Transcriptional regulation of the human lipoprotein lipase gene in 3T3-L1 adipocytes.
Lipoprotein lipase (LPL), a key enzyme in normal lipoprotein metabolism, has a complex pattern of regulation and tissue-specific expression. Several potential binding sites for transcription factors, including the recognition sequences for CCAAT/enhancer-binding protein and octamer-binding proteins (Oct) have been described in the 5'-flanking region of the human LPL gene. To identify elements which regulate the expression of LPL in adipocytes, plasmids containing deletion mutants of the 5'-LPL promoter region and the luciferase reporter gene were transfected in 3T3-L1 adipocytes. Deletions at -724, -565, -461, -368, -232, -167, -92, -35, and -17 relative to the transcriptional start site modified transcription from 100 to 162, 194, 185, 128, 63, 53, 29, and 0%, respectively, indicating the presence of negative (-724 to -565) and positive (-368 to -35) cis-acting regulatory elements. Transfection of HepG2 cells, which do not synthesize LPL, with the same constructs resulted in a similar pattern of expression for the majority of the deletions. However, deletions between -724 and -368 base pairs resulted in a 75-100% increase in transcription in 3T3 adipocytes but not in HepG2 cells, indicating the presence of tissue-specific regulatory element(s) in this region. An important regulatory element affecting LPL transcription in adipocytes was identified by gel mobility shift assays and DNase I footprint analysis. Using these techniques, a nuclear protein(s) in 3T3-L1 adipocytes was shown to bind specifically to a fragment which included the proximal octamer recognition site (from -46 to -39) present in the LPL promoter. The DNA-protein complex comigrates with an electrophoretic band containing the Oct-1-DNA complex in BJA-B nuclear extracts and the DNA-protein complex was selectively competed only by DNA fragments containing the octamer sequence. Preincubation of 3T3-L1 nuclear extracts with an antibody directed against the POU domain of Oct-1 inhibited the formation of the DNA-protein complex. Deletion of the proximal octanucleotide motif from the plasmid containing the -461 fragment of the LPL promoter, resulted in a 79 and 76% decrease in the level of expression in transfected 3T3-L1 adipocytes and HepG2 hepatocytes, respectively. These combined results have established that the expression of LPL in adipocytes is modulated by multiple positive and negative regulatory elements within the 5'-flanking region of the LPL gene. A proximal octamer binding sequence which specifically interacts with a nuclear protein(s) that exhibits the characteristics of Oct-1 has been identified.(ABSTRACT TRUNCATED AT 400 WORDS)
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Molecular Disease Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Parrott
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Santamarina-Fojo
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Brewer
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D016475:3T3 Cells; D000273:Adipose Tissue / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007700:Kinetics; D008071:Lipoprotein Lipase / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic; D014162:Transfection
D004247:DNA; D008071:Lipoprotein Lipase