A nuclear factor-binding domain in the 5'-untranslated region of the amyloid precursor protein promoter: implications for the regulation of gene expression.

BACKGROUND: The extracellular deposition of aggregated amyloid beta-protein is a neuropathological manifestation of Alzheimer disease and Down syndrome. The Amyloid beta-protein is derived from a group of larger differentially spliced proteins, the amyloid protein precursors (APP). Data suggests that the level of APP gene expression could contribute to the pathological processes leading to amyloid depositions. FINDINGS: The 5' untranslated region (UTR) of the APP gene, encompassing 147 base pairs between the transcriptional (+1) and the translational start site, was examined for its role in APP expression. Deletions close to the transcriptional start site reduced expression from the APP promoter in part by transcriptional mechanisms. However, deletions between position +50 and +104 had no effect on transcriptional activity while significantly reducing overall expression from the promoter. A nuclear factor-binding domain designated as DAPB was identified between position +72 and +115 of the 5'-APP-UTR. The binding-recognition sequence was localized between position +96 and +105. The same mutations that eliminated factor-binding also reduced expression from the APP promoter while having no effect on in vitro transcription or the RNA levels transcribed from transfected constructs. CONCLUSIONS: A nuclear factor-binding domain designated as DAPB was identified in the 5'-UTR of the APP gene. Elimination of factor-binding correlated with an overall decline in expression from the APP promoter while in vitro transcription and the total amount of in vivo transcribed RNA remained unaffected. This suggests that the binding-factor may have a function in post-transcriptional regulation, including nuclear export of mRNA.

Two adjacent nuclear factor-binding domains activate expression from the human PRNP promoter.

BACKGROUND: The transmissible spongiform encephalopathies (TSEs) comprise a group of fatal degenerative neurological diseases in humans and other mammals. After infection, the cellular prion protein isoform PrPC is converted to the pathological PrPSC scrapie isoform. The continued conversion of PrPC to PrPSC requires de novo endogenous PrP synthesis for disease progression. The human prion protein gene (PRNP) promoter was therefore investigated to identify regulatory elements that could serve as targets for therapeutic intervention. FINDINGS: The human prion protein gene (PRNP) promoter from position -1593 to +134 relative to the putative transcriptional start site (+1) was analyzed by transient transfection in HeLa cells. Deletions from the 5' end between positions -1593 and -232 yielded little change in activity. A further 5' deletion at position -90 resulted in a decline in activity to a level of about 30% of the full-length value. DNase I footprinting of the region between positions -259 and +2 identified two adjacent protected domains designated as prpA (-116 to -143) and prpB (-147 to -186). Internal deletions combined with mobility shift electrophoresis and methylation interference assays indicated the presence of sequence specific nuclear factor complexes that bind to the prpA and prpB domains and activate expression from the human PRNP promoter in an additive fashion. CONCLUSION: Results from transient transfection, DNase I footprinting, mobility shift electrophoresis, and methylation interference experiments suggest that two DNase I protected domains designated as prpA and prpB are binding sites for as yet unidentified regulatory factors that independently activate expression from the PRNP promoter.