Aryl hydrocarbon (Ah) receptor DNA-binding activity. Sequence specificity and Zn2+ requirement

Abstract

The aryl hydrocarbon (Ah) receptor, also called the xenobiotic or TCDD receptor, mediates transcriptional activation of the cytochrome P-450c (CYP1A1) gene by interacting with Ah or xenobiotic response elements. This paper presents evidence that a metal ion, probably Zn2+, is an essential cofactor for the Ah receptor. This paper also maps in detail the interactions between the Ah response element XRE1 and the Ah receptor from the rat hepatocyte-derived cell line LCS7. Interactions were mapped by three methods, 1) methylation interference footprinting, 2) mobility shift competition experiments, using a series of synthetic oligonucleotides with systematic alterations in the Ah response element core sequence, and 3) orthophenanthroline/Cu+ footprinting. These findings suggest the following consensus sequence for DNA recognition by the Ah receptor: CNA/TNA/TCACGCA/TA/T. The chelators 1,10-phenanthroline and oxalic acid inhibited the sequence-specific DNA-binding activity of the AH receptor in a concentration dependent manner, suggesting that the DNA-binding activity of the receptor requires divalent metal ions. Inhibition was due to metal-chelation, since: 1) inhibition was almost completely prevented by the presence of Zn2+, or other divalent metal ions having high affinity for the chelators used, while metal ions with low affinity did not protect; 2) the DNA-binding activity of the receptor could be restored by dialysis to remove 1,10-phenanthroline, but only in the presence of Zn2+, while dialysis in the absence of metal ions reversed inhibition by the nonchelating isomer 4,7-phenanthroline. The involvement of a divalent cation in receptor function, possibly bound via sulfhydryls, was also suggested by the finding that Cd2+ and Co2+ inhibited DNA-binding activity. Once bound to the XRE1 DNA sequence, the receptor could not be inhibited by 1,10-phenanthroline, suggesting that the essential metal ion must become inaccessible to chelation when the receptor binds DNA. The Zn2+ requirement of the Ah receptor is similar to that of the estrogen and the glucocorticoid receptors and is consistent with the hypothesis that the Ah receptor is a member of the steroid and thyroid hormone receptor superfamily.