Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Transcriptional activation of the murine Cyp1a-1 (cytochrome P(1)450) gene by inducers such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (dioxin) requires the aromatic hydrocarbon (Ah) receptor and the interaction of an inducer-receptor complex with one or more of the Ah-responsive elements (AhREs) located about 1 kb upstream from the transcriptional initiation site. We find that treatment of mouse hepatoma Hepa-1 cells with 2-aminopurine, an inhibitor of protein kinase activity, inhibits CYP1A1 mRNA induction by TCDD as well as the concomitant increase in CYP1A1 enzyme activity. Formation of DNA-protein complexes between the Ah receptor and its AhRE target is also inhibited by 2-aminopurine, as determined by gel mobility shift assays. Phosphorylation is required for the formation of Ah receptor-specific complexes, since in vitro dephosphorylation of nuclear extracts from TCDD-treated Hepa-1 cells abolishes the capacity of the Ah receptor to form specific complexes with its cognate AhRE sequences. To determine whether any one of several known protein kinases was involved in the transcriptional regulation of the Cyp1a-1 gene, we treated Hepa-1 cells with nine other protein kinase inhibitors prior to induction with TCDD; nuclear extracts from these cells were analyzed for their capacity to form specific DNA-protein complexes. Only extracts from cells treated with staurosporine, a protein kinase C inhibitor, were unable to form these complexes. In addition, staurosporine completely inhibited CYP1A1 mRNA induction by TCDD. Depletion of protein kinase C by prolonged treatment with phorbol ester led to the complete suppression of CYP1A1 mRNA induction by TCDD. We conclude that (i) phosphorylation is necessary for the formation of a transcriptional complex and for transcriptional activation of the Cyp1a-1 gene; (ii) the phosphorylation site(s) exists on at least one of the proteins constituting the transcriptional complex, possibly the Ah receptor itself; and (iii) the enzyme responsible for the phosphorylation is likely to be protein kinase C.
Mol Cell Biol 1992 Apr
PMID:Dioxin-dependent activation of murine Cyp1a-1 gene transcription requires protein kinase C-dependent phosphorylation. 131 72

In the rat, expression of the CYP1A1 gene is closely associated with arylhydrocarbon hydroxylase (AHH) enzyme activity. AHH is an inducile enzyme activity known to play an important role in the bioactivation of polycyclic aromatic hydrocarbons (PAHs) to mutagenic and carcinogenic metabolites. PAH-induced expression of the CYP1A1 gene appears to be regulated by several trans-acting factors, including the Ah receptor and the 4S PAH-binding protein. In this study, we used the PAH isomers benzo[a]pyrene (BaP) and benzo[e]pyrene (BeP) to further evaluate the role of the 4S PAH-binding protein in induction of the CYP1A1 gene in H4-II-E rat hepatoma cells. Although BaP is believed to bind to both the Ah receptor and the 4S protein, BeP has been reported to bind exclusively to the 4S protein. The results of the study presented here indicate that BaP and BeP induce the expression of the CYP1A1 gene, as measured by ethoxyresorufin O-deethylase (EROD) activity, in a concentration-dependent manner. However, BaP is about 25 times as potent as BeP in inducing EROD activity in these cells. Slot-blot analysis of total RNA isolated from these cells indicated that BeP, BaP, and 3-methylcholanthrene increased the level of CYP1A1 mRNA expression. Sucrose-gradient analysis of BeP binding activity indicated that BeP bound with high affinity to the 4S PAH-binding protein, but not to the Ah receptor. These results suggest that the 4S protein may play a role in the PAH-induced expression of the CYP1A1 gene in rat H4-II-E cells.
Mol Carcinog 1992
PMID:Induction of CYP1A1 gene expression in H4-II-E rat hepatoma cells by benzo[e]pyrene. 131 59

We have analyzed the function of the CYP1A1 promoter using in vitro transcription. Using nuclear extracts from HeLa cells, we found that transcription from the promoter in vitro was constitutive. Transcription in vitro was not increased by prior exposure of the HeLa cells to the inducer 2,3,7, 8-tetrachlorodibenzo-p-dioxin nor by the inclusion of a dioxin-responsive enhancer in the DNA template. Analyses of mutants revealed that a TATAAA sequence and two recognition motifs for CCAAT box transcription factor/nuclear factor I contributed to the constitutive activity of the promoter in vitro.
Mol Carcinog 1991
PMID:Analysis of CYP1A1 promoter function by transcription in vitro. 204 54

Transcripts of the murine CYP1A1 (cytochrome P1450) mRNA are markedly elevated in mutant hepatoma cell lines that contain missense mutations in the Cyp1a-1 structural gene. This putative derepression extends to other genes in the [Ah] battery. To test whether the Cyp1a-1 gene product is involved in a mechanism of feedback regulation of transcription, we introduced expression plasmids carrying the murine wild-type Cyp1a-1 cDNA into the mutant hepatoma cells. Measurements of steady-state mRNA levels and of transcriptional rates in the transfectants reveal that expression of a functional, exogenous CYP1A1 protein is sufficient to restore the repression of the endogenous gene, as well as restore the inducibility by dioxin, and that this effect takes place primarily at the level of transcription. Similar experiments with expression plasmids that carry the human CYP1A2 cDNA indicate that the CYP1A2 protein (cytochrome P3450) can also function as a transcriptional repressor. In addition, we find that expression of the Nmo-1 [NAD(P)H:menadione oxidoreductase] gene, a third member of the [Ah] gene battery, is also repressed by the exogenous expression of either Cyp1a-1 or CYP1A2 cDNA. These results indicate that the gene product of either member of the mammalian CYP1 family has a previously unrecognized transcriptional regulatory function, which is likely to be exerted by modification of preexisting trans-acting factors. This function may help bring about a fast reprogramming of gene expression, as might be needed during detoxification of toxic foreign chemicals.
Mol Endocrinol 1990 Dec
PMID:The murine Cyp1a-1 gene negatively regulates its own transcription and that of other members of the aromatic hydrocarbon-responsive [Ah] gene battery. 208 80

Three nuclear factors, the Ah receptor, XF1, and XF2, bind sequence specifically to the Ah response elements or xenobiotic response elements (XREs) of the cytochrome P450IA1 (P450c) gene. The interactions of these factors with the Ah response element XRE1 were compared by three independent methods, methylation interference footprinting, orthophenanthroline-Cu+ footprinting, and mobility shift competition experiments, using a series of synthetic oligonucleotides with systematic alterations in the XRE core sequence. These studies established the following (i) all three factors interact sequence specifically with the core sequence of XRE1; (ii) the pattern of contacts made with this sequence by the Ah receptor are different from those made by XF1 and XF2; and (iii) although XF1 and XF2 can be distinguished by the mobility shift assay, the sequence specificities of their interactions with XRE1 are indistinguishable. Further characterization revealed the following additional differences among these three factors: (i) XF1 and XF2 could be extracted from nuclei under conditions quite different from those required for extraction of the Ah receptor; (ii) XF1 and XF2 were present in the nuclei of untreated cells and did not respond to polycyclic compounds, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-napthoflavone, while nuclear Ah receptor was undetectable in untreated cells and rapidly increased in response to TCDD; (iii) inhibition of protein synthesis did not affect the TCDD-induced appearance of the Ah receptor but substantially decreased the constitutive activities of XF1 and XF2, suggesting that the Ah receptor must be present in untreated cells in an inactive form that can be rapidly activated by polycyclic compounds, while the constitutive expression of XF1 and XF2 depends on the continued synthesis of a relatively unstable protein; (iv) the receptor-deficient and nuclear translocation-defective mutants of the hepatoma cell line Hepa1, which are known to lack nuclear Ah receptor, expressed normal levels of XF1 and XF2, suggesting that the former factor is genetically distinct from the latter two; and (v) a divalent metal ion, probably Zn2+, is known to be an essential cofactor for the Ah receptor but was not required for the DNA-binding activities of XF1 and XF2. Together, these findings indicate that the Ah receptor is distinct from XF1 and XF2, while the latter two activities may be related. Because the DNA-binding domains of these three factors overlap substantially, their binding to XREs is probably mutually exclusive, which suggests that the interplay of these factors at Ah response elements may be important to the regulation of CYP1A1 gene transcription. The results of preliminary transfection experiments with constructs harboring XREs upstream of the chloramphenicol acetyltransferase gene driven by a minimal simian virus 40 promoter are presented that are consistent with this hypothesis.
Mol Cell Biol 1990 Dec
PMID:Multiple DNA-binding factors interact with overlapping specificities at the aryl hydrocarbon response element of the cytochrome P450IA1 gene. 217 7

In mouse hepatoma cells, the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases the transcription rate of the CYP1A1 gene, which encodes a cytochrome P-450 enzyme. In this study, we analyzed the DNA region immediately upstream of the CYP1A1 gene. A domain that extends upstream to nucleotide--166 was found to function as a transcriptional promoter. The promoter was silent when uncoupled from the dioxin-responsive enhancer located farther upstream. DNase footprinting experiments indicated that nuclear proteins interact with distinct domains of the promoter in a TCDD-independent fashion. Mutational analyses indicated that the CYP1A1 promoter contains at least three functional domains, including a TATAAA sequence, a CCAAT box transcription factor/nuclear factor I-like recognition motif, and a guanine-rich G box.
Mol Cell Biol 1990 Oct
PMID:Functional analysis of the transcriptional promoter for the CYP1A1 gene. 239 86

The mouse cytochrome P1450 (CYP1A1) gene is responsible for the metabolism of numerous carcinogens and toxic chemicals. Induction by the environmental contaminant tetrachlorodibenzo-p-dioxin (TCDD) requires a functional aromatic hydrocarbon (Ah) receptor. We examined the 5'-flanking region of the CYP1A1 gene in mouse hepatoma Hepa-1 wild-type cells and a mutant line having a defect in chromatin binding of the TCDD-receptor complex. We identified two cis-acting elements (distal, -1071 to -901 region; proximal, -245 to -50 region) required for constitutive and TCDD-inducible CYP1A1 gene expression. Three classes of DNA-protein complexes binding to the distal element were identified: class I, found only in the presence of TCDD and a functional Ah receptor, that was heat labile and not competed against by simian virus 40 (SV40) early promoter DNA; class II, consisting of at least three constitutive complexes that were heat stable and bound to SV40 DNA; and class III, composed of at least three constitutive complexes that were thermolabile and were not competed against by SV40 DNA. Essential contacts for these proteins were centered at -993 to -990 for the class I complex, -987, -986, or both for the class II complexes, and -938 to -927 for the class III complexes. The proximal element was absolutely essential for both constitutive and TCDD-inducible CYP1A1 gene expression, and at least two constitutive complexes bound to this region. These data are consistent with the proximal element that binds proteins being necessary but not sufficient for inducible gene expression; interaction of these proteins with those at the distal element was found to be required for full CYP1A1 induction by TCDD.
Mol Cell Biol 1989 Jun
PMID:Regulation of mouse CYP1A1 gene expression by dioxin: requirement of two cis-acting elements during induction. 254 80

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces, in a receptor-dependent fashion, an increase in the accessibility of CYP1A1 chromatin to restriction endonucleases. The 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced change in chromatin structure occurs rapidly and does not require ongoing RNA or protein synthesis. The increased accessibility of chromatin DNA may facilitate its subsequent interaction with other transcription factors.
Mol Cell Biol 1989 Dec
PMID:2,3,7,8-Tetrachlorodibenzo-p-dioxin-inducible aryl hydrocarbon receptor-mediated change in CYP1A1 chromatin structure occurs independently of transcription. 257 37

We have sequenced the human CYP1A2 (cytochrome P(3)450) gene, 1,906 basepairs (bp) of the 5' flanking region, and 113 bp of the 3' flanking region. The gene spans almost 7.8 kilobases, comprising seven exons and six introns. The transcriptional start site was determined by both primer extension and S1 mapping. Including the first noncoding exon of 55 bp, the entire mRNA is 3,121 bp in length, and the open reading frame, starting with nucleotide 10 of exon 2, encodes 515 amino acids (mol wt = 58,294). Between the human CYP1A2 and CYP1A1 (cytochrome P(1)450) genes, exons 2, 4, 6, and especially 5 are strikingly conserved in both nucleotide similarity and total number of bases. Alignment of the upstream sequences and exon 1 of human CYP1A2 with that of mouse or rat CYP1A2 revealed two possibly significant regions of similarity: 1) 68% in the approximately 150 bases immediately 5' from the mRNA cap site and 2) 80% identify between the human -841 to -758 segment and the mouse -1,529 to -1,439 segment. The canonical 5-bp box (CACGC), found upstream of all mammalian CYP1A1 genes to date and believed to interact with the inducer.aromatic hydrocarbon receptor complex, was not found on either strand in the 1,906 bp of the 5' flanking region of human CYP1A2. In contrast, alignment of the upstream sequences, exon 1, and intron 1 of human CYP1A1 with that of mouse or rat CYP1A1 revealed large, highly conserved regions. Conserved regions were found in intron 1 of the human, mouse, and rat CYP1A2 gene. These data suggest that the regulatory elements controlling the CYP1A2 gene might differ in location from those controlling the CYP1A1 gene. Among 12 human liver samples, striking differences (greater than 15-fold) in the 3.3-kilobase 1A2 mRNA levels were seen. This result may reflect significant genetic differences in constitutive and/or inducible CYP1A2 gene expression that could play an important role in individual risk of environmental toxicity or cancer.
Mol Endocrinol 1989 Sep
PMID:Human CYP1A2: sequence, gene structure, comparison with the mouse and rat orthologous gene, and differences in liver 1A2 mRNA expression. 257 18

The halogenated aromatic hydrocarbon 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) is a persistent, widespread, potentially toxic environmental contaminant, which is a potent inducer of aryl hydrocarbon hydroxylase activity in the liver and other tissues. TCDD induces hydroxylase activity by increasing the rate of transcription of the CYP1A1 gene. Activation of CYP1A1 transcription requires the binding of TCDD to an intracellular protein, the Ah receptor, followed by the binding of the liganded receptor to a dioxin-responsive enhancer that is located upstream from the CYP1A1 gene. The liganded receptor recognizes a specific DNA sequence, which is present in multiple copies within the enhancer. The receptor-enhancer interaction occurs within the major groove of the DNA helix. DNA methylation in vitro interferes with the receptor-enhancer interaction and, therefore, has the potential to inhibit the biological response to TCDD.
Mol Biol Med 1989 Apr
PMID:Induction of hepatic cytochrome P450 gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin. 269 91


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