Species-specific polymorphism in the promoter of the apolipoprotein A-I gene: restoration of human transcriptional efficiency by substitution at positions -189, -144 and -48 bp.

Previous studies indicate that species-specific differences in apolipoprotein A-I (apo A-I) expression could be largely explained by cis-acting factors located within or near the 5' flanking region (-231 to +223 bp, where +1 is the start site of transcription). In the present studies, we have localized 7 sites within the (-231 to -15 bp) region of the African green monkey apo A-I gene that differ from the human apo A-I gene 5' flanking region. To identify which of the 7 polymorphic sites were essential for the species-specific differences in apo A-I gene expression, mutated promoter constructs were transfected into HepG2 cells and reporter gene expression was measured. Each of the 7 sites within a defined 5' flanking region of the human gene was individually mutated to the African green nucleotide sequence found at that position. Three of the sites (-189, -144 and -48) were found to raise the human apo A-I promoter activity to approx. 60-65% of the African green promoter. While double mutations (-144/-48 bp and -189/-144 bp), restored the human apo A-I promoter activity to 100% of that found with the African green monkey promoter. Additional studies revealed similar DNA: protein interactions with DNA probes from either human or African green monkey and HepG2 cell nuclear extract. In conclusion, these studies demonstrate that double and triple nucleotide substitutions within the human apo A-I promoter are sufficient to restore gene expression in HepG2 cells to levels seen with the African green monkey promoter. These data suggest that sites -189, -144 and -48 bp are involved in significantly altering the binding affinity of a nuclear factor determining the species-specific level of apo A-I gene transcription.