Complete genomic sequence of the human ABCA1 gene: analysis of the human and mouse ATP-binding cassette A promoter

Abstract

The ABCA1 gene, a member of the ATP-binding cassette A (ABCA1) transporter superfamily, encodes a membrane protein that facilitates the cellular efflux of cholesterol and phospholipids. Mutations in ABCA1 lead to familial high density lipoprotein deficiency and Tangier disease. We report the complete human ABCA1 gene sequence, including 1,453 bp of the promoter, 146,581 bp of introns and exons, and 1 kb of the 3' flanking region. The ABCA1 gene spans 149 kb and comprises 50 exons. Sixty-two repetitive Alu sequences were identified in introns 1-49. The transcription start site is 315 bp upstream of a newly identified initiation methionine codon and encodes an ORF of 6,783 bp. Thus, the ABCA1 protein is comprised of 2,261 aa. Analysis of the 1,453 bp 5' upstream of the transcriptional start site reveals multiple binding sites for transcription factors with roles in lipid metabolism. Comparative analysis of the mouse and human ABCA1 promoter sequences identified specific regulatory elements, which are evolutionarily conserved. The human ABCA1 promoter fragment -200 to -80 bp that contains binding motifs for SP1, SP3, E-box, and AP1 modulates cellular cholesterol and cAMP regulation of ABCA1 gene expression. These combined findings provide insights into ABCA1-mediated regulation of cellular cholesterol metabolism and will facilitate the identification of new pharmacologic agents for the treatment of atherosclerosis in humans.

Figure 1

The genomic organization of the human ABCA1 gene is illustrated. The location of the 50 exons, two STS markers, and Alu repetitive elements present within the ABCA1 introns are shown.

Figure 2

Comparison of the translation start sites and 5′ UTR of the human ABCA1, mouse ABCA1A, and human ABCR genes. The transcription start site (G⁺¹) is boxed. The three stop codons upstream of the newly identified translation start site at position +315 bp (boxed) are underlined. The previously identified translation start site at position +495 bp (boxed) is also shown. The location of the highly conserved purine residues (−3) and G (+4) relative to both ATG start sites are indicated. The putative 45-aa signal peptide is underlined.

Figure 3

Comparison of the mouse and human ABCA1 promoter sequence. blast2 software was used to search for homologous sequences (highlighted in gray) in the 5′ flanking regions of the mouse and human ABCA1 genes. The proposed transcription start site is indicated (G⁺¹). Exon 1 is boxed.

Figure 4

The sequence of the human ABCA1 promoter is illustrated. The location of some potential binding sites for transcription factors identified by both matinspector (core similarity >1 and matrix similarity > 0.87) and motif programs is shown. Other motifs also identified by using the above criteria but not shown include: USF, myc-max, ARNT, myoD, E47, EVI1, MZF1, LMO2COM, and Nkx2.5. A potential TATA box is boxed. The transcription start site (G) identified by 5′ RACE is indicated as +1. Motifs that are conserved between the mouse and human sequences are highlighted in gray. Highly conserved motifs also are bolded.

Figure 5

Cholesterol and cAMP stimulation of the ABC1 promoter. Luciferase/β-galactosidase activity relative to the −990 bp to +120 bp fragment (F) is shown. Gel mobility shift assays demonstrates competition with 100× molar excess of fragment F, E-box (−147 to −142 bp), and SREBP-2 antibody (Santa Cruz Biotechnology). Data represent four independent studies. *, P < 0.05.