Characterization of the DNA-binding properties of the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin

Abstract

The DNA-binding properties of the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were investigated using chromatography on DNA-cellulose columns. A maximal binding of about 40% of the total receptor complex to DNA-cellulose was observed. In order to interact with DNA, the receptor must first bind TCDD. A heat-activation step followed by gel permeation chromatography using Sephadex G-25 increased the binding of the cytosolic receptor to DNA. The DNA-binding ability of the receptor was almost lost following mild proteolysis using trypsin or a-chymotrypsin, although these treatments did not reduce its ligand binding capacity and had no apparent effect on its size. Furthermore, pre-treatment of the DNA-cellulose column with an intercalating drug, ethidium bromide, resulted in inhibition of the binding of the TCDD-receptor complex to DNA, indicating that not only electrostatic interactions but also the configuration of DNA are of importance in receptor-DNA interactions.In analogy to the endocrine model of gene regulation by steroid hormones, several lines of evidence indicate that intracellular, soluble receptor proteins are essential mediators of the biological responses produced by certain xenobiotics, i. e. halogenated or non-halogenated aromatic hydrocarbons One particularly striking and often-studied biochemical effect produced by TCDD and related compounds is their potency to induce specific isozymes of cytochrome P-450 and the associated monooxygenase activity aryl hydrocarbon hydroxylase [l , 41 together with several non-monooxygenase activities, e. g. UDP-glucuronyltransferase, NAD(P)H dehydrogenase (quinone) (formerly called DT-diaphorase) and ornithine decarboxylase [l]. Several studies have demonstrated that administration of TCDD or TCDD-like inducers leads to a large increase in translatable mRNAs encoding the TCDDinduced isozyme(s) of cytochrome P-450 [5 -71. By in vitro nuclear transcription studies, this process has been shown to reflect an increased rate of transcription of the specific cytochrome P-450 gene(s) [8-lo]. In analogy to the 'twostep' model proposed for the mechanism of action of steroid Correspondence to J.-A. Gustafsson, Institutionen for Medicinsk Naringslara, Huddinge Sjukhus, F69, S-141 86 Huddinge, SwedenAbbreviations. TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDBF, 2,3,7,8-tetrachlorodibenzofuran; Azso -310 unit, the amount of material giving an absorbance at 280nm of 1 more than the absorbance at 310 nm in a 1 -cm pathlength cell; DCC, dextran-coated charcoal.Enzymes (IUB Recommendations 1984). Aryl hydrocarbon hydroxylase (EC 1.14.14.1); a-chymotrypsin (EC hormones [ l l , 121, an accumulation of TCDD-receptor complexes in the cell nucleus following occupation of cytosolic receptor sites has been reported by several authors [13 -171. This classical 'two-step' model has been questioned, however, and some recent reports [18, 191 suggest that unoccupied steroid hormone receptors as well as unoccupied TCDD receptors in the intact cell are contained in the nucleus, and ...

Genetic and Molecular Aspects of 2,3,7,8-Tetra-Chlorodibenzo-P-Dioxin Action

“…The binding of TCDD to the Ah receptor is associated with the accumula tion of the liganded receptor in the nucleus in vivo and with the acquisition of DNA-binding ability in vitro (76)(77)(78)(79)(80). These observations imply that ligand binding converts the receptor to a DNA-binding protein, a process that has been termed "transformation."…”

Specific protein-DNA interactions at a xenobiotic-responsive element: copurification of dioxin receptor and DNA-binding activity.

“…First, the nuclear factor has a high affinity for XREs only after treatment of mouse hepatoma Hepa lclc7 cells with TCDD or TCDF, two well-known inducers of cytochrome P-450c and ligands of the dioxin receptor. Similarly, pretreatment of target cells with cytochrome P450c-inducing compounds is necessary for nuclear translocation of the dioxin receptor (16); more significantly, DNA binding in vitro of the dioxin receptor, as assessed by retention of the receptor on calf thymus DNA-cellulose, is a ligand-dependent event (21). In the case of steroid hormone receptors, the hormone is required for induction of a DNA-binding form of the glucocorticoid receptor both in vivo (22) and in vitro (23).…”

2,3,7,8‐Tetrachlorodibenzo‐p‐dioxin receptors regulate transcription of the cytochrome P1‐450 gene

“…Subsequent studies have demonstrated that the hydrodynamic properties of TCDD receptors and their apparent mechanism of signal transduction are similar to those of steroid receptors [20,21]. For example, both TCDD and steroid receptors (1) are acidic proteins with salt-dissociated monomeric molecular weights around 90,000-120,ooO [see 20,21 for a more thorough discussion of hydrodynamic properties] ; (2) bind DNA-cellulose and heparin-Sepharose [2 I] ; (3) have distinct ligand-binding and DNA-binding domains as demonstrated by limited proteolysis [21,22]; (4) exist in the absence of ligand; as soluble intracellular proteins with a low affinity for the cell nucleus [23,24]; (5) undergo ligandinduced transformation to a state with high affinity for the nucleus [23,25-271; and (6) act at the level of transcription to induce the expression of a target gene [28-301. TCDD receptors differ from steroid receptors in their ligand-binding domain, because steroids do not bind TCDD receptors, nor does TCDD bind to steroid receptors 131,311. Also, genetic analyses of TCDD receptor function using cell fusion reveal that receptor-defective variant cells compose at least two complementation groups .…”

“…This response requires both the formation of TCDD-receptor complexes and an interaction between the inducerreceptor complex and the cell nucleus, because transcriptional activation does not occur in receptor-defective variant cells [29]. Furthermore, the TCDD-receptor complex is a DNA-binding protein [22]. By analogy with the mechanism of action of steroid-receptor complexes [30], it appeared that the TCDD-receptor complex might activate gene expression by interacting with a genomic regulatory element located upstream of the transcriptional promoter for the cytochrome P1-450 gene.…”

“…The binding of TCDD to its receptor increases the affinity of the receptor for the cell nucleus and DNA [22,25]. The activation of cytochrome P1450 gene expression requires both the DRE and functional TCDD-receptor complexes [38,39].…”