Interference with protein binding at AP2 sites by sequence‐specific methylation in the late E2A promoter of adenovirus type 2 DNA

Abstract

The in vitro methylation of the +6, +24, and -215 located 5'-CCGG-3' sequences in the late E2A promoter of adenovirus type 2 (Ad2) DNA abrogates promoter function. 5-Methyldeoxycytidine (5-mC) at positions +6 and +24 in both or either of the two DNA complements in the late E2A promoter abolishes the formation of a high-molecular-mass DNA-protein complex that is essential for promoter function. The formation of this complex can be competed for by an oligodeoxyribonucleotide with a consensus AP2 sequence, but not by AP1, AP3, or CREB sequences. The AP2 sites comprise the +6 and +24 located 5'-CCGG-3' sequences in the late E2A promoter; the AP1, AP3 and CREB sequences are in their immediate vicinity. Methylation of either the +6 or the +24 5' -CCGG-3' sequence also compromises formation of the DNA-protein complex. A 40 nucleotide pair oligodeoxyribonucleotide encompassing the -215 5' -CCGG-3' site in the late E2A promoter can also form DNA-protein complexes which is not affected by the introduction of a 5-mC residue in the -215 position. The data suggest that the AP2 protein together with other proteins is involved in the generation of a transcription-activating complex with the late E2A promoter of Ad2 DNA, and the formation of this complex is completely abolished when both the +6 and +24 5'-CCGG-3' sequences are methylated.