BMC Genomics 2007, 8:99 http://www.biomedcentral.com/1471-2164/8/99 IFdiegnutrifeica3tion of functional regulatory sequences in the proximal promoter region of the HL gene by rVista Identification of functional regulatory sequences in the proximal promoter region of the HL gene by rVista. Of the proximal promoter regions of the rat and human HL genes, 600 bp were submitted to the rVista sequence analysis soft- ware, and searched for conserved clusters of TFBS for a selection of 50 transcription factors known to be expressed in mam- malian liver. A vertical line indicates the position of the conserved TFBS relative to the human sequence (x-axis, numbering is relative to the transcriptional start site). Homology between the rat and human sequence is given as described for figure 1. Three clusters of conserved TFBS are identified, and designated A (-240/-200), B (-80/-40) and C (-25/+5). In human-mouse comparison, these clusters are also evident with an additional cluster at -295/-265. HepG2 cells with non-hepatic HeLa cells (Fig. 7). Pro- moter activity in each cell line was expressed as percentage of that of the rHL-39 construct, because this fragment rep- resents 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. Conse- quently, there was a marked, 3–5-fold difference in rela- tive 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. Discussion Global alignment of the 5'-flanking region of mammalian HL genes revealed three highly conserved elements (P ≤ 10-5) that lie far upstream of the HL promoter (Fig. 1). Two of these elements, at -14 kb and -22 kb, show moder- Page 6 of 13 (page number not for citation purposes)