Binding of the Transcription Factor EBP-80 Mediates the Methylation Response of an Intracistemal A-Particle Long Terminal Repeat Promoter

Abstract

Intracistemal A-particle (IAP) expression in mouse cells has been correlated with hypomethylation of HhaI and HpaII sites in proviral long terminal repeats (LTRs). In a previous study, in vitro methylation of three HhaI sites in the U3 region of the LTR from the cloned genomic IAP element, MIA14, was shown to inhibit promoter activity in vivo. In this study, we found by site-directed mutagenesis that the two more downstream HhaI sites within this LTR were responsible for the methylation effects on promoter activity in vivo; methylation of the other (5′) HhaI site, which lies within a putative SP1 binding domain, did not affect promoter activity. Methylation of the HhaI sites also inhibited promoter activity of the LTR in a cell-free transcription system. Exonuclease III footprinting demonstrated methylation-induced changes in protein binding over the region encompassing the downstream HhaI site, designated the Enh2 domain. The protein that interacts specifically with this domain, EBP-80, was characterized in a previous study (M. Falzon and E. L. Kuff, J. Biol. Chem. 264:21915-21922, 1989). We show here that the presence of methylcytosine in the HhaI site within the Enh2 domain inhibited binding of EBP-80 in vitro. The methylated MIA14 LTR construct was much less responsive to added EBP-80 in an in vitro transcription system than was the unmethylated construct. These data suggest that CpG methylation within the Enh2 domain may exert its effect on transcription in vivo by altering the interaction between EBP-80 and its cognate DNA sequence.