upstream region hHL promoter region sense ** * ** -685 300 200 100 0 300 ) % ( y t iv200 i t c a r e o100 t m o r p 0 +13 anti-sense LUC * * BMC Genomics 2007, 8:99 http://www.biomedcentral.com/1471-2164/8/99 no -10K -14K -22K no -10K -14K -22K upstream region PFoigsusirbele2enhancer activity of the conserved sequences in the far upstream HL regulatory region Possible enhancer activity of the conserved sequences in the far upstream HL regulatory region. HepG2 cells were transiently transfected with the indicated promoter-reporter constructs. At 48 h post-transfection, transcriptional activ- ity was determined as the firefly over renilla luciferase activity. Data are expressed as percentage of the ratio measured in the hHL-685luc – transfected cells. Data are means ± SD from 3–4 independent experiments, each performed in quadruplicate. *and **: P < 0.05; and P < 0.01, respectively. between modules A and B (rHL-86 and rHL-75) had no significant effect on CAT expression. In contrast, addi- tional removal of most of module B in rHL-39 reduced transcriptional activity by approximately 60 %. With rHL- 23, in which the remainder of module B as well as the putative TATA-box has been deleted, CAT expression decreased further. Despite absence of the TATA-box, CAT expression of the rHL-23 construct was significantly higher than of promoter-less pCAT-Basic, which may be due to residual promoter activity of module C. Comparison with the proximal human HL promoter region Similar promoter-reporter assays were performed with the -685/+13 region of the orthologous human HL gene, except that the luciferase gene was used as reporter (Fig. 6). Luciferase activity of the hHL-306 construct was simi- lar to hHL-685, whereas activity of the hHL-79 construct was slightly, but not significantly, higher. This is in line with the rat promoter data, which show little effect of module A, and of the intervening sequence between mod- ules A and B, on basal transcriptional activity in HepG2 cells. The luciferase activity of the hHL-36 construct, in which entire module B has been removed, was only 25 % of the hHL-79 construct. The transcriptional activity of hHL-36, which contains a bona fide TATA box and entire module C, was 7-fold higher than background. This con- firms that modules B and C are crucial for basal transcrip- tional activity in HepG2 cells, with module B being most important. Role of module B in liver cell-specific HL transcription To test whether modules A and B are involved in liver-spe- cific expression of the HL gene, we compared transcrip- tional activity of different rat HL promoter fragments in Page 5 of 13 (page number not for citation purposes)