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Characterization of the human ABCG1 gene: liver X receptor activates an internal promoter that produces a novel transcript encoding an alternative form of the protein.
Journal of Biological Chemistry
The human ABCG1 gene encodes a member of the ATP-binding cassette (ABC) superfamily of transporter proteins and is highly induced when macrophages are incubated with oxysterols. Using mRNA from oxysterol-treated human THP-1 cells together with 5'-rapid amplification of cDNA ends and polymerase chain reaction, we identified a novel ABCG1 transcript that encodes a putative protein of 786 residues containing a new amino terminus of 203 amino acids. Characterization of the genomic organization and structure of the human ABCG1 gene demonstrates that: (i) the gene consists of 23 exons spanning 98 kilobase pairs (kb) on chromosome 21q22.3, (ii) the 203 amino acids are encoded on three previously unidentified exons, 8-10, and (iii) a promoter, containing a TATA box and two liver X receptor (LXR) alpha response elements (LXREs), is located upstream of exon 8. Northern analysis using exon-specific probes confirms that oxysterol treatment results in >10-fold induction of ABCG1 transcripts that are derived from either exons 8-23 or exons 5, 7, and 11-23. Electromobility shift assays demonstrate that LXRalpha and retinoid X receptor alpha bind to the two LXREs in intron 7. Cells were transiently transfected with reporter luciferase constructs under the control of either (i) 9 kb of genomic DNA corresponding to intron 7 and part of exon 8 and containing either wild-type or mutant LXREs or (ii) two copies of the wild-type or mutant LXRE. In all cases, the wild-type construct was regulated in an LXR- and oxysterol-dependent manner, and this regulation was attenuated when the LXREs were mutated. In conclusion, the human ABCG1 gene contains multiple promoters, spans more than 98 kb and comprises 23 exons that give rise to alternative transcripts encoding proteins with different amino-terminal sequences. Elucidation of the various roles of different ABCG1 isoforms will be important for our understanding of mammalian cholesterol homeostasis.
ATP-Binding Cassette Transporters/biosynthesis, ATP-Binding Cassette Transporters/chemistry, Algorithms, Amino Acid Sequence, Animals, Base Sequence, Blotting, Northern, Cell Line, Cholesterol/biosynthesis, Cholesterol/chemistry, Chromosomes, Human, Pair 21, DNA, Complementary/metabolism, DNA-Binding Proteins, Dimerization, Enzyme Activation, Exons, Gene Expression Regulation, Genes, Reporter, Humans, Luciferases/metabolism, Macrophages/metabolism, Models, Genetic, Molecular Sequence Data, Orphan Nuclear Receptors, Polymerase Chain Reaction, Promoter Regions, Genetic, Protein Binding, Protein Isoforms, Protein Structure, Tertiary, RNA, Messenger/metabolism, Receptors, Cytoplasmic and Nuclear, Receptors, Retinoic Acid/metabolism, Receptors, Steroid/metabolism, Receptors, Thyroid Hormone/metabolism, Reverse Transcriptase Polymerase Chain Reaction, Sequence Analysis, DNA, Transfection
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