Reactivation of the methylation-inhibited late E2A promoter of adenovirus type 2 by a strong enhancer of human cytomegalovirus

Abstract

Promoter inactivation by sequence-specific methylation was demonstrated by using a construct which contained the late E2A promoter of adenovirus type 2 (Ad2) DNA and the prokaryotic gene for chloramphenicol acetyltransferase (CAT) as indicator. After the in vitro methylation of 5′-CCGG-3′ sequences at positions −215, +6, and +24 relative to the cap site of the promoter, the construct was inactive upon transfection into mammalian cells. The same pAd2E2ALCAT construct was active in the unmethylated form. Promoter inactivation could be overcome when the strong immediate early enhancer of human cytomegalovirus DNA, which lacked 5′-CCGG-3′ sites, was inserted into the construct either in a position immediately antecedent to the promoter or in a location several thousand nucleotides remote from it. Reactivation of the 5′-CCGG-3′ methylated pAd2E2AL-CAT construct entailed initiation of transcription at the authentic cap site of the late E2A promoter and maintenance of methylation at least during the duration of the transient expression experiment. Reactivation of the methylated late E2A promoter had also been demonstrated by the trans-activating 289 amino acid protein which was encoded in the El A region of adenoviruses (B. Weisshaar et al., 1988, J. Mol. Biol. 202, 255–270). Thus there are several ways in which a methylated and silenced promoter can be reactivated in mammalian cells.