Two 21-hydroxylase [P450(C21)] genes have been isolated from a human genomic library using a bovine P-450(C21) cDNA. The insert DNAs containing the P450(C21) genes were also hybridized with the sequences of the 5' or 3' end regions of human C4 cDNA, indicating a close linkage of the P450(C21) gene to the C4 gene. Sequence analysis has revealed that the two P450(C21) genes are both 3.4 kilobases long and split into 10 exons. Comparing the two sequences, we found that the two genes are highly homologous including their introns and flanking sequences, but that three mutations render one of the two P450(C21) genes nonfunctional-i base insertion, an 8-base deletion, and a transition mutation-all of which may cause premature termination of the translation.

The DNA element governing the inducible expression of drug-metabolizing P-450c gene by xenobiotic treatments was investigated by gene transfer methods. A variety of dissected fragments from -844 to -1140bp region which was essential for the inducibility of P-450c gene were placed on the heterologous SV40 promoter for testing the inducibility. Mapping studies in combination with gel retardation assay defined the presence of the two xenobiotic responsive elements (XRE, XRE1, -1007 - -1021bp; XRE2, -1088 - -1092bp) composed of about 15 nucleotides which expressed the enhancer activity in response to xenobiotic inducers. The two XREs share 10 nucleotides in common out of 15 as expressed in the sequence CG/CTG/CC/TTG/CTCACGCT/AA and are arranged in the inverse orientation. They are different from DREs (drug responsive element) proposed previously (Sogawa, K. et al. Proc. Natl. Acad. Sci. 83, 8044-8048 (1986] and expressed a strong enhancer activity in response to 3-methylcholanthrene. The XRE shows a significant homology with glucocorticoid regulatory elements and apparently needs normal functions of a putative xenobiotic receptor for the inducible enhancer activity.

Transcription of the drug-metabolizing cytochrome P-450c gene is induced by 3-methylcholanthrene or 2,3,7,. Previously, we dermed two xenobiotic responsive elements (XREs) of 15 base pairs, both of which activate transcription in cis in response to these xenobiotics. Using a gel mobility shift assay, we have identified a factor that specifically binds to the XREs. This factor appears in nuclei of mouse hepatoma cell line Hepa-1 only when the cells are treated with the xenobiotics, while the factor is undetectable in the nuclei of a 3-methylcholanthrenetreated mutant of Hepa-1 with defective function of a xenobiotic receptor. In addition, the nuclear factor bound to the XRE in the gel was found to be associated with [3H]TCDD when the cells were treated with it, suggesting that the xenobiotic receptor is at least a component of the DNA-binding factor. The cytoplasmic fraction from nontreated Hepa-1 cells also contains the factor as a cryptic form and prominently reveals its DNA-binding activity by incubation with 3-methylcholanthrene in vitro. These results not only suggest the involvement of the XRE-binding factor in transcriptional activation via XREs but also provide evidence that the binding of ligands to the preexisting factor in a cryptic form induces its XRE-binding activity, which is probably followed by its translocation from cytoplasm to nucleus.

The synthesis of cytochrome P-450c is induced remarkably in cultured cells as well as animal tissues in response to added chemicals such as 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzodioxin. To study this mechanism, we joined the sequence of 5'-flanking and upstream regions of the P-450c gene to the structural gene for chloramphenicol acetyltransferase. The fusion gene was introduced into Hepa-1 cells for the assay of the expressed acetyltransferase activity. At least three cis-acting regulatory regions that are responsible for the inductive expression were determined in the sequences from nucleotide -3674 to -3067, from -1682 to -1429, and from -1139 to -1029, relative to the transcription start site, by external deletion analysis. Further detailed analysis of the region (nucleotides -1139 to -1029) most influential on the inducibility revealed that a regulatory element consisting of 10 base pairs termed a drug regulatory element (DRE) and its homologues were tandemly arranged in this region. The consensus sequence deduced from DREs is 5'-CNGGGCTGGG-3'. The regulatory sequence from nudeotide -1140 to -844 is capable of conferring inducibility on a heterologous promoter in a manner independent of its orientation and distance from the subordinate promoter.A family of hemoproteins, collectively termed cytochrome P-450, occurs in liver microsomal membranes, and its members metabolize a number of endogenous substrates such as steroids, prostaglandins, and fatty acids and an almost infinite number of exogenous substances. The wide substrate specificity of the P-450 system results from multiple molecular forms of P-450, each of which shows different but sometimes overlapping substrate specificities (1). Of these P-450s, P-450c especially catalyzes the hydroxylation reaction of polycyclic aromatic hydrocarbons such as benzo[a]-pyrene and 3-methylcholanthrene (MC), and it is also present in various extrahepatic tissues (2, 3). This species of P-450 is strongly induced by a number of chemicals, including MC and 2,3,7,8-tetrachlorodibenzodioxin (TCDD). The induction of P-450c is known to be controlled at the level of transcription (4, 5). In the previous study (6), we described a transient assay system that mimics the induction process of P-450c in vivo in animal tissues by the administration of MC. We present in this paper the location of three cis-acting regulatory sequences in the 5' flanking region of the P-450c gene, using the transient assay system. From the analysis of the proximal region, sequence elements of 10 base pairs (bp) that were deduced to be responsible for the induction by MC were found. MATERIALS AND METHODSConstruction of Deletion Plasmids. Fusion genes with various external deletions were constructed as follows. pMC6.3k (6) DNA Sequence Analysis. Sequences were determined by the chain-termination method as described (8,9). RESULTS Location of Sequences Responsible for Drug Induction. To identify functional regions responsible for the induction in the 5' flanking sequence of the P-450...

Gene expression of rat cytochrome P-45Oc (P-45Oc) depends upon inducible enhancers scattered in the 5'-upstream region of the gene. We show that expression of the P-45Oc gene is repressed by contransfection with adenovirus Ela DNA, regardless of the presence or absence of inducers, in a transient expression system of HeLa cells. Since cotransfection of either 13s or 12s Ela cDNA was effective in the repression, the region necessary for repression could be separated from that of transactivation of other adenovirus early genes. Moreover, we investigated the regions responsible for the inhibitory activity using in-frame deletion mutants lacking internal or external portions of the Ela proteins. The sequence responsible for the repression was located in the aminoterminal half of the Ela proteins. The inducible expression of the chimeric plasmid containing a 24-base-pair enhancer sequence of the P-45Oc gene placed in a heterologous promoter of SV40 was repressed by cotransfection with Ela DNA, suggesting strongly that the inhibitory effect of the Ela proteins upon P-45Oc gene expression was caused by blocking the enhancer activity.Cytochrome P-450 (P-450) constitutes a superfamily and plays an important role in oxidative metabolism of exogenous and endogenous substrates as a terminal oxidase in an NADPH-dependent electron pathway of liver microsomes [l]. Of these P-450s, P-45Oc (new nomenclature: P-450IA1 [2]) is distributed fairly ubiquitously in various tissues in rats and is induced by the administration of inducers such as 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzodioxin [3,4]. Severallaboratories have attempted to reveal the molecular mechanisms of the induction phenomena, using the DNA transfection technique [5 -91. From these studies, a cis-acting DNA element, responsible for the inducible expression of the gene, was identified within short segments in the upstreamflanking region of the P-45Oc gene and designated XRE (xenobiotic responsive element) [7], although the element is also indispensable for the basal level expression of the P-45Oc gene. Furthermore, this element was found to operate also on a heterologous promoter of SV40 in a manner relatively independent of distance and orientation and is, therefore, classified into the category of inducible enhancers. Several lines of evidence strongly suggest that the inducible expression of the P-45Oc gene after administration of inducers is mediated Correspondence to K. Sogawa, Department of Chemistry, Faculty of Science, Tohoku University, Aobayarna, Sendai-shi Miyagi-ken, Japan 980Abbreviations. P-450c, cytochrome P-45Oc; CAT, chloramphenicol acetyltransferase; XRE, xenobiotic responsive element; Ad, adenovirus.Enzymes. T4 DNA ligase (EC 6.5. [18], were activated by the Ela proteins, while activities of viral enhancers, including SV40 and polyoma virus and cellular enhancer elements such as immunoglobulin-heavychain and insulin genes, were repressed [19-221. We are interested in the mechanism whereby the inducible enhancer of the P-45Oc gene is infl...