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Figure 1 Alignment of the 5'-flanking region of four mammalian HL genes . From the rat, mouse, macaque and human HL genes, exon-1 and 30 kb of upstream sequence was aligned by the MLAGAN algorithm of the mVista program. The sequences of rat (A), mouse (B) and macaque (C) are aligned to the human HL sequence (x-axis); numbering is relative to the transcriptional start site. Conserved regions (> 70% homology over 100 bp window) are shaded. The boxes indicate conserved regions among the four sequences, as determined by RankVista (P ≤ 10-5), with the P-values given above.
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Figure 2 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 activity 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
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Figure 3 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 software, and searched for conserved clusters of TFBS for a selection of 50 transcription factors known to be expressed in mammalian 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.
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Figure 4 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
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Figure 5 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
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Figure 6 Effect of serial 5'-deletions of the human HL promoter region on transcriptional activity in HepG2 cells. HepG2 cells were transiently transfected with the indicated promoter-reporter constructs. At 48 h post-transfection, transcriptional activity 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 4 independent experiments, each performed in quadruplicate. *: P
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Figure 7 Transcriptional activity of the rat proximal HL promoter region in HepG2 and HeLa cells. HepG2 (hatched bars) and HeLa cells (open bars) were transiently transfected with the indicated promoter-reporter constructs. At 48 h post-transfection, transcriptional activity was determined as the ratio of CAT over β-galactosidase expression level. Data are expressed as percentage of the ratio measured in the rHL-39 CAT – transfected cells. Data are means ± SD from 3 independent experiments, each performed in triplicate. *,+: P
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