that Bm3R1 can recognize all three operator sites indicates that the P450 and P450 genes are coordinately regulated in the same regulon. Results in our previous reports also implied that P450 and P450 are coordinately regulated. Thus, BB(1) and BB(3) were recognized by the same positive factors (6) and Bm3R1 interactions with BB(1) or BB(3) were inhibited by Bm1P1 and Bm1P2 proteins(7) .

CAT assays with the heterologous CAT constructs of the regulatory regions of P450 genes (Fig. 10) and the Western immunoblotting analyses (Fig. 11) on the expression of P450 cytochromes in wild-type and the G39E mutant B. megaterium cells confirm that Bm3R1 is a repressor of both P450 and P450 genes in vivo. The functions of Bm3R1 were also characterized by DNA-protein interaction assays in vitro. Bm3R1 can specifically bind to the operator sites and Barbie box elements located on the 5` regulatory regions of P450 genes. Since Barbie box elements are found in essentially all of the 5`-flanking regions of the eukaryotic genes that encode barbiturate-inducible proteins(6) , it should be interesting to see whether barbiturate-inducible P450 genes in eukaryotes also have the Barbie box cognate binding factor analogous to Bm3R1. In rat, Barbie box sequences in CYP2B1/B2 were reported (18) to interact with proteins from liver cell nuclear extracts from untreated rats to form a Barbie box-protein interaction complex. Recently, Dr. R. A. Lubet and his co-workers (^2)at the National Cancer Institute also made a similar observation in the rat CYP2B1 system. They found that a protein in rat liver nuclear extracts from animals untreated by barbiturates could bind to a 30-bp DNA fragment from the 5`-flanking region of the CYP2B1 gene. This protein disappeared, however, and was replaced by a smaller Barbie box-binding protein in liver nuclear extracts from phenobarbital-treated animals. As shown by Padmanaban and his co-workers(18, 19) , this fragment contained a Barbie box sequence and functioned as a positive cis-acting element in regulating the transcription of the CYP2B1/B2 genes and in mediating the inductive effects of phenobarbital. Together, these data imply that, in the absence of barbiturate inducers, a protein binds to Barbie box elements that represses the expression of CYP2B1/B2. In the presence of barbiturates, this repressor protein appears to be displaced from the Barbie box by a positive factor with the concomitant activation of CYP2B1/B2 transcription. Very recently, Padmanaban et al.(20) reported that the positive factor binding to the Barbie box in the presence of barbiturates was derived by phosphorylation of the Barbie box-binding repressor. Although these reports generally agree with our previous suggestion (6) that significant aspects of the mechanism of barbiturate-mediated induction of P450s (and related enzymes) are conserved between prokaryotes and eukaryotes and that the cis-acting Barbie box sequences and its cognate protein factors are important conserved components, the regulation of barbiturate-mediated induction of P450s in eukaryotes may be much more complex. In addition to the Barbie box elements, several other cis-acting elements also have been reported. Hahn et al.(21) reported a phenobarbital-responsive enhancer domain between positions 5.9-1.1 kb of the chicken CYP2H1 gene. Jaiswal et al.(22) found a functional glucocorticoid response element located approximately 1.3 kb upstream of the transcription initiation site of the CYP2B2 gene. Fernandez, Shervington, and their co-workers (23, 24) identified a DNA sequence located between positions 199 and 183 of the CYP2B2 promoter that interacted with nuclear proteins and was involved in basal and phenobarbital-induced transcription of the CYP2B2 gene.

Based primarily on the results of our experiments showing that the barbiturate-mediated induction of cytochrome P450s in B. megaterium involve coordinately regulated and mechanistically related derepressions, we propose the following model for the transcriptional regulation of the P450 and P450 genes. In the absence of inducers, Bm3R1 exists in a conformation that binds to operator and Barbie box sites and in so doing enhances DNA loop formation in the 5`-flanking region of the P450 structure gene by protein-protein interaction between the Bm3R1 molecules. As a consequence, the P450 gene promoters are not accessible to positive factors, and RNA polymerase and transcription of the P450 genes is inhibited. In the presence of barbiturates, Bm3R1 assumes another allosteric form that can still bind to O(I), O, and Barbie box elements, but not to O(3) . However, under the influence of Bm1P1 and Bm1P2(7) , the Bm3R1-inducer complexes dissociate from O(I), O, and Barbie box sequences and DNA looping is reversed. The positive factors and RNA polymerase can now access the promoters of the P450 genes to greatly accelerate the rate of synthesis of the P450 mRNAs and cause the observed induction of the P450 genes by barbiturates.


FOOTNOTES

*
This 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) X16610 [GenBank]and JO4832.

§
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; BB, Barbie box.

^2
R. Lubet, personal communication.


ACKNOWLEDGEMENTS

We appreciate helpful discussions with Dr. Harvey Herschman and Dr. Kevin McEntee. We especially thank Jian-Sen He from this laboratory for providing the pBM1-385 constructs and Lisha Chen and Keynes Tong, also from this laboratory, for their excellent technical assistance in several of the experiments reported here.


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