Isolation, sequencing, and functional analysis of the TATA-less human ATPase II promoter

Abstract

Multiple lines of evidence indicate that the P-type Mg 2+-ATPase, termed ATPase II, could play an important role in apoptosis. With the long-term objective of studying the regulation of this protein during apoptosis, we delineated the exon–intron organization of the human ATPase II gene (within chromosome 4). Subsequently, we used RNA ligase-mediated rapid amplification of cDNA ends to identify a major transcription start site at position −143 with respect to the translation start site. Luciferase reporter analysis of a 1.2-kb 5′-flanking sequence (−1222 to +94 with respect to the transcription start site) revealed strong promoter activity in three human cell lines, human oligodendroglioma (HOG), SHSY5Y (hybrid neuroblastoma), and EA.hy926 (endothelial cell line). Serial deletions from the 5′ end of this sequence up to nucleotide −291 yielded some decrease in activity only in the EA.hy926 cells. Further deletion to −217 caused a drastic decrease in activity in all three cell lines, but a −148 fragment showed preferential reduction in activity in the EA.hy926 cells. The promoter activity was nearly equal in two sequence variants of the promoter, one of which (designated as Variant 2) contained a 15-bp direct repeat within a GC-rich region. Additionally, there were several single base-pair changes from the sequence reported by the human genome project. Despite the presence of enhancer/repressor elements, such as Sp1 and NFκB, relatively small differences in promoter activity were observed in the three cell lines. However, it is likely that such sequence elements could cause major regulation of promoter activity in cells subjected to conditions that trigger apoptosis. The ATPase II promoter sequence will provide valuable clues to the regulation and role of the ATPase II protein.