Promoter function of the human ap-endonuclease

The cDNA of the major apurinic/apyrimidinic endonuclease (APE), named APE-1, has been cloned, not only on the basis of its nuclease activity in base excision repair pathway but also because of its role as an activator of AP-1 transcription factor and as a represser of parathyroid hormone (PTH) gene. APE-1 was shown to bind two negative Ca²⁺-dependent response elements (nCaRE) in the PTH promoter. In a systematic effort to characterize regulatory elements in the APE-1 promoter, we constructed a series of expression plasmids in which the luciferase coding sequence is fused to overlapping segments of the APE-1 promoter. Transient expression studies of luciferase in electroporated human cells showed -10-fold lower expression of the reporter from plasmids containing a 3.9 kb promoter than that from a plasmid in which a 2.0 kb upstream fragment was deleted. This result suggested the presence of a negative regulatory sequence in the upstream region. A more detailed deletion analysis of the reporter plasmid narrowed the activity to a 800 bp region that is needed for repression of luciferase. Sequencing of this DNA region reveals the presence of an Alu sequence and two nCaRE sequences. Because APE-1 binds to nCARE, the possibility of its feedback autoregulation is being investigated.