We have sequenced the complete human ABCA1 gene including 1153bp of the promoter, 150,000 bp of introns and exons and 1 kb of the 3' flanking region. The ABCA1 gene spans 149 kb and consist of 50 exons interrupted by 49 introns. Twenty-two alu repeats were identified in introns 1-49 of the ABCA1 gene. Analysis of the 1153 bp of the ABCA1 gene promoter reveals a TATA box and potential binding sites for transcription factors SP1, AP1, AP2, AP4, NF-kB as well as 3 E-Box motifs. Binding sites for potential transcription factors involved in monocyte/macrohpage differentiation and liver-specific expression including STAT c-myb, GATA and HNF3b were also present. To determine the effect of cholesterol on ABCA1 gene expression we generated constructs containing the luciferase reporter cDNA under the control of the human ABCA1 promoter fragments -990, -473, -295, -200 and -80 bp relative to the transcription start site and transfected them in Hepa1-6, RAW and human embryonal kidney 293 cells. The proximal 990 bp sequence of the human ABCA1 promoter induced a 3-fold increase in the expression of the luciferase reporter gene in all 3 cell types after cholesterol loading. Gel shift analysis of a -110 bp fragment of the ABCA1 promoter that confers cholesterol regulation demonstrated that binding the proteins from THP and Hela cell nuclear extracts was competed by DNA fragments 20 bp or greater containing intact AP1 binding sequences. Summary: 1) the complete human ABCA1 gene has been sequenced and consists of 50 exons and 49 introns spanning 149 kb; 2) Analysis of 1153 bp of the human ABCA1 promoter reveals multiple potential binding motifs for transcription factors involved in monocyte/macrohpage differentiation and liver specific gene expression; 3) A regulatory element that confers cholesterol regulation in all 3 cell types is present within the proximal 990 bp of the human ABCA1 promoter; and 4) The proximal 200 bp of the ABCA1 promoter contains motifs that are involved in cholesterol modulation of ABCA1 gene expression.
