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

Hepatic cell-specific expression of the human apolipoprotein B (apoB) ene I s controlled by at least four cis-acting I elements located within the -128 to +122 promoter region (S.S. Chuang, H.K. Das, Identification of trans-ac ting factors that interact with cia-acting elements present in the first no ntranslated 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 negat ive element (+20 to +40) and a strong positive element (+33 to +53) are loc ated in the first non-translated exon of the human apolipoprotein B gene. T rans-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 ex periments 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 mutagenesi s of the downstream positive element +43 to +53 and transfection of the mut ant promoter constructs in HepG2 cells reveal that nucleotide G at position +51 is essential for the strong positive activity of the element +43 to +5 3. 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 v ivo, of nucleotide G to either A or T at position +51 in the downstream pos itive promoter element +43 to +53 may potentially cause hypobetalipoprotein emia, a heterozygous form of an autosomal-dominant disorder. (C) 1999 Elsev ier Science B.V. All rights reserved.