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 significant 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 identical among the three primate sequences.

To test the potential enhancer function of two of the most conserved elements in the far upstream regulatory region, promoter-reporter assays were performed with human HL promoter constructs in transiently transfected HepG2 hepatoma cells. The -14 kb element (679 bp) and the -22 kb element (387 bp) were inserted in the hHL-685Luc plasmid. As a control, we also tested the activity of the non-conserved -10 kb sequence (502 bp). The transcriptional activity of the human HL -685/+13 promoter region was increased 3- and 2-fold with the -14 kb and -22 kb elements inserted in the sense orientation, respectively (Fig. 2). In contrast, the -10 kb sequence slightly but significantly reduced HL promoter activity (n = 3; P < 0.05). Qualitatively similar effects were obtained when the elements were inserted in the anti-sense orientation (Fig. 2). Hence, the conserved sequences at -14 kb and -22 kb have moderate enhancer activity of the proximal HL promoter region.

Submission of 2-kb upstream sequence of the rat HL gene to the MatInspector software program (core similarity > 0.75; matrix similarity > 0.70) returned over 2000 potential TFBS, randomly distributed over the entire sequence. A similar number of sites was predicted for the orthologous mouse, human and rhesus macaque sequences. When we searched for clustered TFBS motifs that are conserved between the rat and human sequence, using the web-tool rVista, three separate modules were identified within the proximal promoter region (Fig. 3). Module A (-240 to -200 in the human sequence relative to the transcriptional start site), for which AP1, AP2, CAAT, COUP, C/EBP, HNF4α, PPAR and USF binding sites are predicted, corresponds to the DR1 site recently identified by Rufibach et al. 25. Module B (-80 to -40) potentially contains AP2, CAAT, C/EBP, HNF1, HNF4, PPAR and Sp1 sites, and corresponds to the previously characterized HNF1 site 222324. Module C (-25 to + 5), which may bind AP2, C/EPB, HNF4, PPAR and USF factors, contains the transcription start site preceded by a conserved pyrimidine-rich motif, and therefore likely represents the Inr involved in binding of the transcription initiation complex. These three modules were also found to be conserved among the human and mouse HL gene. The human-mouse comparison revealed an additional, conserved module (-295 to -265), with potential binding sites for AP2, C/EBP, HNF1, HNF4, PPAR and Sp1, and which partly overlaps the DR4-site recently described by Rufibach et al. 25. Similar results were obtained in pairwise comparisons between orthologous sequences of macaque and rat, and of macaque and mouse. Despite the high homology in the intervening sequence between modules A and B, the rVista program did not recognize conserved clusters of TFBS among the human, macaque, rat and mouse. Irrespective of which transcription factors actually bind to these sites, the results of the interspecies sequence comparison by rVista suggest that the three highly conserved sequence modules in the proximal HL promoter region are involved in common features of transcriptional regulation. This is further supported by the fact that these three modules correspond to distinct DNA footprints of the human HL sequence in rat liver 22 and human HepG2 cells 23.

To corroborate the in silico results, promoter-reporter assays were performed with promoter fragments of the rat HL gene in transiently transfected HepG2 cells. Plasmids were constructed with progressively 5'-deleted promoter fragments spanning the -2287/+9 region of the rat HL gene in front of the CAT reporter gene. Compared to the SV40 promoter, the rHL-2287 construct showed low CAT expression (Fig. 4). Upon deleting the 5'-end of the HL promoter fragments to position -1048, CAT expression became even lower, and was no longer significantly different from promoter-less pCAT-Basic, suggesting that there is weak enhancer activity between nucleotides -1697 and -2287. Further deletion to position -754 slightly increased promoter activity to levels significantly above background. Shortening the insert from -754 to -446 resulted in a 5-fold increase in promoter activity, suggesting the presence of a strong negative regulatory element in this region of the rat HL gene. CAT expression was not significantly affected by deleting the insert from -446 to -211. The presence of the weak enhancer element between -2287 and -1697, and the negative element between -754 and -446 corresponds to positive and negative elements in the human HL upstream regulatory region observed by Oka et al. 23. We assume, therefore, that both these elements are present in homologous parts of the rat and human gene. Indeed, the global alignment of the four species by mVista detected homology at these parts of the gene (Fig. 1), but homology did not exceed the 70 % over 100 bp mark used as threshold in this analysis. Apparently, potentially important elements may be missed due to the high stringency of the conservation rule in the mVista program.

To test the importance of the conserved sequence modules within the -220 to +9 region, further 5'-deletions in the rat HL promoter region were made (Fig. 5). Transcriptional activity of the rHL-127 construct, in which module A has been removed, was not significantly different from that of the rHL-446 or rHL-221 constructs. Similarly, removal of the highly conserved intervening sequence between modules A and B (rHL-86 and rHL-75) had no significant effect on CAT expression. In contrast, additional removal of most of module B in rHL-39 reduced transcriptional activity by approximately 60 %. With rHL-23, in which the remainder of module B as well as the putative TATA-box has been deleted, CAT expression decreased further. Despite absence of the TATA-box, CAT expression of the rHL-23 construct was significantly higher than of promoter-less pCAT-Basic, which may be due to residual promoter activity of module C.

Similar promoter-reporter assays were performed with the -685/+13 region of the orthologous human HL gene, except that the luciferase gene was used as reporter (Fig. 6). Luciferase activity of the hHL-306 construct was similar to hHL-685, whereas activity of the hHL-79 construct was slightly, but not significantly, higher. This is in line with the rat promoter data, which show little effect of module A, and of the intervening sequence between modules A and B, on basal transcriptional activity in HepG2 cells. The luciferase activity of the hHL-36 construct, in which entire module B has been removed, was only 25 % of the hHL-79 construct. The transcriptional activity of hHL-36, which contains a bona fide TATA box and entire module C, was 7-fold higher than background. This confirms that modules B and C are crucial for basal transcriptional activity in HepG2 cells, with module B being most important.

To test whether modules A and B are involved in liver-specific expression of the HL gene, we compared transcriptional activity of different rat HL promoter fragments in HepG2 cells with non-hepatic HeLa cells (Fig. 7). Promoter activity in each cell line was expressed as percentage of that of the rHL-39 construct, because this fragment represents the minimal promoter with the TATA-box and transcription start site. In the hepatoma cells, the activity of the rHL-75 construct was 1.5–2 fold higher than the minimal promoter construct. In HeLa cells, contrastingly, transcriptional activity of rHL-75 was 2–4 fold lower than the minimal promoter construct in HeLa cells. Consequently, there was a marked, 3–5-fold difference in relative promoter activity between these two cell lines. Similar results were obtained with the longer rat HL constructs that all contained module B. The data were minimally affected by the simultaneous presence of module A (Fig. 7). Qualitatively similar results were obtained with human HL promoter fragments (data not shown). We conclude therefore, that module B plays a pivotal role in liver-restricted expression of the HL gene, by moderately activating transcription in liver cells, and simultaneously suppressing activity in non-hepatic cells.

The annotated data of the mammalian genome sequence projects were accessed through the Ensembl genome server (e!42: Dec 2006) 30. The exon-1 and 5'-upstream regulatory sequence of the hepatic lipase gene was available only for human (ENSG00000166035), rat (ENSRNOG00000015747), mouse (ENSMUSG00000032207), hedgehog (ENSETEG00000015177), chimpanzee (ENSPTRG00000007115) and rhesus macaque (ENSMMUG00000009566). Multiple sequence alignment was performed with DNAMAN software package version 3.2 (Lynnon BioSoft, Quebec, Canada). Global sequence alignments were performed with the publicly available web-based tool mVista 1231 using the MLAGAN algorithm. A search for potential TFBS in the upstream regulatory region of a particular HL gene was performed online at Genomatix using the MatInspector program 232. Clusters of TFBS that are conserved among the rat, mouse, human and macaque HL promoter regions were identified by the publicly available web-tool rVista 111231.

A rat genomic library in λ DASH II (Stratagene, La Jolla, CA, USA) was used for isolation of the HL promoter region, using a HL cDNA probe corresponding to exons-1 and -2. The probe was generated by RT-PCR on 1 μg rat liver RNA using the oligonucleotides (5'-GGT AAG ACG AGA GAC ATG G-3', nt 1–19; numbering according to 33) and (5'-CCC GTG GAT GAT CAT GAC AA-3', nt 285–266) as forward and reverse primers, respectively. The RT-PCR product was isolated by agarose gel electrophoresis, and radiolabeled using [α³²-P]dCTP and the Megaprime kit from Amersham (Amersham, UK). Filters containing 10⁶ plaques were hybridized overnight at 42°C with 50 ng of the labeled cDNA probe in hybridization buffer (50 % (v/v) formamide, 0.5 % (w/v) SDS, 0.1 mg/ml denaturated herring sperm DNA and 2 × PIPES buffer; 34). After washing in 0.2 × sodium chloride/sodium citrate/0.5% SDS at 65°C for 5 min, the filters were exposed to autoradiography film. Two positive clones were identified, which were plaque-purified three times. One of these clones was selected for further analysis. Phage DNA was isolated and digested with EcoRI. A 6 kb fragment 35 was subcloned into pBluescript KS^- (pBsE6) and its identity with the 5'-regulatory region of the rat HL gene was verified by sequence analysis.

The clone in pBluescript containing the 6 kb EcoRI fragment of the rat HL gene (pBsE6) was used to generate promoter-reporter constructs in pCAT-Basic (Promega, Madison, WI, USA). By digestion with PstI and XbaI, a 1.85-kb PstI/PstI, a 0.32-kb PstI/XbaI and a 0.15-kb XbaI/XbaI fragment was isolated. First, the 0.32-kb PstI/XbaI (-446/-127; numbering according to 35) fragment was cloned into pCAT-Basic. From this construct, the rHL-446 CAT plasmid was generated by insertion of the 0.15-kb XbaI (-127/+9) fragment. Subsequently, rHL-2287 CAT was generated by insertion of the 1.85-kb PstI (-2287/-446) fragment into rHL-446. The rHL-127 CAT construct was made by subcloning the 0.15-kb XbaI (-127/+9) fragment into pCAT-Basic.

From the rHL-2287 CAT vector, the 5'-truncated rHL-1697, rHL-1041 and rHL-747 constructs were generated by PCR using HindIII-restriction site-containing oligonucleotides 3F, 4F and 5F as upstream primer, respectively, and the CAT-gene specific oligonucleotide CATrev2 as downstream-primer (Table 2). After digestion of the PCR products with HindIII and PstI, the DNA fragments were purified by electrophoresis through agarose gel, and subsequently ligated into the rHL-446 CAT plasmid that had been digested with the same restriction enzymes. Similarly, the rHL-211 construct was generated from the rHL-446 CAT by PCR using oligonucleotides 9F and CATrev2 as upstream and downstream primer, respectively, followed by ligation into the HindIII and XbaI sites of rHL-446 CAT. Finally, the rHL-75, rHL-39 and rHL-23 constructs were generated from pBsE6 using 7F, 6F and 11F as upstream, and T3Primer as downstream primer, respectively, followed by digestion and ligation into the HindIII and PstI sites of pCAT-Basic; subsequently, the resulting plasmids were digested with XbaI followed by self-ligation.

Human HL promoter constructs were made in the pGL3-Basic luciferase reporter plasmid (Promega, Madison, WI, USA), starting from the hHL(-685/+13)-CAT plasmid described previously 36. An upstream SacI restriction site was introduced by PCR using the HHL-685Sac primer (Table 2) and the downstream HHL+13Xba primer. After digestion with SacI and XbaI, the gel-purified DNA products were ligated into the SacI and NheI sites of pGL3, thus generating the hHL-685Luc plasmid. Similarly, hHL-306Luc, hHL-79Luc and hHL-36Luc plasmids were generated by using HHL-306Nhe, HHL-79Kpn, and HHL-36Kpn as upstream primers, respectively.

To test the enhancer activity of conserved upstream sequences, the -14 kb, -22 kb and -10 kb elements were inserted into the enhancer site of the hHL-685Luc plasmid using the BamHI and SalI restriction sites. Human genomic DNA was isolated from a buffy coat, and the -14 kb, -22 kb and -10 kb elements were PCR amplified using specific primers (Table 2). The PCR products were cloned into the pGEM T-easy vector (ProMega, Madison, WI, USA). After digestion of the plasmids with BamHI and SalI, the inserts were cloned into hHL-685Luc in either sense or anti-sense orientation.

All clones were verified by DNA sequencing using the Thermo-sequenase dye terminator kit (Amersham, UK) and the ABI 377 sequencer (Applied Biosystems, Foster City, CA, USA).

HepG2 hepatoma cells and HeLa cells were cultured at 37°C and 5% CO₂ in Dulbecco's modified Eagle's medium (ICN, Costa Mesa, CA, USA) supplemented with 10% (v/v) fetal calf serum (Gibco, Breda, Netherlands) and penicillin/streptomycin. Transfection of HepG2 cells with CAT-reporter constructs was performed by the calcium-phosphate co-precipitation method. At 24 h before transfection, the cells were plated in 6-wells plates at 20–30 % confluence. At 3 h before transfection, the medium was refreshed. Cells were co-transfected with 2.5 μg/well of the CAT reporter test plasmid and 0.2 μg/well of control RSV β-galactosidase expression plasmid (Promega) 36. Parallel transfections with SV40-CAT-Control and empty pCAT-Basic plasmids were used as controls. Fourty-eight hours post-transfection, cell lysates were prepared. CAT and β-galactosidase were determined by ELISA (Roche). Promoter activity was expressed as pg CAT/ng β-galactosidase to correct for differences in cell number and transfection efficiency.

Transfections of HepG2 and HeLa cells with the luciferase-reporter constructs were performed in 24-wells plates with Lipofectamine Plus (Invitrogen, Groningen, Netherlands) using 0.4 μg of the luciferase-reporter construct and 20 ng of pRL-CMV (Promega) per well 37. Cell extracts were prepared at 48 h post-transfection. The luciferase activity in the cell extracts was determined with the FireLight kit (Perkin-Elmer, Boston MA, USA) and the Packard Top Count NXT luminometer. Data were normalized for the Renilla activity measured in the same sample.

Experimental data are expressed as mean ± SD. Differences were tested for statistical significance by paired Student's t-test.