BMC Genomics 2007, 8:99 http://www.biomedcentral.com/1471-2164/8/99 Table 1: Pairwise sequence comparison of the coding and the 5'-flanking sequence of mammalian HL genes. cDNA sequence 81 79 79 85 85 85 76 76 82 82 82 53 92 79 80 79 61 64 63 96 96 57 55 57 94 99 56 59 57 94 98 hedgehoga mouse rat rhesusa chimp human upstream regulatory sequence 53 86 79 80 79 rat rabbitb hedgehog mouse rhesus chimp human Sequence identity (%) was determined by pairwise alignment using the DNAMAN software package (optimal alignment; gap open penalty 10.0, gap extension penalty 5.0). cDNA sequence was from the translation start ATG up till the stopcodon; the upstream regulatory region was from -1400 up till the translation start ATG. a: Because of a gap in upstream regulatory region of the public sequence of the rhesus monkey and hedgehog HL gene (Ensembl e!42: Dec 2006), alignments with the rhesus and hedgehog sequence were done with the sequence from -900 and -1000 up till the start ATG, respectively. b:the rabbit HL cDNA sequence was taken from [38]. 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 transcrip- tional 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, respec- tively (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. Comparative genomics of the proximal 2 kb HL upstream regulatory 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 poten- tial TFBS, randomly distributed over the entire sequence. A similar number of sites was predicted for the ortholo- gous mouse, human and rhesus macaque sequences. When we searched for clustered TFBS motifs that are con- served 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 tran- scriptional start site), for which AP1, AP2, CAAT, COUP, C/EBP, HNF4α, PPAR and USF binding sites are pre- dicted, corresponds to the DR1 site recently identified by Rufibach et al. [25]. Module B (-80 to -40) potentially con- tains AP2, CAAT, C/EBP, HNF1, HNF4, PPAR and Sp1 sites, and corresponds to the previously characterized HNF1 site [22-24]. 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 pyrimi- dine-rich motif, and therefore likely represents the Inr involved in binding of the transcription initiation com- plex. These three modules were also found to be con- served among the human and mouse HL gene. The human-mouse comparison revealed an additional, con- served 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 Rufi- bach 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 con- served 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]. Functional characterization of the rat HL promoter region 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 differ- Page 3 of 13 (page number not for citation purposes)