Comparison of the promoters of the mouse (APEX) and human (APE) apurinic endonuclease genes

Abstract

We investigated the minimal promoter of APEX, which encodes mouse apurinic DNA repair endonuclease. A 1.85-kb fragment with APEX upstream sequences and ∼290 bp of the transcribed region linked to a chloramphenicol acetyltransferase (CAT) reporter gene was assayed by transient transfection in NIH-3T3 cells. The minimal APEX promoter was comprised of ∼190 bp of upstream and ∼170 bp of transcribed DNA (exon 1 and most of intron 1). This ∼360-bp region contains two CCAAT boxes and other consensus protein binding sites, but no TATA box. Deletion of the 5′-most CCAAT box decreased activity ∼5-fold. The second CCAAT box (situated in exon 1) may play an independent role in APEX expression. Transcription start sites have been identified downstream of the second CCAAT box, and DNase I footprinting demonstrated NIH-3T3 nuclear proteins binding this region, including an Sp1 site located between the CCAAT boxes. Electrophoretic mobility-shift assays indicated binding by purified Sp1. Mouse proteins did not bind three myc-like (USF) sites in the APEX promoter, in contrast to the APE promoter. The APEX and APE promoter had similar activity in Hela cells, but in mouse cells, the murine promoter had ∼5-fold higher activity than did the human promoter. Both the APEX and APE promoters exhibited bidirectional activity in their cognate cells.