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 non-translated 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 from of an autosomal-dominant disorder.
authors.affiliation: Department of Pharmacology, University of North Texas Health Science Center at Fort Worth 76107, USA.
authors.forename: S S
authors.initials: SS
authors.lastname: Chuang
authors.forename: H K
authors.initials: HK
authors.lastname: Das
mesh_terms: D001055:Apolipoproteins B / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA / Q000235:genetics; D005091:Exons; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D006995:Hypobetalipoproteinemias / Q000235:genetics; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic; D014162:Transfection