Michael S. Denison

Species-specific binding of transformed Ah receptor to a dioxin responsive transcriptional enhancer

The Ah receptor (AhR) mediates many, if not all, of the toxic and biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related halogenated aromatic hydrocarbons. Although wide variations in species sensitivity to these compounds have been observed, numerous biochemical and physiochemical characteristics of the AhR appear similar among species. We have examined the ability of cytosolic AhR, from a variety of species (rat, rabbit, guinea pig, hamster, mouse, cow, sheep, fish, chicken and human), to transform and bind to its cognate DNA recognition sequence, the dioxin responsive enhancer (DRE), to evaluate the importance of these events in species variations in TCDD responsiveness. Gel retardation analysis using a murine DRE oligonucleotide has revealed that cytosolic AhR from a wide variety of species can transform in vitro and bind to the DRE and demonstrates that all of the factors necessary for AhR transformation and DNA binding are present in cytosol. In addition, DNA-binding analysis using a series of mutant DRE oligonucleotides has indicated no apparent species- or ligand-dependent, nucleotide-specific difference in AhR binding to the DRE. These studies support a highly conserved nature of the DRE and AhR (at least in DNA binding) and imply that a sequence closely related to the murine consensus DRE sequence is responsible for conferring AhR-dependent, TCDD responsiveness in each of these species.

Characterization of the interaction of transformed rat hepatic cytosolic Ah receptor with a dioxin responsive transcriptional enhancer

Many of the biological and toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), a highly toxic environmental contaminant, are mediated by a soluble intracellular protein (the Ah receptor (AhR)). Following a poorly defined process of transformation, during which the TCDD:AhR complex acquires the ability to bind to DNA with high affinity, TCDD:AhR complexes activate gene transcription by binding to dioxin responsive enhancers (DREs) adjacent to the responsive gene. Here we have utilized gel retardation analysis to study the interaction of rat hepatic cytosolic TCDD:AhR complexes, transformed in vitro, with dioxin responsive enhancer DNA. Cytosol contains a protein(s) that binds to the DRE in a TCDD-inducible, sequence-specific, time- and temperature-dependent manner and exhibits AhR ligand binding specificity. These results imply that this inducible protein-DNA complex represents the binding of liganded:AhR complex to the DRE. The TCDD:AhR complex bound to the DRE with an equilibrium dissociation constant of 1.2 +/- 0.1 nM, an affinity at least 3800-fold stronger than that for binding to nonspecific DNA. Assuming one DNA binding site per AhR molecule, the total concentration of transformed AhR in these studies was approximately 56.1 +/- 6.6 fmol/mg protein (representing transformation of 45% of the total amount of AhR present in the same cytosolic preparations). Inhibition of AhR transformation, but not ligand or DNA binding, by EDTA and EGTA suggests that a chelatable divalent cation(s) may play a critical role in the transformation process.

The binding of transformed aromatic hydrocarbon (Ah) receptor to its DNA recognition site is not affected by metal depletion.

The biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), a potent environmental contaminant, are mediated by a soluble intracellular protein, the aromatic hydrocarbon (Ah) receptor (AhR). TCDD:AhR complexes activate gene transcription by binding to specific DNA sequences termed dioxin-responsive elements adjacent to TCDD-responsive genes. Analogies between the AhR and receptors for steroid hormones imply similarities in their mechanism of action. The presence of chelatable, protein-bound metal(s), presumably zinc, is required for DNA binding of several proteins, including steroid hormone receptors and the transcription factor SP1. Utilizing gel retardation and DNA-cellulose binding assays we have investigated the importance of metal in DNA binding of transformed TCDD:AhR complexes. Here, we report that although 1,10-phenanthroline, a metal ion chelating agent, inhibited the DNA binding of SP1 and transformed glucocorticoid receptor, no inhibition of transformed AhR was observed. EDTA was similarly ineffective in inhibiting DNA binding of transformed AhR. Our findings suggest that the AhR, although similar to steroid receptors, appears not to require metals for binding to its specific DNA recognition sequence.

The hepatic Ah receptor for 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin : species differences in subunit dissociation

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin) produces many of its biological effects by binding to a soluble, intracellular protein (the Ah receptor (AhR]. The hepatic AhR, from a variety of species, is present in low salt cytosol as a form which sediments at 8-10 S. High salt (0.4 M KCL) dissociates the rat, guinea pig, and rabbit cytosolic TCDD:AhR complex to a form which sediments at 5-6 S. In contrast, high salt conditions failed to dissociate the 8-10 S TCDD:AhR complex present in any of the mouse strains studied. Incubation of cytosol with heparin resulted in a shift of the [3H]TCDD:AhR complex to a smaller sedimenting form in all species. Mouse TCDD:AhR complex sedimented at 8-10 S when cytosol was simultaneously incubated with high salt and heparin, indicating that the interaction of heparin with the AhR was electrostatic in nature. Incubation of heparin-dissociated mouse TCDD:AhR complex (5-6 S) with high salt resulted in reassociation of AhR to a form which sediments at 8-10 S. Our data suggests that the resistance of mouse AhR to salt-mediated dissociation may be due to a property of the receptor protein itself and also indicates that mouse hepatic cytosolic AhR is distinctly different from that present in all other species examined to date.

Nuclear Ah receptor from mouse hepatoma cells: Effect of partial proteolysis on relative molecular mass and DNA-binding properties☆

The nuclear Ah receptor from mouse hepatoma (Hepa-1c1c9) cells is a 176-kDa multimeric protein which is stable under conditions of up to 1 M KCl. Under denaturing conditions, the Hepa-1 nuclear receptor can be dissociated into a ligand-binding subunit of Mr approximately 91,000. The identity of subunits that compose the nuclear Ah receptor is currently unknown. We used partial proteolysis under nondenaturing conditions as an approach to study the domain organization of the nuclear form of Ah receptor from Hepa-1c1c9 cells treated with [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in culture. Low concentrations of trypsin (0.5 microgram/mg nuclear protein) generated heterogeneous fragments with the main fragment having a Stokes radius (Rs) approximately 6 nm. More discrete ligand-binding fragments of Mr approximately 84,000 (Rs approximately 4 nm/approximately 5 S) and Mr approximately 16,000 (Rs approximately 2 nm/approximately 2 S) could be generated using higher concentrations of trypsin (5 micrograms/mg nuclear protein). The relative concentration of the 84 and 16-kDa fragment was dependent on duration of protease treatment; formation of the 16-kDa fragment was accompanied by some loss in [3H]TCDD binding. Treatment of nuclear Ah receptor with alpha-chymotrypsin (1 microgram/mg nuclear protein) generated a single, apparently homogeneous ligand-binding fragment of Mr approximately 101,000 (Rs approximately 5 nm/approximately 5 S). When analyzed by DNA-cellulose chromatography, the chymotryptic fragment eluted at a significantly higher KCl concentration (462 mM) compared to native untreated nuclear Ah receptor (385 mM). Despite this increased affinity for DNA-cellulose columns, the ligand-binding fragment generated by chymotrypsin treatment was unable to interact with a dioxin responsive element in a gel retardation assay. DNA-cellulose binding ability, therefore, does not appear to be a reliable indicator of specific DNA interactions for these protease-modified fragments.

DNA sequence-specific binding of transformed ah receptor to a dioxin responsive enhancer: Looks aren't everything

Abstract Gel retardation analysis has been utilized to examine the ability of transformed hepatic cytosolic TCDD:AhR complex to bind to its specific DNA recognition site, the dioxin responsive element (DRE). Although DRE sequence alignment has identified a conserved DNA recognition sequence of C/GNNNC/GTNGCGTGNC/GT/ANNNC/G, site-directed mutagenesis has demonstrated that not all of these bases are important for DNA binding. A putative TCDD:AhR DRE-binding consensus of GCGTGNNA/TNNNG/C has been derived from our studies and it is highly conserved among species. In addition, we present a model for the DNA-binding of transformed TCDD:AhR complex.