Analysis of the 5'-flanking region of the β-amyloid precursor protein gene that contributes to increased promoter activity in differentiated neuronal cells

Debomoy Lahiri, Weihong Song, Yuan Wen Ge

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

To study the transcription control of the β-amyloid precursor protein (βAPP) in Alzheimer's disease (AD), we functionally characterized the βAPP 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 βAPP 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 βAPP 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 β-amyloid protein involved in AD. (C) 2000 Elsevier Science B.V.

Original languageEnglish
Pages (from-to)185-198
Number of pages14
JournalMolecular Brain Research
Volume77
Issue number2
DOIs
StatePublished - May 5 2000

Fingerprint

Amyloid beta-Protein Precursor
5' Flanking Region
Base Pairing
PC12 Cells
Nerve Growth Factor
Genes
Chloramphenicol
Transferases
Alzheimer Disease
Amyloidogenic Proteins
Nucleic Acid Regulatory Sequences
5' Untranslated Regions
Transcription Initiation Site
Reporter Genes
Genetic Promoter Regions
Introns
Transfection
Exons
Mutation

Keywords

  • β-Amyloid precursor protein
  • Alzheimer's disease
  • Gene regulation
  • Nerve growth factor
  • Neuronal differentiation
  • PC12 cells
  • Promoter
  • Transfection

ASJC Scopus subject areas

  • Molecular Biology
  • Cellular and Molecular Neuroscience

Cite this

Analysis of the 5'-flanking region of the β-amyloid precursor protein gene that contributes to increased promoter activity in differentiated neuronal cells. / Lahiri, Debomoy; Song, Weihong; Ge, Yuan Wen.

In: Molecular Brain Research, Vol. 77, No. 2, 05.05.2000, p. 185-198.

Research output: Contribution to journalArticle

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N2 - To study the transcription control of the β-amyloid precursor protein (βAPP) in Alzheimer's disease (AD), we functionally characterized the βAPP 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 βAPP 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 βAPP 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 β-amyloid protein involved in AD. (C) 2000 Elsevier Science B.V.

AB - To study the transcription control of the β-amyloid precursor protein (βAPP) in Alzheimer's disease (AD), we functionally characterized the βAPP 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 βAPP 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 βAPP 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 β-amyloid protein involved in AD. (C) 2000 Elsevier Science B.V.

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