In situ protein-DNA interactions at a dioxin-responsive enhancer associated with the cytochrome P1-450 gene.

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2,3,7,8-Tetrachlorodibenzo-p-dioxin induces, in a receptor-dependent fashion, an increase in the accessibility of CYPIAl 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.Gene transcription, induced by a ligand-receptor complex, may be accompanied by a change in chromatin structure (1,2,17,28,29,33). For example, the DNase I hypersensitive region (HSR) near the glucocorticoid-response element of the mouse mammary tumor virus long terminal repeat appears minutes after exposure to dexamethasone and depends upon continued presence of inducer (33). However, the temporal relationship between onset of transcription and appearance of HSRs in inducible systems is not established.We have been studying the mechanism by which the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces the transcription of a cytochrome P-450 (CYPIAI) gene. TCDD elicits a variety of biological effects (24,26) that are mediated by an intracellular protein, the aryl hydrocarbon receptor (10, 23). Activation of CYPIAJ gene transcription, in wild-type (Hepa lclc7) mouse hepatoma cells, involves the formation of a TCDDreceptor complex and the binding of the complex to upstream DNA elements that function as dioxin-responsive enhancers (DREs) (5,6,7,16). The response to TCDD is rapid, and half-maximal transcription occurs within 20 min (14). Here, we have studied changes in CYPIAJ chromatin structure that occur rapidly in response to TCDD. Our initial studies, with DNase I, revealed no discrete TCDD-induced HSRs. Since a number of other enzymes and chemical reagents cut DNA within HSRs (8), we asked whether TCDD alters the susceptibility of restriction endonuclease sites in CYPIAJ chromatin. Wild-type mouse hepatoma cells were grown in medium containing 0.1% dimethyl sulfoxide (DMSO) (-TCDD) or 0.1% DMSO plus 1 nM TCDD (+TCDD). After 24 h, the cells were collected and nuclei were prepared by Dounce homogenization, as previously described (7). Nuclei, at a DNA concentration of about 1 mg/ml, were digested for 60 min at 30°C with 300 U/ml of the indicated restriction enzymes, in 10 mM Tris hydrochloride (pH 7.5)-100 mM NaCl-3 mM MgCl2-0.1 mM EDTA-1 mM 2-mercaptoethanol (19). Nuclear DNA was purified, digested to completion with 30 U of HindlIl, fractionated by agarose * Corresponding author. gel electrophoresis, and transferred to a Nytran membrane, as previously described (27). The immobilized DNA was hybridized (15) to a 32P-labeled 300-base-pair fragment of cloned DNA that is homologous to the 5' end of the HindlIl fragment (designated 5'-probe in Fig. 1B). The results (Fig. 1A) show that exposure of wild-type cells to TCDD is associated with increased nuclease susceptibility at all restriction sites examined between the RsaI site located at -167 nucleotides and the Stul s...

supporting

confidence: 46%

mentioning

confidence: 99%

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confidence: 99%

See 1 more Smart Citation

2,3,7,8-Tetrachlorodibenzo-p-dioxin-inducible aryl hydrocarbon receptor-mediated change in CYP1A1 chromatin structure occurs independently of transcription.

Durrin¹,

Whitlock²

1989

Self Cite

“…It has been reported that dioxin treatment induces an exonuclease III stop site in mouse cytochrome P-450IA1 chromatin (11). However, this site does not correspond to any functional dioxin-responsive and dioxin receptor-binding sequences in the mouse cytochrome P-4501A1 gene (31), nor does it correspond to any of the mapped DNase I-hypersensitive sites in the rat gene.…”

Section: Discussionmentioning

confidence: 99%

Liver cells contain constitutive DNase I-hypersensitive sites at the xenobiotic response elements 1 and 2 (XRE1 and -2) of the rat cytochrome P-450IA1 gene and a constitutive, nuclear XRE-binding factor that is distinct from the dioxin receptor.

Hapgood¹,

Cuthill²,

Söderkvist³

et al. 1991

Dioxin stimulates transcription from the cytochrome P-450IA1 promoter by interaction with the intracellular dioxin receptor. Upon binding of ligand, the receptor is converted to a form which specifically interacts in vitro with two dioxin-responsive positive control elements located in close proximity to each other about 1 kb upstream of the rat cytochrome P-450IA1 gene transcription start point. In rat liver, the cytochrome P-450IA1 gene is marked at the chromatin level by two DNase I-hypersensitive sites that map to the location of the response elements and exist prior to induction of transcription by the dioxin receptor ligand 0-naphthoflavone.In addition, a DNase I-hypersensitive site is detected near the transcription initiation site and is altered in nuclease sensitivity by induction. The presence of the constitutive DNase I-hypersensitive sites at the dioxin response elements correlates with the presence of a constitutive, labile factor which specifically recognizes these elements in vitro. This factor appears to be distinct from the dioxin receptor, which is observed only in nuclear extract from treated cells. In conclusion, these data suggest that a certain protein-DNA architecture may be maintained at the response elements at different stages of gene expression.Transcription of the cytochrome P-450IA1 gene is dramatically induced in response to the environmental contaminant dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) or related compounds. The induction process is mediated by the intracellular dioxin receptor protein and initiated by binding of the inducing chemical to the receptor, which in its non-ligandoccupied state most probably is located in the cytoplasmic compartment of target cells (for a review, see reference 29). In the absence of ligand, the cytosolic dioxin receptor is recovered as a latent, non-DNA-binding species (30). However, upon exposure to ligand in vivo, the dioxin-receptor complex undergoes an activation process involving a poorly understood structural alteration that enables it to translocate to the nucleus (29). In vitro, the ligand-activated dioxin receptor has been shown to represent a DNA-binding protein (references 5 and 47 and references therein) which specifically recognizes dioxin-responsive positive control elements (xenobiotic response elements [XREs]) found in the promoter and upstream regions of the cytochrome P-450IA1 and glutathione S-transferase Ya genes (9,16,17,21,31,33 and 48) in that (i) both categories of receptors directly transduce an extracellular signal to the target transcription units they regulate and (ii) the dioxin and steroid receptors require ligand for function.A striking property of the dioxin receptor is that the ligand strictly controls the activation of the receptor from a latent species to a DNA-binding form also under cell-free conditions (7,17,30). In the case of steroid hormone receptors, the glucocorticoid response element of the rat tyrosine aminotransferase gene is protected against methylation by dimethyl sulfate in hepatoma cells only ...

“…Moreover, CYPlAl is an essential enzyme in the detoxification of chemicals responsible for activation of the CYPIAI gene, and paradoxically, the enzyme also potentiates promutagens and procarcinogens by converting them into reactive intermediates (cf. references 53 and 60 and references therein from the transcriptional initiation site (14,19,26,28,40,55,56). As a consequence of gene activation, the rates of CYPlAl mRNA and protein synthesis are increased 20-to 100-fold (for a review, see reference 54).…”

mentioning

confidence: 99%

Dioxin-dependent activation of murine Cyp1a-1 gene transcription requires protein kinase C-dependent phosphorylation.

Carrier¹,

Owens²,

Nebert³

et al. 1992

146

Transcriptional activation of the murine Cypla-l (cytochrome P1450) 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 CYPlAl mRNA induction by TCDD as well as the concomitant increase in CYPlAl 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 Cypla-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 CYPlAl mRNA induction by TCDD. Depletion of protein kinase C by prolonged treatment with phorbol ester led to the complete suppression of CYPlAl mRNA induction by TCDD. We conclude that (i) phosphorylation is necessary for the formation of a transcriptional complex and for transcriptional activation of the Cypla-) 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.The mammalian CYPJAl gene encodes an enzyme expressed endogenously during cell division, embryogenesis, and differentiation, suggesting that this gene as well as certain other P450 genes might be involved in distinct critical life processes (51,53). The CYPlAl enzyme also plays an important role in the detoxification of numerous polycyclic aromatic hydrocarbons, including benzo[a]pyrene, a major component in the production of cigarette smoke and other combustion processes. Moreover, CYPlAl is an essential enzyme in the detoxification of chemicals responsible for activation of the CYPIAI gene, and paradoxically, the enzyme also potentiates promutagens and procarcinogens by converting them into reactive intermediates (cf. references 53 and 60 and references therein from the transcriptional initiation site (14,19,26,28,40,55,56). As a consequence of gene activation, the rates of CYPlAl mRNA and protein synthesis are increased 20-to 100-fold (for a review, see reference 54). Posttrans...