Specific Factors Binding to the Late E2A Promoter Region of Adenovirus Type 2 DNA: No Apparent Effects of 5′-CCGG-3′ Methylation

In the regulation of eukaryotic gene expression, the interactions of several protein factors with specific signals in the promoter sequence can play a decisive role. In addition, the methylation of specific promoter sequences causes the long-term inactivation of eukaryotic promoters. The function of the regulatory signal 5-methylcytidine is complex and can be overcome by a number of factors, e.g., by the E1 proteins of adenoviruses. We studied the inactivation of the late E2A promoter of adenovirus type 2 (Ad2) DNA by the methylation of three 5′-CCGG-3′ sequences at positions −215, +5, and +23, relative to one of the cap sites of this promoter. One would like to understand the mechanisms by which 5-methylcytidine residues are capable of interfering with regulatory functions in the late E2A promoter of Ad2. We have identified six different promoter sequences by DNase I protection analyses, and have shown that these sites bind specifically to host proteins (binding sites I-VI). Binding of these factors to unmethylated or 5′-CCGG-3′ methylated late E2A promoter sequences was compared by gel migration delay assay or DNase I protection (footprinting) analyses. Protein binding does not appear to be affected by late E2A promoter methylation. Even after partial purification of some of these factors by chromatography on heparin–Sepharose, differences in binding to the unmethylated and the 5′-CCGG-3′ methylated promoter were not observed. Even though striking differences in host factor binding were not detectable at late E2A promoter sites, it is conceivable that the functionality of promoter-bound host proteins is altered when the three 5′-CCGG-3′ sequences are methylated.