title: Analysis of the 5'-flanking region of the beta-amyloid precursor protein gene that contributes to increased promoter activity in differentiated neuronal cells.
abstract: To study the transcription control of the beta-amyloid precursor protein (betaAPP) in Alzheimer's disease (AD), we functionally characterized the betaAPP gene promoter in differentiated cells. PC12 cells were first differentiated with nerve growth factor (NGF) and then transient transfection analysis was done with a series of 5'-deletion constructs, that extended as far upstream as -7900 down to +104 base pair (bp) relative to the transcription start site (+1). The truncated regions of the promoter were linked upstream to a reporter gene, chloramphenicol acetyl transferase (CAT). The CAT assay was performed to compare promoter activity of different 5'-flanking and intronic regions of the betaAPP gene. Our results suggest that the longest (-7900/+104) and one of the shortest (-47/+104) regions possessed significantly higher levels of promoter activity than the promoterless vector in NGF-differentiated PC12 cells. A deletion of about 7600 bp region from the -7900 to +104 construct resulted in 50% loss of original promoter activity. A deletion of all but 47 bp from the -7900 to +104 construct resulted in the loss of 66% (and retention of 34%) promoter activity. The region -3416/+104 bp displayed the strongest promoter activity whereas +1/+104 bp showed the least activity among all deletion constructs studied. The upstream region -5529 to -3416 contains a negative regulatory element and -3416 to -1131 contains a positive regulatory element. The very upstream region, -7900 to -3411, lacks independent functional activity. The 5'-UTR region (+1 to +104) showed minimum activity and the -75 to +104 region constitutes the basic promoter element. The first exon or a large part of the first intron (+99 to +6200) did not display any significant promoter activity. Thus, several positive and negative regulatory elements influence the basal level of betaAPP promoter activity in NGF-differentiated PC12 cells. We speculate that any structural alteration(s) due to a specific mutation in these regulatory regions can potentially alter the transcriptional machinery, and that can perhaps affect the level of beta-amyloid protein involved in AD.
authors.affiliation: Laboratory of Molecular Neurogenetics, Institute of Psychiatric Research, Department of Psychiatry, 791 Union Drive, Room No.: Pr-313, Indiana University School of Medicine, Indianapolis, IN 46202-4887, USA. dlahiri@iupui.edu
authors.forename: D K
authors.initials: DK
authors.lastname: Lahiri
authors.forename: W
authors.initials: W
authors.lastname: Song1
authors.forename: Y W
authors.initials: YW
authors.lastname: Ge
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D016564:Amyloid beta-Protein Precursor / Q000235:genetics*; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D005091:Exons / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D007438:Introns / Q000235:genetics; D020932:Nerve Growth Factor / Q000494:pharmacology; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D016716:PC12 Cells; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D020218:Response Elements / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D014162:Transfection
chemical_list: D020121:5' Untranslated Regions; D016564:Amyloid beta-Protein Precursor; D020932:Nerve Growth Factor