BMC Genomics 2007, 8:99 http://www.biomedcentral.com/1471-2164/8/99 Background Understanding transcriptional regulation of gene expres- sion is a major challenge in molecular biology. In eukary- otes, regulation of gene expression is achieved through the complex interaction of transcription factors, which bind to specific DNA sequence motifs. These motifs are pre- dominantly 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 data- base [1], and programs such as MatInspector enable pat- tern 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 pre- dicted binding sites is biologically relevant [3]. Recently, new strategies for the ab initio identification of function- ally significant cis-acting regulatory sequences have been developed, based on the assumption that regulatory ele- ments 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 compar- ing 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 sur- face 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 regu- lated 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 moder- ate regulation by hormones and nutrition, the almost complete restriction of HL gene expression to differenti- ated 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 regula- tory elements responsible for liver-specific expression are conserved among mammals. We therefore searched the upstream regulatory region of the rat, mouse, rhesus mon- key and human genes for the presence of conserved clus- ters 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. Results 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]. Pairwise alignment of the HL coding sequences shows the expected, high degree of sequence identity (Table 1). This high homology also extends into the 5'-UTR and upstream-regulatory region, with sequence identity ranging from 53 to 98 % over the proximal 0.9–1.4 kb. Multiple sequence alignment of 30- kb of the 5'-flanking region available for five HL genes (all except for the hedgehog, which known sequence is too short) was performed by the mVista web-tool (Fig. 1). The chimp and macaque sequences are highly homologous to the human sequence, as 95 % and 64 % of the 30-kb region showed at least 95 % sequence identity over a 100- bp window, respectively. Similarly, 5 % of the rat sequence showed this high degree of sequence identity with the orthologous mouse sequence. Because of the near-identity of the chimp to the human sequence, we only included the latter sequence in subsequent in silico analysis. The global genomic sequence comparison showed a particularly high conservation among the four genes immediately upstream of the transcriptional start site (P = 7*10-7). Three additional islands of highly signif- icant homology (P < 10-5) were identified further upstream. Conservation of a 475 bp element at -14 kb was even more significant (P = 4*10-11) than the proximal HL promoter region (Fig. 1). The element at -22 kb (P = 2*10- 6) contained a 173 bp sequence that was completely iden- tical among the three primate sequences. To test the potential enhancer function of two of the most conserved elements in the far upstream regulatory region, Page 2 of 13 (page number not for citation purposes)