Guanine 6‐O‐Methylation Pattern within the Dioxin Responsive Element of the CYP1A1 Enhancer Shows Two Critical Guanines for AhR/ARNT Binding

Abstract

The core-recognition motif for TCDD-liganded AhR/ARNT complex of the dioxin-responsive element (DRE) contains four guanine residues, three on the antisense (5'-T(T)/(A)GCGTG-3') and one on the sense (5'-CACGC(A)/(T)A-3') strand. It has been reported that, in methylation-protection and methylation-interference assays, the TCDD-liganded AhR/ARNT contacts all four guanine residues. On the other hand, it is known that some anticancer drugs, and various environmental and workplace chemicals, including strongly human carcinogenic nitrosamines, lead to the highly miscoding 6-O-methylation of guanine. In the present study, we have investigated whether specific methylation of guanine at the 6-O-position interferes with the binding of TCDD-liganded AhR/ARNT complex to its recognition motif in the CYP1A1 enhancer, and how individual 6-O-methylated guanines contribute to this interference. We found that only two 6-O-methylguanine residues are critical: the closest to the 5'-end within the three-nucleotide sequence (5'-GTG-3'), identical to a half-site E-box element, on the antisense strand, and the only guanine on the sense strand. In contrast, the 6-O-methylguanine closest to the 5'-end (well) and the one closest to the 3'-end (to a lesser extent), both on the antisense strand, were still able to bind the TCDD-liganded AhR/ARNT complex. This shows that the 6-O-methylation of guanine in the core sequences of CYP1A1 enhancer interferes with the binding of the ligand-activated AhR/ARNT complex in a differentially selective manner; it fully impedes binding of this complex to DRE (the prerequisite of most of the toxic effects of TCDD) only when one of the two particular guanines is methylated at the 6-O-position.