MOLECULAR

AND

Vol. 12, No. 6

CELLULAR BIOLOGY, June 1992, p. 2847-2854

0270-7306/92/062847-08$02.00/0 Copyright ©) 1992, American Society for Microbiology

Role of the Liver-Enriched Transcription Factor DBP in Expression of the Cytochrome P450 CYP2C6 Gene MASAHIKO YANO,' EILEEN FALVEY,2 AND FRANK J.

GONZALEZ'*

Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892,1 and Department of Molecular Biology, University of Geneva, CH-1211 Geneva 4, Switzerland2 Received 14 January 1992/Accepted 17 March 1992

these

Cytochromes P450 (P450) are a superfamily of enzymes, of which are expressed in liver and are involved in oxidation of foreign compounds (10, 21). These are the primary enzymes responsible for metabolism of drugs and activation of chemical carcinogens. Three families and numerous subfamilies of foreign compound-metabolizing P450s exist in mammals. Certain P450 genes within these families are expressed at low levels in untreated animals and are inducible by drugs and chemical carcinogens (20, 27). Other P450 genes are constitutively expressed in hepatocytes. Most interestingly, expression of these genes is developmentally controlled; some, like CYP2EJ, are activated immediately after birth (23), while others, such as CYP2C6 and CYP2C7, are transcriptionally activated when rats reach puberty (30). Still other P450 genes are sex dependent. CYP2A2, CYP2Cll, and CYP2C13 are expressed only in adult male rats, while CYP2C12 is expressed in female rats (30). CYP2A1 is expressed in prepubertal males and females, and its expression in males is extinguished at puberty (18). The related P450 gene, CYP3A2, on the other hand, is expressed in young males and females, and its expression is extinguished in females at puberty (11). Differences in serum growth hormone levels between males and females are responsible in part for the sex-dependent expression of P450s (30). However, the molecular mechanisms by which *

genes are

activated during development is currently

not understood.

many

Control of gene expression has been studied for a number of genes specifically transcribed in rat hepatocytes. In many cases, it appears that such genes are controlled by transcription factors that themselves accumulate preferentially in liver cells (15). Transcription factor DBP was identified by its ability to bind the D site of the albumin promoter, one of six cis-acting control elements that have been identified in this gene (17, 19). This factor is expressed in highest abundance in livers of adult rats, and its expression is similar to that of the CYP2C6 and CYP2C7 genes. We therefore examined the possible role of DBP in the activation of the CYP2C6 promoter in postpubertal rats. MATERIALS AND METHODS

Preparation of nuclear extracts. Rat liver nuclear extracts prepared according to the method of Gorski et al. (13) essentially as described previously (25). Extracts were prepared from rats killed at either 9 a.m. or 8 p.m. because of the known circadian expression of the transcription factor DBP (29). DNase I footprinting. DNase I footprinting was performed essentially as described by Cereghini et al. (3) as detailed earlier (25). A fragment from CYP2C6 was single end labeled at the XhoI site artificially introduced at -103, using [-y-32P]ATP (6,000 Ci/mol; New England Nuclear Corp.,

were

Corresponding author. 2847

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The CYP2C6 gene becomes maximally transcriptionally activated in livers of postpubertal rats. We examined the role of upstream DNA and liver-specific transcription factors in regulation of this promoter by use of transient transfection of heterologous chloramphenicol acetyltransferase gene constructs and vectors containing cDNAs encoding the liver-enriched transcription factors HNF-ba, C/EBP, and DBP. Only DBP was able to activate the CYP2C6 promoter in HepG2 cells. Transactivation was not observed in one mouse and two human nonhepatic origin cell lines tested. Analysis of various constructs in which CYP2C6 upstream DNA was deleted revealed that DNA between -38 to -103 was involved in DBP-mediated activation. A partially purified preparation of DBP produced a footprint between -43 and -64 bp upstream of the transcription start site. A 32P-labeled double-stranded oligonucleotide, containing sequence information corresponding to -40 to -65, bound to both partially pure DBP and extracts from livers of rats as young as 1 week and as old as 25 weeks of age, as assessed by gel mobility shift analysis. This binding was eliminated by coincubation with excess unlabeled -40/-65 double-stranded oligonucleotide and by an oligonucleotide corresponding to the D site of the rat albumin gene. A gel mobility shift-Western immunoblot analysis revealed that the -40/-65 sequence bound to DBP only in liver nuclear extracts from rats older than 3 weeks; maximal binding was observed by 7 weeks of age, and no binding was detected from 1-week-old rat liver extracts. Interestingly, the DBP-binding regions of both CYP2C6 and albumin bind to C/EBP, but this factor is capable of transactivating only the latter gene. Although the DBP-binding regions in these two genes share no obvious sequence similarities, the CYP2C6 region contains consensus palindromic half sites for DBP-related binding proteins and affinity for recombinant DBP of 17-fold greater than that of the D site of albumin. This difference in affinity is probably responsible for the markedly lower amounts of DBP required for half-maximal activation of the CYP2C6 promoter, as compared with the albumin promoter, in transactivation transfection assays. These data indicate that the CYP2C6 gene may be regulated, at least in part, by DBP, a liver transcription factor produced when rats reach puberty that may also be involved in maintenance of albumin gene transcription.

2848

YANO ET AL.

MOL. CELL. BIOL.

TABLE 1. Effect of cotransfection with various liver-enriched transcription factor expression vectors on expression of the CYP2C6 and albumin promoters linked to the CAT genea Expression (mean pmol/min/mg of protein ± SD) after cotransfection with:

CAT vector

pSVOCAT (1) pSV2CAT (1)

p2C6(-1225/+14)-CAT (3) pAlb-CAT (3) p2D5(-810/+73)-CAT (3)

pCMV4

C/EBP

HNF-la

DBP

0.20 6.44 0.47 + 0.07 2.10 ± 0.40 1.06 + 0.15

0.42 + 0.02 6.07 + 1.75 0.68 ± 0.04

0.60 ± 0.12 6.46 ± 1.81 0.43 ± 0.12

2.18 ± 0.48 6.98 ± 0.65 0.81 ± 0.18

a Transfections were carried out by using 10 Fg of CAT vector plasmids and 10 p.g of pCMV-4, pHNF-la (HNF-la), pSCT-DBP (DBP), or pMSV-C/EBP wt (C/EBP). The number of experiments is shown in parentheses.

cation of the appropriate-size fragments, and ligation to pSVOCAT. HepG2 cells were grown to 50% confluence and transfected with 10 ,ug of the CAT vector containing various lengths of upstream DNA of the CYP2C6 gene, 10 ,ug of pSCT-DBP, an expression vector containing the DBP cDNA driven by the cytomegalovirus promoter (19), or a pCMV-4 vector with no cDNA (1) and 2 ,ug of pRSV-LA5', a luciferase reporter gene driven by a Rous sarcoma virus promoter (5). The latter was used as an internal standard for transfection efficiency. Transfections of HepG2 cells were carried out by calcium phosphate precipitation (14), using 75-cm2 flasks. Nonhepatoma cell lines were transfected by using Lipofectin (Life Sciences Inc., Gaithersburg, Md.) as described previously (7). Cells were incubated with this reagent for 20 h and harvested 48 h after addition of DNA. For titration experiments with pSCT-DBP, total DNA amounts were adjusted to 22 ,ug by using pCMV-4. Cells

301A 21 2

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6 I-

5 4 3 a 2 11

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15

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FIG. 1. Transactivation of the CYP2C6 promoter by DPB (A) and C/EBP (B). Cotransfection of pSCT-DBP into HepG2 cells was carried out with pCYP2C6(-1225/+14)CAT (-), pAIb-CAT (-), and pCYP2D5(-810/+73)CAT (0). Ten micrograms of the CAT constructs and 2 ,ug of pRSV-LA&5' were mixed with various amounts of pSCT-DBP and pCMV-4 and transfected into HepG2 cells. The relative CAT activity was calculated by dividing the CAT/luciferase value of each transfection by that of the transfection with no pSCT-DBP. Relative CAT activities of 1 for the CYP2C6, albumin, and CYP2D5 promoters are 0.22, 1.28, and 7.63, respectively.

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Boston, Mass.) and T4 polynucleotide kinase. This fragment extended to + 14. The lower strand was labeled at an EcoRI site artificially introduced at +14 and was isolated by digestion with RsaI, which cleaves at position -170. Partially purified recombinant DBP (0.1 to 1.0 jig), prepared as described earlier (19), or rat liver nuclear extract protein (50 to 100 ,ug) was incubated in 50 RI of 25 mM Tris-HCI (pH 7.9)-50 mM KCI-6.25 mM MgCl2-0.5 mM EDTA-1 mM dithiothreitol-10% (vol/vol) glycerol-2 ,ug of poly(dI-dC) for 10 min on ice. The end-labeled fragment was added to the mixture, incubation continued at 24°C for 20 min, and then 50 ,ul of a Ca-Mg solution (5 mM CaCl2, 10 mM MgCl2) was added. The mixture was incubated for 1 min at room temperature, and the sample was digested with diluted DNase I for 1 min at room temperature. The reactions were stopped by addition of 200 ,u1 of 50 mM EDTA-0.2% (wt/vol) sodium dodecyl sulfate-100 ,ug of tRNA per ml-100 ,ug of proteinase K per ml. The mixture was incubated at 42°C for 45 min, extracted with a 1:1 phenol-chloroform solution, and precipitated with ethanol. The sample was denatured and subjected to electrophoresis on a 50% (wt/vol) urea-containing polyacrylamide sequencing gel. Gel mobility shift analysis. Gel mobility shift assays were performed essentially as described by Cereghini et al. (2) as detailed earlier (25). 2P-labeled oligonucleotides (0.25 ng for gel mobility shift or 20 ng for gel mobility shift-Western immunoblot analysis) were incubated with 10 to 100 ng of recombinant DBP or C/EBP or 4 to 20 ,ug of nuclear extract proteins for 30 min at 24°C in a 20-,ul solution of binding buffer as described previously (25). After incubation, the mixtures were subjected to electrophoresis in a 5% nondenaturing acrylamide gel. In some cases, the gels were transferred to a nitrocellulose membrane (24). The filters were developed by using rabbit anti-rat DBP antibody and 125I-labeled protein A (New England Nuclear) (29). Construction and expression of CYP2C6 promoter chloramphenicol acetyltransferase (CAT) constructs. An EcoRI fragment containing the first exon, a portion of the first intron and 1,225 bp of DNA upstream of the RNA polymerase II start site (26) was inserted into the vector pGBT518. A new EcoRI site at +14 was generated according to the protocol supplied by Alter Gene (Gold Biotechnology, Inc., St. Louis, Mo.). This fragment was inserted into the HindIll site of pSVOCAT (12) by conversion of the EcoRI and HindIlI sites in the insert and vector, respectively, to blunt ends by using DNA polymerase Klenow fragment followed by ligation. Constructs -900/+14 and -500/+14 were prepared by using SacI and HindIII, respectively. Other deletion mutants, -345/+14, -234/+14, -103/+14, and -38/+14, were made by Bal 31 digestion at the -500 HindIII site, treatment with DNA polymerase Klenow fragment, agarose gel purifi-

CONTROL OF CYP2C6 TRANSCRIPTION BY DBP

VOL. 12, 1992

2849

TABLE 2. Expression of pCYP2C6(-12251+14) CAT in various cell lines cotransfected with pSCT-DBPa Expression (mean pmol/min/mg of protein + SD)

Plasmid

pSVOCAT + pCMV-4 pSV2CAT + pCMV-4 pCYP2C6(-12251+14) + pCMV-4 pCYP2C6(-1225/+14) + pSCT-DBP

A549

H441

TK-

CV-1

HepG2

0.86 (1) 10.36 (1) 1.00 + 0.29 (5) 1.07 ± 0.57 (5)

30.8 (1) 612.7 (1) 7.5 + 1.4 (5) 4.8 ± 1.6 (5)

0.40 (1) 13.20 (1) 0.24 ± 0.05 (5) 0.30 + 0.08 (5)

2.3 (1) 109.2 (1)

0.15 (1) 7.14 (1)

2.1 2.2

+ +

1.0 (5) 0.9 (5)

0.50 (2) 3.92 (2)

a Transfections were carried out with 2 ,ug each of pCYP2C6(-1225/+14)CAT and pSCr-DBP. Lipofectin was used for the nonhepatoma cell lines A549, H441, TK-, and CV-1. The number of experiments is shown in parentheses.

lated compounds [dpm]/acetylated and nonacetylated compounds [dpm]) x 100/luciferase activity/107. RESULTS Transactivation of the CYP2C6 promoter by DBP. To determine whether any of the known hepatocyte transcription factors play a role in regulating CYP2C6, cotransfection studies were carried out, using the CYP2C6 promoter (-1225 to + 14) linked to the CAT gene and plasmids containing DBP, HNF-lot, and C/EBP cDNAs expressed from heterologous promoters. The pAlb-CAT, containing -787 to +8 bp of the rat albumin promoter, was used as a positive control. Transfection of HepG2 cells revealed that only DBP was able to activate the CYP2C6 promoter. HNF-1a and C/EBP were ineffective (Table 1). All factors were able to activate the albumin promoter, and none were capable of activating the CYP2D5 promoter (-810 to +73). To further verify the extent of activation of the CYP2C6 promoter by DBP, titration experiments were carried out (Fig. 1A). Increasing amounts of the transcription factor cDNA-containing plasmid were mixed with a fixed amount of CAT reporter plasmid and transfected into HepG2 cells. Expelment I

Expewiment I normalied CAT acivity +DBP -DBP

told Induction by DBP

fold Induction by DBP

@ 14

-1225

I

-M0

I

H_S I=H

1.08±0.22

6S8 ± 0.23

6.0

6.5

3.64 ± 0.61

13.4 ± 2.0

3.7

3.5

3.87±1.51

13.6±1.6

3.7

4.9

2.55±1121

13.3 2.1

5.2

5.5

4.06±0.78

14.2±1.8

3.5

3.0

6.00 ± 0.4

16.1 ± 4.7

2.7

1.6

7.07 ± 2.23

5.06 0.32

0.7

0.7

1.76

0.89

0.5

65.5

72.5

1.1

500

-103

D[}@ [}

FIG. 2. Delineation of DBP-responsive sequences in the CYP2C6 promoter. Various deletions of the pCYP2C6-CAT constructs (10 pLg) were cotransfected with 10 ,ug of either pCMV-4(-DBP) or pSCT-DBP(+DBP) and 2 jig of pRSV-LA5', and the CAT/luciferase values were determined. The normalized CAT activities are the average values plus standard deviations of triplicate transfections and assays. Experiment II shows a summation of fold induction by DBP, using separately prepared preparations of all DNAs. SV2, simian virus 2.

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were lysed by freezing and thawing five times in 0.25 M Tris-HCl (pH 7.5)-l mM dithiothreitol. These experiments were also carried out with a CYP2D5 P450 promoter that shows a pattern of developmental expression different from that of the CYP2C6 gene and with pAlb-CAT, which is known to be under control by this factor (19). The latter plasmid, which was provided by Peter F. Johnson, Frederick Cancer Research and Development Program, contains -787 to +8 of the rat serum albumin gene plus 12 nucleotides from the herpes simplex virus thymidine kinase (TK) gene 5' leader (28). Plasmid pCMV-HNF-la was prepared by polymerase chain reaction amplification of rat liver mRNA to generate the coding region of HNF-lao. This fragment was inserted into pCMV-4. pMSV-C/EBP wt was obtained from Steven L. McKnight and Alan D. Friedman, Carnegie Institution of Washington, Baltimore, Md. CAT assays were performed by using [14C]chloramphenicol and thin-layer chromatography as described previously (12). The radioactive spots were excised and counted by liquid scintillation. Luciferase assays were carried out as described previously (5), using a Monolight 2010 luminometer (Analytical Luminescence Laboratory). CAT activities were normalized by luciferase activity as follows: (acety-

2850

YANO ET AL.

Maximal CAT activity was observed at a ratio of about 0.4 pSCT-DBP/pCYP2C6(-1225/+14)CAT, while activity from pAlb-CAT was still increasing at a ratio of 1.0 pSCT-DBP/ pAlb-CAT (Fig. 1A). These results indicate that more DBP is required to maximally activate the albumin promoter than the CYP2C6 promoter. In contrast, expression of the CYP2D5 promoter was not increased by DBP. Half-maximal activation of CYP2C6 and the albumin promoter required ratios of 0.15 and >0.45 pSCT-DBP/CAT vector, respectively. Results displayed in Fig. 1B revealed that even though C/EBP could activate the albumin promoter, it was ineffective in activating the CYP2C6 promoter.

ma-specific posttranslational processing event. Delineation of the region of CYP2C6 gene that binds DBP. To determine which region of the CYP2C6 promoter is required for the transactivation by DBP, deletion constructs were made and subjected to cotransfection with pSCT-DBP. In the absence of pSCT-DBP, a 3.5-fold difference in CAT activity was observed between the -1225 and -900 deletion constructs (Fig. 2). The former construct had a relative CAT activity of 1.08 when normalized to cotransfected pRSVLA5'-generated luciferase activity which was increased sixfold by pSCT-DBP. The -900 construct had a relative activity of 3.64 in the absence of pSCT-DBP which was increased 3.6-fold by the DBP expression plasmid. Activities of the -500, -345, and -234 constructs ranged from 2.55 to 4.06, and all were activated by pSCT-DBP. Transactivation was slightly reduced and constitutive activity increased to 6.0 by deletion to -103 bp upstream of the transcription start site (Fig. 2). Transactivation was eliminated when DNA upstream of -38 was deleted. These data indicate that the CYP2C6 promoter region required for DBP activation lies between -103 and -38. DNase I footprinting of the CYP2C6 promoter. DNase I footprinting was carried out by using partially purified bacterially expressed DBP and liver nuclear extracts from 8-week-old rats killed at 8 p.m. Footprints were obtained on both strands between -64 and -43 by using DBP (Fig. 3, lanes 3 and 7). More extensive protection of the CYP2C6 upstream DNA that included the -64 to -43 region, and a few additional hypersensitive sites, was obtained with nuclear extracts prepared from livers of adult male rats (Fig. 3, lanes 4 and 8). DBP is not expressed in liver of immature rats and is expressed in adult rats at the highest level at night (19, 29). We therefore examined DNase I footprints from male and female rats of different ages killed at 8 p.m., a time of maximal DBP expression (data not shown). One-week-old rat liver nuclear extracts contained no immunodetectable DBP (see below) yet still produced a footprint similar to that seen in 8-week-old rats killed at 8 p.m., indicating that proteins other than DBP specifically interact with this region of the promoter. The same result was obtained with 4-weekold male and female and 25-week-old male liver nuclear extracts prepared from rats killed at 8 p.m. which do contain

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1 2 34 FIG. 3. DNase I footprinting of the promoter region of the CYP2C6 gene, using partially pure DBP (lanes 3 and 7) and liver nuclear extracts from 8-week-old male rats (lanes 4 and 8). The coding (lanes 1 through 4) and noncoding (lanes 5 through 8) strands were labeled with 32p, incubated consecutively with protein and DNase I, and then subjected to electrophoresis in parallel with the same fragment that had been partially cleaved at G and A residues (lanes 1 and 5). Protein-free DNA was included as a nonprotected control (lanes 2 and 6).

DBP. A footprinting pattern indistinguishable from that in Fig. 3, lane 8, was also obtained with 4-week-old rats killed at 9 a.m., at which time DBP is not significantly expressed, regardless of age. Therefore, as in the case of the albumin promoter element D, the DBP-binding site appears to be recognized by other proteins found in liver nuclear extracts. Gel mobility shift analysis of the CYP2C6 and albumin DBPbinding regions. A double-stranded oligonucleotide, designated oligo-2C6(-40/-65), corresponding to the DBP-binding region (5'-TAGTCAATTATGCAATA1TGATlTCAG-3') was labeled with 32P and analyzed for its binding to partially pure recombinant DBP and adult liver nuclear extracts from rats killed at 8 p.m. DBP bound to this oligonucleotide, and the binding was inhibited only by a vast excess of the oligo2C6(-40/-65) double-stranded oligonucleotide unlabeled competitor and even a larger amount of the DBP-binding

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To determine whether transactivation of the CYP2C6 promoter by DBP was hepatocyte-derived cell type specific, transfection of other cell lines was carried out. Transactivation of the CYP2C6 promoter by DBP was not found when the human lung carcinoma cell lines A549 and H441, a human embryoblast TK- cell line, and a mouse kidney cell line CV-1 were transfected with pSCT-DBP and pCYP2C6 (-1225/+14)CAT (Table 2). Thus, similar to what had been suggested for the albumin promoter (19), the CYP2C6 promoter requires some additional factors found in hepatoma cells for DBP activation, or perhaps DBP requires a hepato-

MOL. CELL. BIOL.

VOL. 12, 1992

A

Adult Liver

DBP

OligoOligoOligoOligo2C6(-40/-65) Alb-D 2C6(-40/-65) Alb-D 0 20100 0 1880400 Com petitor 020100500 0 80400 (ng)

f

1

t

B

Competitor (ng)

Adult Liver

DBP

OligoOligoOligoOligoAlb-D 2C6(-40J-65) Alb-D 2C6(-40/-65) 0 16 80 0 5 20100 0 16 80400 0 20100

I?

1

2 3 4 5 6 7

2851

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Z;

:,

:L..:-:., -, s:

f,

A A.

8 9 10 111213 14

1 2 3 4 5 6

7

8 9 1011 1213 14

FIG. 4. Gel mobility shift analysis of partially pure DBP and adult rat liver nuclear extract proteins to the oligo-2C6(-40/-65). 32P-labeled oligo-2C6(-40/-65) (0.25 ng) (A) and oligo-Alb-D (0.20 ng) (B) were incubated with 2 pg recombinant DBP protein (lanes 1 through 7) or 4 pLg of extract from 8-week-old male rats killed at 8 p.m. (lanes 8 through 14) in the presence or absence of various amounts of competitor as indicated and subjected to acrylamide gel electrophoresis and autoradiography.

region of the albumin gene designated oligo-Alb-D (5'TGGTATGAT'lTTlGTAATGGGG-3') (Fig. 4A). When the amount of recombinant DBP used was reduced to from 2 ,ug to 10 ng, competition with unlabeled oligonucleotides was much more efficient. However, still a much larger excess of unlabeled oligo-Alb-D than of unlabeled oligo-2C6(-40/-65) was needed for efficient competition. When adult rat liver extract was mixed with labeled oligo-2C6(-40/-65), we detected a series of bands that migrated slower than the recombinant DBP-bound oligonucleotide and were eliminated by addition of a moderate excess of either oligo-Alb-D or oligo-2C6(-40/-65). The differences in mobilities of the recombinant DBP-bound oligonucleotides and the nuclear extract-bound oligonucleotides may be due to other proteinprotein interactions in the crude nuclear extracts. Similar results were obtained when oligo-Alb-D was labeled with 32p (Fig. 4B). Again, because of the large amount of recombinant DBP used, a large excess of competitor was needed to eliminate labeled oligo-Alb-D binding. Unlabeled oligo-2C6(-40/-60) was more efficient than unlabeled oligoAlb-D at competing with labeled oligo-Alb-D. Again, bands of lower mobility were detected when the oligonucleotide was mixed with adult rat liver nuclear extract as compared with recombinant DBP, and the binding was eliminated by only a moderate excess of unlabeled oligo-Alb-D and oligo2C6(-40/-65). The differences in unlabeled-oligonucleotide competition between the nuclear extracts and recombinant DBP could be explained by the concentration differences between DBP in the crude extracts and the recombinant preparations and the presence of other DNA-binding proteins in the crude nuclear extracts. The relative DNA-binding affinities of oligo-Alb-D and oligo-2C6(-40/-65) were measured by saturation binding assays using gel mobility shift analysis in which a constant amount of recombinant DBP was titrated with increasing amounts of labeled oligo-Alb-D and oligo-2C6(-40/-65). Both probes saturated DBP, with oligo-2C6(-40/-65) dis-

playing an affinity 17-fold higher than that of oligo-Alb-D (Fig. 5). These results suggest that the reason for the inefficient competition of oligo-Alb-D for the labeled oligo2C6(-40/-65) as shown in Fig. 4A is differences in binding affinities for recombinant DBP. On the basis of gel mobility shift analysis, it is impossible to determine whether DBP binds to the -40 to -65 region of the CYP2C6 gene when crude nuclear extracts are used as the source of binding protein, since several alternative factors may recognize the same element. We therefore performed gel mobility shift assays using extracts from rats of different ages killed at 8 p.m., transferred the proteins to nitrocellulose membranes, and developed the membranes by using rabbit anti-DBP antibodies (29). Several distinct retarded bands were detected from the nuclear extract samples, as revealed by autoradiography (Fig. 6A). Staining of the membrane for DBP revealed that the fastest-migrating band (Fig. 6A, arrow) bound to the transcription factor in 5-week-old male and female rats and 25-week-old male rats. DBP was detected in rats as young as 3 weeks old at a level of about one-third that found in 7-week-old rats but was not found in immature 1-week-old rats (Fig. 7), as predicted from earlier studies (29). DBP was not found in livers of rats killed between 9 a.m. and 10 a.m. regardless of age. The increase in DBP during development correlated with increases in the CYP2C6 mRNA (Fig. 7). Since C/EBP is capable of transactivating the albumin promoter and can bind to the D site, we examined whether it could bind to oligo-2C6(-40/-65). Recombinant C/EBP was, in fact, capable of binding the CYP2C6 DBP-binding sequence, and this binding was abolished by unlabeled homologous oligonucleotide and two other C/EBP-binding oligonucleotides (Fig. 8). Considerably more unlabeled oligo-Alb-D and oligo-SV40 C/EBP was required to compete with oligo-2C6(-40/-65), suggesting major differences in affinity for C/EBP between the latter oligonucleotide and oligo-Alb-D. Indeed, C/EBP exhibited Kds of 170 and 26 nM

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sIW.U

CONTROL OF CYP2C6 TRANSCRIPTION BY DBP

2852

MOL. CELL. BIOL.

YANO ET AL.

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2 0

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e

8

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0 0 0.2 0.1

0 0

5

10

15

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BOUND (f mole /I Ong protein)

BOUND (f mole / IOOng protein)

FIG. 5. Measurements of relative binding affinities of DBP for oligo-2C6(-40/-65) (A and B) and oligo-Alb-D (C and D). The gel mobility shift assay was performed by using a fixed amount of DBP (100 ng/20-,ul reaction) or C/EBP (25 ng/20-,ul reaction) and increasing amounts of labeled oligonucleotide (0.1 to 100 ng/20-pJl reaction). The shifted bands were identified by autoradiography and excised from the gel for liquid scintillation spectroscopy. The amount of protein-bound probe was plotted as a function of input DNA, and saturation was accomplished for both oligonucleotides (A and C). The data were plotted by the method of Scatchard (22). Kd = -1/slope.

for oligo-Alb-D and oligo-2C6(-40/-65), respectively, indicating that the latter oligonucleotide has a higher affinity for C/EBP (data not shown).

DISCUSSION Early studies revealed that transcription of activation of the rat hepatic CYP2C6 gene commences at around 3 weeks of age and that maximal levels of mRNA are reached at about the age of 4 weeks, or just at the onset of puberty (11). On the basis of results in the present report, CYP2C6 may be controlled in large part by the transcription factor DBP, or a closely related factor, which itself is maximally expressed in postpubertal rats. Unlike albumin expression, the accumulation of CYP2C6 mRNA follows more closely expression of DBP, indicating that this factor may have a key role in CYP2C6 expression. In contrast, the albumin gene is already efficiently expressed before DBP reaches its maximal level. Thus, in prepubertal rats, the albumin gene may be regulated to a large degree by other factors such as HNF-la (and HNF-13) and C/EBP (19). In prepubertal rats, CYP2C6 mRNA can be induced to levels similar to those of adults by

administration of phenobarbital (8), indicating that an overriding transcription activation can occur in young animals by inducer treatment. Since DBP expression follows a circadian rhythm (29), we expected to see circadian control of CYP2C6 transcription. Unfortunately, due to cross-hybridization of closely related genes in the complex rat CYP2C subfamily, transcription rates of CYP2C6 cannot accurately be measured. Therefore, we do not yet know whether the CYP2C6 gene is transcribed in a circadian fashion. DBP may play a major role in the transcriptional regulation of two genes, CYP2C6 and the albumin gene, whose products have completely different functions in the organism. Interestingly, the DBP recognition sequences of these two genes do not share any overt similarities to one another. The DBP recognition sequence of CYP2C6 5'-GTCAAT TATGCAATATTGATT-3' contains two almost perfect half sites, left GTCAATTATG and right GTTATAACTA (complement), which partially conform to the minimal DNAbinding consensus sequence 5'-(T/C)(AIG)(A/G)TTA(T/C) (A/G)-3' for DBP-related proteins (6). Interestingly, the CYP2C6 sequence, having the two near perfect half sites,

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DBP (nM) 0.02

CONTROL OF CYP2C6 TRANSCRIPTION BY DBP

VOL. 12, 1992 A

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FIG. 6. Gel mobility shift-Western blot analysis of DBP in liver nuclear extracts isolated from rats of various ages killed at 8 p.m. Gel mobility shift assays were performed by using 20 ng of 32plabeled (A) and unlabeled (B) oligo-2C6(-40/-65) complexed with 1 ,ug of recombinant DBP or 20 pg of nuclear extract protein from male rats of the indicated ages. The gel was cut in half; the half containing the unlabeled oligonucleotide (B) was electroblotted to nitrocellulose paper and treated with anti-DBP antibody, and the DBP protein was stained by using '25I-labeled protein A. The other half of the gel was dried and subjected to autoradiography. The arrow designates the shifted band that reacts with the anti-DBP

antibody.

has a markedly higher affinity for DBP than does the albumin D site. This difference in affinity might be the basis for the differences in half-maximal transactivation by DBP of the CYP2C6 and albumin promoters. Gel mobility shift analyses with both oligonucleotides also revealed binding of proteins in nuclear extracts that do not contain DBP. Multiple bands were detected, only one of which was found to bind to DBP in 3-week-old and older rats killed at 8 p.m. The identities of the other proteins binding to both CYP2C6 and albumin oligonucleotides are unknown, but they may be identical to or related to C/EBP (19) or other bZIP DNA-binding proteins such as CRP1, CRP3 (28), or LAP (4). Indeed, recombinant C/EBP is capable of binding the DBP recognition site of CYP2C6, and the binding, as assessed by DNase I footprinting using recombinant C/EBP, is qualitatively identical to that found with recombinant DBP (data not shown). Surprisingly, however, C/EBP can bind the albumin D site and also transactivate transcription (9), whereas this factor could not transactivate the CYP2C6 promoter even though the latter has a higher-affinity binding site for C/EBP than does the D site of albumin. These data suggest a functional difference in the albumin and CYP2C6 DBP-binding sites. The mechanism of this lack of correlation between binding and transactivation is not currently understood. Perhaps the CYP2C6 site is not activated by C/EBP in the context of its own promoter element but would function with another promoter. This possibility awaits further experimentation. A recent report described a new bZIP transcription factor, designated thyrotroph embryonic factor (TEF), that is expressed on embryonic day 14 in the region of the rat

FIG. 7. Correlation between expression of DBP and CYP2C6 mRNA in developing rats. DBP levels were measured as described for Fig. 6 after scanning of the Western blots with a Molecular Dynamics series 300 computing densitometer. CYP2C6 mRNA levels were determined by Northern (RNA) blot analysis and scanning quantitation of autoradiographic bands. Liver nuclear extracts and total RNAs were prepared from three pooled livers taken from 3-week-old, 5-week-old, and 7-week-old rats. El, gel shift-Western assay (DBP); E, Northern assay (CYP2C6 mRNA); N.D., not determined.

pituitary, giving rise to thyrotrophs (6). This factor exhibits high sequence similarity to DBP, in particular, a segment called proline acid rich that is unique to these two factors among the bZIP proteins. DBP and TEF can also form heterodimers and bind the same recognition sequence. It is unknown whether TEF can transactivate the albumin promoter. These data indicate that other members of this bZIP Oligo- Oligo- OligoSV40 2C6 (-40/-65) Alb-D C/EBP Com petitor

0 50 250 40 200 60 300

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FIG. 8. Gel mobility shift analysis of partially purified C/EBP binding to oligo-2C6(-40/-65). Partially purified C/EBP (10 ng) was incubated with 0.25 ng of 32P-labeled oligo-2C6(-40/-65), and competition was carried out with the indicated amounts of unlabeled homologous oligonucleotide oligo-Alb-D, and oligo-SV40 C/EBP. The latter oligonucleotide sequence (5'-TGTCAGTTAGGGTGTG GAAAGTCCCAGGCT-3') was taken from Johnson et al. (16).

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Age (Week)

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YANO ET AL.

subfamily exist, and we cannot presently rule out that they may play a role in CYP2C6 regulation in the liver, although no other DBP-like liver-enriched factors have been described. In conclusion, these studies suggest that CYP2C6 may be under control of the hepatocyte-enriched, adult-specific transcription factor DBP. The lack of transactivation in nonhepatoma cells and the binding of other nuclear proteins in extracts lacking DBP to the cis-acting region indicates that DBP itself may not be sufficient for CYP2C6 transcription but likely works in concert with other regulatory factors. Further studies will be required to delineate the sequence requirements of this factor and the complex interplay between various transcription factors to control developmental

MOL. CELL. BIOL.

12. 13. 14. 15. 16.

regulation of albumin and CYP2C6. The mechanism by which phenobarbital activates the latter gene also remains a 17.

ACKNOWLEDGMENTS

18.

We thank Steven L. McKnight for pMSV-C/EBP wt and recombinant C/EBP, and we thank Peter F. Johnson for pAIb-CAT. We also thank Ueli Schibler for use of pSCT-DBP, helpful suggestions, and critical review of the manuscript. 19. 1.

2.

3. 4.

5. 6.

7.

8.

9.

10. 11.

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