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.