BMC Genomics 2007, 8:99 http://www.biomedcentral.com/1471-2164/8/99 FEfifgeuctreof4serial 5'-deletions of the rat HL upstream region on transcriptional activity in HepG2 cells Effect of serial 5'-deletions of the rat HL upstream region on transcriptional activity in HepG2 cells. HepG2 cells were transiently transfected with the indicated promoter-reporter constructs. At 48 h post-transfection, cells were lysed and expression of CAT and β-galactosidase protein was determined. Data are expressed as the ratio of CAT over β-galactosidase expression. Data are means ± SD from 4–7 independent experiments, each performed in triplicate. *: P < 0.05; **: P < 0.01; n.s.: not significant. ate enhancer activity in HepG2 cells. What discriminates the conserved -14 kb and -22 kb elements from the non- functional, non-conserved -10 kb sequence is unclear, as all three sequences contain a similar repertoire of TFBS for liver-expressed transcription factors (data not shown). Further studies are required to clarify the mechanism responsible for the enhancer activity of the two highly conserved elements in the HL gene. The finding of two hitherto unknown enhancers supports the hypothesis that conserved non-coding sequences may identify functional regulatory elements. Experimentally, we also found a pos- itive and a negative regulatory sequence between -2.2 and -0.4 kb of the rat HL gene that coincided with homology peaks, but were not recognized by the Rankvista analysis of the sequence comparison. Rubin's group recently dem- onstrated strong in vivo enhancer activity for almost half of the elements that are ultra-conserved among human/ mouse/rat [8,27]. Our study further illustrates the power of the approach, and suggests that gene regulatory func- tions may also reside in somewhat less conserved ele- ments among mammalian genomes. We also tested whether global genome comparisons can also aid in identification of functional regulatory ele- ments within highly conserved sequences, using the prox- imal HL promoter region as a model. Within this proximal promoter region, three modules are identified with conserved clusters of TFBS motifs. These modules A, B and C correspond with the previously identified regula- tory elements DR1 [25], HNF1 [22-24] and Inr [22-24], Page 7 of 13 (page number not for citation purposes)