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).

The Extracellular Regulated Kinase-1 (ERK1) Controls Regulated α-Secretase-mediated Processing, Promoter Transactivation, and mRNA Levels of the Cellular Prion Protein

“…Measurements of PrP c Promoter Transactivation-The 1543-bp 5Ј promoter region of the human PrP c gene or serial 5Ј-truncated constructs (1303-, 909-, 567-, 284-, and 131-bp constructs) were subcloned into the luciferase reporter vector pGL3 basic and used to measure PrP c promoter transactivation, as extensively described (25). Cells grown in 12-well plates were co-transfected with full-length or mutant PrP c promoterluciferase, ␤-galactosidase (to normalize transfection efficiencies), and the indicated cDNAs with Lipofectamine (HEK293 cells) or with the Amaxa Nucleofector TM kit (mouse embryonic fibroblasts).…”

“…10A). We first examined whether these two putative ERK1/2-regulated sites indeed behaved as PrP c transcription activators by means of deletion analysis of the 5Ј PrP c promoter region (25). Deletion of the Sp1 binding site does not affect luciferase activity (compare Ϫ1543 and Ϫ1303 constructs; Fig.…”

p53-Dependent Transcriptional Control of Cellular Prion by Presenilins

“…However, little is known about the molecular pathways underlying these regulatory events. The functional characterization of the human prion promoter (Funke-Kaiser et al, 2001) identified two regulatory regions where sequence analysis revealed consensus sequences for AP-1, Sp1, and Sp2 factors (Mahal et al, 2001;Bellingham et al, 2009). Our in silico analysis of the human PRioN protein (PRNP) gene promoter also revealed a motif partly matching the binding sequence targeted by the oncogene p53.…”

“…Molecular cloning of the human PRNP gene promoter facilitated the understanding of the transcriptional regulation of PrP c (Funke-Kaiser et al, 2001;Mahal et al, 2001). Thus, the 5Ј-flanking region of PRNP revealed several putative binding sites for transcription factors including Sp1, AP1, AP2, c-REL, and Nkx2-5, suggesting stimulus-dependent and cellspecific mechanisms of transcriptional regulation.…”

Ovine PRNP untranslated region and promoter haplotype diversity

“…Other studies have previously identified TFBSs that are potentially associated with PRNP gene regulation in sheep, cattle, humans and rats (Saeki et al, 1996;Inoue et al, 1997;Baybutt & Manson, 1997;Hills et al, 2001;Funke-Kaiser et al, 2001;McCormack et al, 2002;Sander et al, 2004Sander et al, , 2005Premzl et al, 2005). Using Biobase MATCH software to search the TRANSFAC database (Matys et al, 2003), nine TFBSs were identified that were gained or lost in the promoter haplotypes (Supplementary Table S5).…”