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- Comparative genomics and experimental promoter analysis reveal functional liver-specific elements in mammalian hepatic lipase genes

Understanding transcriptional regulation of gene expression is a major challenge in molecular biology. In eukaryotes, 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. Over the last decades, numerous transcription factors have been identified, each with its own specific DNA binding sequence (TFBS). 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]. Unfortunately, most transcription factors bind to short, degenerate sequences, which occur very frequently in the eukaryotic genome. Only a very small fraction of all predicted binding sites is biologically relevant [3]. Recently, new strategies for the ab initio identification of functionally significant cis-acting regulatory sequences have been developed, based on the assumption that regulatory elements are conserved among multiple species [4-8], and that multiple TFBS tend to specifically cluster together [9,10]. The rVista computational tool for identification of functional regulatory elements combines the comparative sequence analysis of orthologous genes with the analysis of clustering of predicted TFBS [11,12]. In this study, 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.

Hepatic lipases (HL) are synthesized and secreted almost exclusively by hepatocytes [13-15]. Although synthesis of HL has been shown to occur in mouse adrenals [16], and in mouse and human macrophages [17], this is negligible compared to expression in liver. The HL activity present in adrenals and ovaries [18] originates predominantly from liver, and is transported through the circulation to these organs [19,20]. In liver, the enzyme is bound to cell surface proteoglycans within the sinusoids, from where it can be released by heparin. Hepatic lipase plays an important role in plasma lipoprotein metabolism and intracellular lipid homeostasis [21], by mediating cholesterol influx into liver cells from high-density lipoproteins (HDL), and clearance of remnant lipoproteins from the circulation by the liver. HL is an important determinant of plasma HDL cholesterol levels, and is implicated in the protection against development of premature atherosclerosis by HDL [21]. HL gene expression in humans and rodents is regulated by various hormones and nutritional states mainly at the transcriptional level, but up- or downregulation is limited to about two-fold [15]. In contrast to this moderate regulation by hormones and nutrition, the almost complete restriction of HL gene expression to differentiated liver cells is highly conspicuous [13,14]. Several groups have pointed to the HNF1 and HNF4α binding sites in the proximal promoter of the HL gene to explain this liver-specificity in humans [22-25]. Since the liver-restricted expression is a common feature of most, if not all, mammalian HL genes, we hypothesize that the regulatory elements responsible for liver-specific expression are conserved among mammals. We therefore searched the upstream regulatory region of the rat, mouse, rhesus monkey and human genes for the presence of conserved clusters of TFBS motifs, and combined the in silico data with experimental promoter-reporter assays in cultured cells of hepatic versus non-hepatic origin. This unbiased approach led to the identification of two putative enhancer elements in the far upstream region, and of highly conserved sequence modules within the proximal promoter of the HL genes.

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