A single in vitro point mutation in the first non-translated exon silences transcription of the human apolipoprotein B gene in HepG2 cells

Samuel S. Chuang, Hriday Das

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Hepatic cell-specific expression of the human apolipoprotein B (apoB) gene is controlled by at least four cis-acting elements located within the - 128 to +122 promoter region (S.S. Chuang, H.K. Das, Identification of trans- acting factors that interact with cis-acting elements present in the first nontranslated exon of the human apolipoprotein B gene, Biochem. Biophys. Res. Commun. 220 (1996) 553-562). Two cis-acting positive elements (-104 to -85; - 84 to -60) are located upstream from the start of transcription. A negative element (+20 to +40) and a strong positive element (+43 to +53) are located in the first non-translated exon of the human apolipoprotein B gene. Trans- acting factors BRF-2, BRF-1, BRF-3, and BRF-4 interact with the above four cis-acting elements respectively. In this study, we examine the roles of the upstream positive elements -104 to -85 and -84 to -60 in modulating transcriptional regulation of the apoB gene by downstream elements +20 to +40 and +43 to +53. Using in vitro mutagenesis and transient transfection experiments in HepG2 cells, the cis-acting element -84 to -60 has been found to be absolutely necessary for the function of the upstream element -104 to - 85 and downstream elements +20 to +40 and +43 to +53. In vitro mutagenesis of the downstream positive element +43 to +53 and transfection of the mutant promoter constructs in HepG2 cells reveal that nucleotide G at position +51 is essential for the strong positive activity of the element +43 to +53. A single substitution point mutation of nucleotide G to either A or T at position +51 reduces apolipoprotein B gene transcription substantially in HepG2 cells. These results suggest that a single substitution mutation in vivo, of nucleotide G to either A or T at position +51 in the downstream positive promoter element +43 to +53 may potentially cause hypobetalipoproteinemia, a heterozygous form of an autosomal-dominant disorder.

Original languageEnglish
Pages (from-to)600-605
Number of pages6
JournalBiochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
Volume1436
Issue number3
DOIs
StatePublished - 4 Jan 1999

Fingerprint

Hep G2 Cells
Apolipoproteins B
Point Mutation
Exons
Trans-Activators
Nucleotides
Genes
Mutagenesis
Transfection
Hypobetalipoproteinemias
Genetic Promoter Regions
Hepatocytes
In Vitro Techniques
Mutation

Keywords

  • Apolipoprotein B gene
  • Downstream
  • Promoter activity
  • Transcription factor

Cite this

@article{58fffce626d84f00a6e2ed75f05bceff,
title = "A single in vitro point mutation in the first non-translated exon silences transcription of the human apolipoprotein B gene in HepG2 cells",
abstract = "Hepatic cell-specific expression of the human apolipoprotein B (apoB) gene is controlled by at least four cis-acting elements located within the - 128 to +122 promoter region (S.S. Chuang, H.K. Das, Identification of trans- acting factors that interact with cis-acting elements present in the first nontranslated exon of the human apolipoprotein B gene, Biochem. Biophys. Res. Commun. 220 (1996) 553-562). Two cis-acting positive elements (-104 to -85; - 84 to -60) are located upstream from the start of transcription. A negative element (+20 to +40) and a strong positive element (+43 to +53) are located in the first non-translated exon of the human apolipoprotein B gene. Trans- acting factors BRF-2, BRF-1, BRF-3, and BRF-4 interact with the above four cis-acting elements respectively. In this study, we examine the roles of the upstream positive elements -104 to -85 and -84 to -60 in modulating transcriptional regulation of the apoB gene by downstream elements +20 to +40 and +43 to +53. Using in vitro mutagenesis and transient transfection experiments in HepG2 cells, the cis-acting element -84 to -60 has been found to be absolutely necessary for the function of the upstream element -104 to - 85 and downstream elements +20 to +40 and +43 to +53. In vitro mutagenesis of the downstream positive element +43 to +53 and transfection of the mutant promoter constructs in HepG2 cells reveal that nucleotide G at position +51 is essential for the strong positive activity of the element +43 to +53. A single substitution point mutation of nucleotide G to either A or T at position +51 reduces apolipoprotein B gene transcription substantially in HepG2 cells. These results suggest that a single substitution mutation in vivo, of nucleotide G to either A or T at position +51 in the downstream positive promoter element +43 to +53 may potentially cause hypobetalipoproteinemia, a heterozygous form of an autosomal-dominant disorder.",
keywords = "Apolipoprotein B gene, Downstream, Promoter activity, Transcription factor",
author = "Chuang, {Samuel S.} and Hriday Das",
year = "1999",
month = "1",
day = "4",
doi = "10.1016/S0005-2760(98)00117-9",
language = "English",
volume = "1436",
pages = "600--605",
journal = "Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids",
issn = "1388-1981",
publisher = "Elsevier",
number = "3",

}

TY - JOUR

T1 - A single in vitro point mutation in the first non-translated exon silences transcription of the human apolipoprotein B gene in HepG2 cells

AU - Chuang, Samuel S.

AU - Das, Hriday

PY - 1999/1/4

Y1 - 1999/1/4

N2 - Hepatic cell-specific expression of the human apolipoprotein B (apoB) gene is controlled by at least four cis-acting elements located within the - 128 to +122 promoter region (S.S. Chuang, H.K. Das, Identification of trans- acting factors that interact with cis-acting elements present in the first nontranslated exon of the human apolipoprotein B gene, Biochem. Biophys. Res. Commun. 220 (1996) 553-562). Two cis-acting positive elements (-104 to -85; - 84 to -60) are located upstream from the start of transcription. A negative element (+20 to +40) and a strong positive element (+43 to +53) are located in the first non-translated exon of the human apolipoprotein B gene. Trans- acting factors BRF-2, BRF-1, BRF-3, and BRF-4 interact with the above four cis-acting elements respectively. In this study, we examine the roles of the upstream positive elements -104 to -85 and -84 to -60 in modulating transcriptional regulation of the apoB gene by downstream elements +20 to +40 and +43 to +53. Using in vitro mutagenesis and transient transfection experiments in HepG2 cells, the cis-acting element -84 to -60 has been found to be absolutely necessary for the function of the upstream element -104 to - 85 and downstream elements +20 to +40 and +43 to +53. In vitro mutagenesis of the downstream positive element +43 to +53 and transfection of the mutant promoter constructs in HepG2 cells reveal that nucleotide G at position +51 is essential for the strong positive activity of the element +43 to +53. A single substitution point mutation of nucleotide G to either A or T at position +51 reduces apolipoprotein B gene transcription substantially in HepG2 cells. These results suggest that a single substitution mutation in vivo, of nucleotide G to either A or T at position +51 in the downstream positive promoter element +43 to +53 may potentially cause hypobetalipoproteinemia, a heterozygous form of an autosomal-dominant disorder.

AB - Hepatic cell-specific expression of the human apolipoprotein B (apoB) gene is controlled by at least four cis-acting elements located within the - 128 to +122 promoter region (S.S. Chuang, H.K. Das, Identification of trans- acting factors that interact with cis-acting elements present in the first nontranslated exon of the human apolipoprotein B gene, Biochem. Biophys. Res. Commun. 220 (1996) 553-562). Two cis-acting positive elements (-104 to -85; - 84 to -60) are located upstream from the start of transcription. A negative element (+20 to +40) and a strong positive element (+43 to +53) are located in the first non-translated exon of the human apolipoprotein B gene. Trans- acting factors BRF-2, BRF-1, BRF-3, and BRF-4 interact with the above four cis-acting elements respectively. In this study, we examine the roles of the upstream positive elements -104 to -85 and -84 to -60 in modulating transcriptional regulation of the apoB gene by downstream elements +20 to +40 and +43 to +53. Using in vitro mutagenesis and transient transfection experiments in HepG2 cells, the cis-acting element -84 to -60 has been found to be absolutely necessary for the function of the upstream element -104 to - 85 and downstream elements +20 to +40 and +43 to +53. In vitro mutagenesis of the downstream positive element +43 to +53 and transfection of the mutant promoter constructs in HepG2 cells reveal that nucleotide G at position +51 is essential for the strong positive activity of the element +43 to +53. A single substitution point mutation of nucleotide G to either A or T at position +51 reduces apolipoprotein B gene transcription substantially in HepG2 cells. These results suggest that a single substitution mutation in vivo, of nucleotide G to either A or T at position +51 in the downstream positive promoter element +43 to +53 may potentially cause hypobetalipoproteinemia, a heterozygous form of an autosomal-dominant disorder.

KW - Apolipoprotein B gene

KW - Downstream

KW - Promoter activity

KW - Transcription factor

UR - http://www.scopus.com/inward/record.url?scp=0033521705&partnerID=8YFLogxK

U2 - 10.1016/S0005-2760(98)00117-9

DO - 10.1016/S0005-2760(98)00117-9

M3 - Article

C2 - 9989290

AN - SCOPUS:0033521705

VL - 1436

SP - 600

EP - 605

JO - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids

JF - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids

SN - 1388-1981

IS - 3

ER -