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Studies on the biochemical mechanism of promoter inhibition or inactivation by sequence-specific promoter methylations necessitated the development of a cell-free transcription system that responded to in vitro promoter methylations. Such systems were hitherto not available. In nuclear extracts from HeLa cells, the activities of two adenovirus type 2 promoters in the nonmethylated and methylated forms were compared. The late E2A promoter in vitro methylated at three 5'-CCGG-3' (HpaII) sequences at nucleotides -215, +6, and +24, or the major late promoter in vitro methylated at nucleotide -52 in the 5'-CCGG-3' sequence or at nucleotide -13 in the 5'-GCGC-3' (HhaI) sequence exhibited strikingly lower activities than did the nonmethylated constructs or exhibited no activity at all. The designated nucleotide positions were calculated relative to the cap sites of the two promoters. Upon in vitro transcription, the methylation pattern of the E2A late promoter was shown to be stable. For the inhibitory effects by sequence-specific methylations to be elicited, circular templates had to be used, the DNA titers had to be at critical levels for each extract, and high protein concentrations had to be maintained. When a template mixture of the nonmethylated major late promoter and the 5'-CCGG-3' methylated late E2A promoter of adenovirus type 2 DNA was used, the major late promoter was active and the methylated late E2A promoter was inhibited or inactivated. Activity levels of the two different promoters could be assessed simultaneously in the same assay due to differences in lengths between the products of transcription from the late E2A and major late promoters. Thus inhibition in the cell-free system could be proven to be specific for the methylated promoter. We are currently pursuing the hypothesis that cellular factors are crucial in recognizing methylated promoters and somehow participate in their inactivation.

Inactivation by sequence-specific methylations of adenovirus promoters in a cell-free transcription system