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

Abstract

Upon binding of 2,3,7,8-tetrachlorodibenzop-dioxin (called dioxin or TCDD), the dioxin receptor exhibits increased affinity for the cell nucleus in vivo and for DNA in vitro. To derme the recognition sequence of the dioxin receptor and its relationship with that of the glucocorticoid receptor, oligonucleotides derived from dioxin-responsive elements of the rat cytochrome P-450c gene were tested for their ability to form specific protein-DNA complexes in a gel retardation assay. We found that a previously defined sequence motif that is similar to the glucocorticoid-responsive element and exhibits strong enhancer activity in response to dioxin receptor ligands bound a dioxin-inducible factor with high specificity but was not recognized by the DNA-binding domain of the glucocorticoid receptor. Binding to this element was only observed in nuclear extracts of wild-type mouse hepatoma cells in a time-and dose-dependent manner and not in nuclear extracts from a nonresponsive mutant cell line deficient in DNA binding of the dioxin receptor. The specific DNA-binding activity in wild-type nuclear extracts comigrated in a Superose size-exclusion column and cosedimented on sucrose gradients with the in vivo labeled dioxin receptor. These experiments strongly suggest that the dioxin receptor is a sequence-specific DNA-binding protein and is not only biochemically but also functionally similar to the steroid receptor family.The effect of dioxin on specific cytochrome P-450c gene transcription is mediated by an intracellular receptor protein to which dioxin and related compounds bind with high affinity and selectivity (1,2). In analogy to the mechanism of action of steroid hormones, binding of dioxin to its receptor induces an increased affinity ofthe receptor for nuclear target sites in vivo and for nonspecific DNA in vitro (ref. 3 and references therein). Several biochemical properties of the dioxin receptor are similar to those of the glucocorticoid receptor (refs. 4 and 5). However, the dioxin and glucocorticoid receptors do not appear to share any common ligandbinding specificity (6), and the endogenous ligand for the dioxin receptor, if any, has not yet been identified. Although steroid hormone receptors are known to activate gene expression by binding to specific hormone-dependent enhancers, little is known about the function of the dioxin receptor. Attempts to determine whether the dioxin receptor binds directly to specific D1A sequences have been hampered by difficulties in purifying the receptor (7). While this work was in progress, dioxin-inducible protein-DNA interactions with an unidentified sequence motif in the 5' flank of the murine cytochrome P1-450 gene were reported (8).In the rat cytochrome P-450c gene, two classes of sequence elements have been defined by deletion analysis to mediate dioxin induction of gene expression. The "drug regulatory elements" (DREs; ref. 9) exhibit a rather weak enhancer activity, whereas the second class of elements, referred to as "xenobiotic-responsive elements" (XREs),...

Glycine N -Methyltransferase Tumor Susceptibility Gene in the Benzo( a )pyrene-Detoxification Pathway

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“…The low (Ϫ9.10 kcal/mol) binding energy between the dimeric form of GNMT and BaP suggests that BaP may displace (Table 2). Accordingly, several GNMT amino acid residues (including Thr 37 (Fig. 5C).…”

Cloning and functional characterization of the bile acid-sensitive methotrexate carrier from rat liver cells

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The MurineIL-2Promoter Contains Distal Regulatory Elements Responsive to the Ah Receptor, a Member of the Evolutionarily Conserved bHLH-PAS Transcription Factor Family

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