Database analysis
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 [12,31] 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 [2,32]. 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 [11,12,31].
Isolation of exon-1 and the 5'-flanking region of the rat HL gene
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.
Construction of reporter plasmids
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).
Promoter reporter assays
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.
Statistics
Experimental data are expressed as mean ± SD. Differences were tested for statistical significance by paired Student's t-test.
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