BMC Genomics 2007, 8:99 http://www.biomedcentral.com/1471-2164/8/99 EFfifgeuctreof5serial 5'-deletions of the rat HL promoter region on transcriptional activity in HepG2 cells Effect of serial 5'-deletions of the rat HL promoter region on transcriptional activity in HepG2 cells. Experiments were performed as described in the legends to figure 4. Data were expressed as percentage of the ratio measured in the rHL- 446 CAT – transfected cells, and are means ± SD from 3–5 independent experiments, each performed in triplicate. *: P < 0.05; n.s.: not significant. respectively. However, we missed an additional module (- 295 to -265) that has recently been identified as a func- tional DR4 site [25]. The cluster of TFBS within this mod- ule appeared to be conserved among human and mouse, but not among human and rat. Despite the relatively high homology between the mouse and rat over the proximal 5'-flanking region of the HL gene (Table 1), the outcome of the genomic sequence analysis differed whether the rat or the mouse sequence was used. Hence, although search- ing genomic sequences for conserved clusters of TFBS is a valuable tool in predicting functionally important regula- tory elements, this approach is sub optimal. For two of the modules that are conserved among the four species, a significant contribution to basal transcription was confirmed by promoter-assays in HepG2 cells. For module C (-25/+5), this is not surprising since it contains the transcriptional start site itself, as well as a pyrimidine- rich stretch that may serve as an initiator region (Inr). Module B (-80 to -40) overlaps with a protected region in DNAse footprinting in rat liver [22] as well as in HepG2 cells [23], and contains a HNF1 binding site that has been implicated in liver-specific expression of the human HL gene by other groups [22-24]. Experimentally, we could not confirm a major role for module A (-240 to -200) in determining basal transcription activity in HepG2 cells. This is surprising since it corresponds to a functional DR1 site [25], and perfectly matches with a protected region in DNAse footprinting in rat liver and human HepG2 nuclear extracts [22,23], suggesting that this part of the HL promoter is occupied by transcription factors under basal conditions. Similarly, we could not confirm the role of the Page 8 of 13 (page number not for citation purposes)