Functional characterization of the 5' flanking region of the BACE gene: identification of a 91 bp fragment involved in basal level of BACE promoter expression.
Pathological characteristics of Alzheimer's disease (AD) include amyloid-beta (Abeta) plaques. Abeta is derived from the Abeta peptide precursor protein (APP) by gamma- and beta-secretases, the latter known as beta-site APP-cleaving enzyme 1 (BACE1, or herein BACE). We have also described potentially important regions in the promoter of BACE, which may regulate its activity (1). Herein, we have functionally dissected the regulatory regions within the BACE promoter into areas containing positive and negative regulatory elements. The 4.1 kb promoter region (-3765/+364, +1 being the transcription start site [TSS]) includes positive regulatory element in the -2975 to -2062 region flanked on either side by negative regulatory elements at -3764/-2975 and -2062/-1056. This is separated from the minimal promoter and 5' UTR by a neutral region of roughly 700 base pairs (bp). A 91 bp fragment (224/314) is the shortest region with a significant reporter gene activity and constitutes the minimal promoter element for BACE. Neuronal preference of the promoter was apparent in the 141 bp fragment (224/364) that contained the 91 bp fragment. Gel shift results also suggest the strongest signal of DNA-protein interaction with the 91 bp fragment in neuronal nuclear extracts. This interaction was strongly blocked by an activator protein (AP)2 binding oligomer. Super gel shift assays suggest that both the AP2-binding oligomer and the 91 bp fragment interfered with each other's binding capacity. An AP2 sequence is predicted to occur within the 91 bp fragment. We also found stimulating protein (SP)1-binding sites in this region of the promoter. Thus, functional and gel shift analysis indicate that the 91 bp fragment containing the AP2 and SP1 binding sites constitutes the core promoter region of BACE. Changes in the activity of this region could play an important role in regulating BACE activity in neurons.
Ge
Yuan-Wen
YW
Department of Psychiatry, Institute of Psychiatric Research, Indiana University School of Medicine, Indianapolis, Indiana, USA.
Maloney
Bryan
B
Sambamurti
Kumar
K
Lahiri
Debomoy K
DK
D024506:5' Flanking Region / Q000235:genetics*; D020121:5' Untranslated Regions / Q000235:genetics; D000544:Alzheimer Disease; D053829:Amyloid Precursor Protein Secretases; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D001483:Base Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D010450:Endopeptidases / Q000235:genetics*; D005786:Gene Expression Regulation / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D008957:Models, Genetic; D009687:Nuclear Proteins / Q000378:metabolism; D009928:Organ Specificity; D016716:PC12 Cells; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D020218:Response Elements / Q000235:genetics*; D017384:Sequence Deletion / Q000235:genetics; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism
D020121:5' Untranslated Regions; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; C509281:Bace1 protein, rat