Functional characterization of the human prion protein promoter in neuronal and endothelial cells

Abstract

Human prion diseases such as Creutzfeld-Jakob disease and kuru are of major medical and biological importance because of their fatal course, epidemic potential, and unique pathophysiology. Endogenous expression of the normal cellular prion protein (PrP^C) is necessary for infection and prion replication. However, knowledge of human PrP^C gene regulation is rudimentary. We therefore cloned1543 bp of the 5′ untranslated and promoter region of the PrP gene. Using transient transfection assays, the full-length promoter and serial deletion mutants subcloned in a luciferase reporter vector were analyzed in neuronal (KELLY) and endothelial (EA.hy926) cell lines, which both express PrP^C as shown by RT/PCR. Analysis of promoter constructs in KELLY cells indicated two activating regions at –131/–284 and –1303/–1543, relative to the 3′-terminal end of exon 1, and also two repressing elements at –254/–567 and –567/–909 in neuronal cells. In EA.hy926 cells, activating elements were identified at –131/–284 and –284/–567, and one repressing region was localized at –567/–909. In addition, transcriptional start sites were determined by 5′-RACE reaction and RNase protection assay, revealing one major transcriptional start site located at –47 (in KELLY cells), –53 (in human thalamus) and at about –55 (in EA.hy926 cells).