Abstract
BackgroundMammalian hepatic lipase (HL) genes are transcribed almost exclusively in hepatocytes. The basis for this liver-restricted expression is not completely understood. We hypothesized that the responsible cis-acting elements are conserved among mammalian HL genes. To identify these elements, we made a genomic comparison of 30 kb of 5'-flanking region of the rat, mouse, rhesus monkey, and human HL genes. The in silico data were verified by promoter-reporter assays in transfected hepatoma HepG2 and non-hepatoma HeLa cells using serial 5'-deletions of the rat HL (-2287/+9) and human HL (-685/+13) promoter region.ResultsHighly conserved elements were present at the proximal promoter region, and at 14 and 22 kb upstream of the transcriptional start site. Both of these upstream elements increased transcriptional activity of the human HL (-685/+13) promoter region 2–3 fold. Within the proximal HL promoter region, conserved clusters of transcription factor binding sites (TFBS) were identified at -240/-200 (module A), -80/-40 (module B), and -25/+5 (module C) by the rVista software. In HepG2 cells, modules B and C, but not module A, were important for basal transcription. Module B contains putative binding sites for hepatocyte nuclear factors HNF1α. In the presence of module B, transcription from the minimal HL promoter was increased 1.5–2 fold in HepG2 cells, but inhibited 2–4 fold in HeLa cells.ConclusionOur data demonstrate that searching for conserved non-coding sequences by comparative genomics is a valuable tool in identifying candidate enhancer elements. With this approach, we found two putative enhancer elements in the far upstream region of the HL gene. In addition, we obtained evidence that the -80/-40 region of the HL gene is responsible for enhanced HL promoter activity in hepatoma cells, and for silencing HL promoter activity in non-liver cells.
Highlights
Mammalian hepatic lipase (HL) genes are transcribed almost exclusively in hepatocytes
Regulation of gene expression is achieved through the complex interaction of transcription factors, which bind to specific DNA sequence motifs
Interspecies comparison of genomic HL sequences Of the mammalian HL genes, genome sequence including part of the 5'-flanking region is available for human, chimpanzee, rhesus monkey, rat, mouse and hedgehog (Ensembl e!42:Dec 2006)[26]
Summary
Mammalian hepatic lipase (HL) genes are transcribed almost exclusively in hepatocytes. Regulation of gene expression is achieved through the complex interaction of transcription factors, which bind to specific DNA sequence motifs. These motifs are predominantly located in the upstream region of genes. Transcription factors that are potentially involved in the regulation of a particular gene are usually identified by the presence of the specific DNA binding motif in the upstream regulatory region. These binding motifs are compiled in libraries such as the Transfac database [1], and programs such as MatInspector enable pattern recognition with the entries in this database [2]. We tested the validity of this approach to identify functional TFBS for the mammalian hepatic lipase genes, by comparing the in silico data with experimental promoter-reporter assays
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