Functional identification of the promoter of the gene encoding the Rhesus monkey beta-amyloid precursor protein.
Misregulation of the transcription of the beta-amyloid precursor protein (betaAPP) gene is implicated in the pathogenesis of Alzheimer's disease (AD). Here we characterize the 5'-flanking region, the first exon and intron of the betaAPP gene of the Rhesus monkey (rhbetaAPP). For functional analysis, transient transfection in PC12 cells was performed with a series of 5'-deletion constructs (fused with a reporter gene), that extended as far upstream as -7900 down to -1bp. Chloramphenicol acetyltransferase/promoter fusion assays showed that both -7900/+104 and -75/+104-bp regions possessed strong promoter activity. However, -2542/+104bp had the strongest promoter activity, whereas -204/+104bp showed a major reduction in activity and -47/+104bp showed almost a complete loss of activity. A region from -75 to +104bp was essential for minimal basic promoter activity because mutation at the activating site of an upstream stimulator factor (USF) within this region abolished the promoter activity. The very upstream region (-5529/-3416bp) displayed a negative effect on promoter activity. Two blocks of the sequence, 641bp (-1131 /-490) and 105bp (-309/-204), acted as positive regulators for promoter activity. Another 61-bp block (-204/-143) acted as a negative regulator. Gel shift assay indicated that the -249-242-bp region contains a binding domain for the AP-2 transcription factor. No second promoter or bidirectional promoter was observed. A region spanning the first exon and part of the first intron (+99 to +6800bp) acted as a negative regulator. These results suggest that a region of -75 to +104bp, which contains the pyrimidine-rich initiator element, the 5'-untranslated region and the binding site for USF, constitute the minimal promoter element and that interactions between multiple positive and negative elements, the USF and initiator element are crucial for transcription of the TATA-less betaAPP promoter.
Song
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Program in Medical Neurobiology, Institute of Psychiatric Research, Department of Psychiatry, 791 Union Drive, Indianapolis, IN 46202, USA.
Lahiri
D K
DK
D016564:Amyloid beta-Protein Precursor / Q000096:biosynthesis / Q000235:genetics*; D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D007438:Introns; D008253:Macaca mulatta / Q000235:genetics*; D016716:PC12 Cells; D010957:Plasmids; D011401:Promoter Regions, Genetic; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D017384:Sequence Deletion; D014158:Transcription, Genetic; D014162:Transfection
D016564:Amyloid beta-Protein Precursor; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase