Sequence-specific methylation in a downstream region of the late E2A promoter of adenovirus type 2 DNA prevents protein binding.

In studies on the promoter-inhibitory effect of sequence-specific DNA methylations, the late E2A promoter of human adenovirus type 2 (Ad2) has been used as an experimental tool. Upon the in vitro methylation of 5'-CCGG-3' (HpaII) sequences at nucleotides +24, +6 and -215 relative to nucleotide + 1, the site of transcriptional initiation, promoter inhibition or inactivation has been demonstrated in transient expression tests in Xenopus laevis oocytes (Langner et al., 1984), in mammalian cells (Langner et al., 1986), after the genomic fixation of the promoter in conjunction with a reporter gene in mammalian cells (Müller & Doerfler, 1987), and in a cell-free transcription system employing nuclear extracts of human HeLa cells (Dobrzanski et al., 1988). Possible explanations for the inhibitory effect of three 5-methyldeoxycytidine (5-mC) residues in a promoter sequence are structural alterations in DNA or the positive or negative modulation of the sequence-specific binding of proteins. This modulation could be indirect at the level of protein-protein interactions. A synthetic oligodeoxyribonucleotide of 50 base-pairs (bp) or a restriction endonuclease fragment of 73 bp in length, which comprised the +24 and +6 5'-CCGG-3' sequences of the late E2A promoter, were methylated or hemimethylated at these two sites, or were left unmethylated and were subsequently incubated with a partly purified (heparin-Sepharose) nuclear extract of human HeLa cells. Protein binding was monitored by electrophoretic migration delay of the 32P-labeled 50 bp oligodeoxyribonucleotide or the 73 bp fragment on polyacrylamide gels. The formation of one of the DNA-protein complexes in this analysis was compromised when 5'-CCGG-3' methylated oligodeoxyribonucleotides were used in the binding assays. Similar results were obtained when the 50 bp oligodeoxyribonucleotide was hemimethylated in either complement.The formation of the same complex could also be obliterated by adding the same non-methylated oligodeoxyribonucleotide as competitor to the reaction mixture. The methylated oligodeoxyribonucleotide did not act as a competitor, nor did a randomly composed oligodeoxyribonucleotide of identical length. The results show that protein binding is abolished by methylation of those sequences in the late E2A promoter whose methylation inhibits promoter function. The abrogation of protein binding has been observed with a 50 bp or 73 bp fragment. With a 99 bp or a 377 bp fragment, binding differences between the unmethylated and the 5'-CCGG-3' methylated late E2A promoter are not apparent.