In mouse hepatoma cells, the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, or dioxin) induces Cyp1A1 gene transcription, a process that requires two basic helix-loop-helix regulatory proteins, the aromatic hydrocarbon receptor (AhR) and the aromatic hydrocarbon receptor nuclear translocator (Arnt). We have used a ligation-mediated PCR technique to analyze dioxin-induced changes in protein-DNA interactions and chromatin structure of the Cyp1A1 enhancer-promoter in its native chromosomal setting. Dioxin-induced binding of the AhR/Arnt heteromer to enhancer chromatin is associated with a localized (about 200 bp) alteration in chromatin structure that is manifested by increased accessibility of the DNA; these changes probably reflect direct disruption of a nucleosome by AhR/Arnt. Dioxin induces analogous AhR/Arnt-dependent changes in chromatin structure and accessibility at the Cyp1A1 promoter. However, the changes at the promoter must occur by a different, more indirect mechanism, because they are induced from a distance and do not reflect a local effect of AhR/Arnt binding. Dose-response experiments indicate that the changes in chromatin structure at the enhancer and promoter are graded and mirror the graded induction of Cyp1A1 transcription by dioxin. We discuss these results in terms of a TCDD-induced shift in an equilibrium between nucleosomal and nonnucleosomal chromatin configurations.
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...
We used an in situ exonuclease III protection technique (C. Wu, Nature [London] 309:229, 1984) to analyze protein-DNA interactions at a dioxin-responsive enhancer. Our results imply that the 2,3,7,8-tetrachlorodibenzo-p-dioxin-receptor complex interacts with the dioxin-responsive enhancer to activate transcription of the cytochrome Pl-450 gene.Halogenated aromatic hydrocarbons are environmental contaminants that produce diverse biological effects (11,12). In mouse hepatoma cells, the prototypical compound, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) activates transcription of the cytochrome P1-450 gene (3,4,16). Studies of receptor-defective cells reveal that this response requires the formation of a TCDD-receptor complex and an interaction between the inducer-receptor complex and the nucleus (3, 4). A cis-acting dioxin-responsive element (DRE) located upstream of the cytochrome P1-450 gene mediates the action of the TCDD-receptor complex (2, 5, 7). The DRE contains two TCDD-responsive domains, which have properties typical of transcriptional enhancers and require TCDD-receptor complexes for their function (5-7). Here, we use an exonuclease protection technique (18,19) to show that both the DRE and the TCDD-receptor complex participate in the formation of a stable nucleoprotein complex within the cell nucleus. These results imply that the TCDDreceptor complex interacts with the DRE in vivo. Figure 1 shows the relevant restriction sites in the DNA upstream of the mouse cytochrome P1-450 gene. The hatched boxes indicate the position of the two DREs (8). To examine this region for resistance to exonuclease III (Exo III), nuclei were prepared by Dounce homogenization and centrifugation (1) and were digested with BamHI (Bethesda Research Laboratories, Inc.) (300 U/ml, 30 min, 30°C) to nick the DNA downstream of the proximal DRE. Next, chromatin (at a DNA concentration of about 1 mg/ml) was digested for 30 min with various concentrations of Exo III (BRL) as previously described (9). The BamHI-Exo IIIdigested DNA was purified by phenol-chloroform extraction, digested with Si nuclease (BRL) (4,000 U/ml, 15 min, 30°C) as previously described (18), and then digested to completion with HindIII (BRL). The repurified and ethanolprecipitated DNA was fractionated by agarose gel electrophoresis, transferred to a nylon membrane (15), and hybridized to a nick-translated (13) probe complementary to the 5' end of the HindIll-HindIII fragment. The 32P-labeled bands were detected by autoradiography, with Kodak XAR-5 film and an intensifying screen.The results in Fig. 2 reveal that BamHI digested the cytochrome P1-450 regulatory region in nuclei from TCDDinduced cells (Fig. 2B, lane b) to a greater extent than in nuclei from uninduced cells (Fig. 2A, Fig. 2A, lane b). However, digestion with Exo III (Fig. 2A, lanes c to g) failed to generate a smaller HindlIlExo III subband(s). In contrast, Exo III digestion of nuclei from TCDD-induced cells generated a 0.67-kilobase HindlIlExo III subband (Fig. 2B, lanes c to g)
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.