Figure 6

Functional identification of HNF1 binding sites in the promoter region of lhx1 in HEK293 cells and in animal caps. (A) HEK293(HNF1B) cells [45] were transfected with lhx1 gene firefly luciferase fusion constructs and HNF1B expression was induced by adding doxycycline. On the left panel schematic drawings of transfected lhx1 promoter luciferase fusion constructs (not to scale) are given. Lines represent promoter and intron regions, boxes are exons with protein coding (filled) or untranslated (open) regions (adapted from [42]). Arrows mark potential HNF1 binding sites identified by JASPAR [44]. The arrow head marks the ARE (activin response element) in intron 1 [42]. The Ex1(-120/+3) construct contains the complete HNF1 binding site in the promoter region of lhx1, whereas the binding site in the Ex1(-117/+3) construct is partially deleted (compare panel C). The HNF-4a P2-285 construct contains the P2-promoter of the HNF-4a gene which is regulated by HNF1B [46]. The fold change of luciferase activity of transfected constructs in doxycycline induced HEK293(HNF1B) versus untreated cells is given using the renilla luciferase reporter pRL-Con as an internal control. The Student's t-test was used and *, ** and *** refer to p-value of ≤ 0.05, ≤ 0.01 and ≤ 0.001, respectively and n.s. means not significant. N is the number of independent transfections made. (B) The luciferase reporter constructs (50 pg each) were tested in animal caps of controls or HNF1B (150 pg) injected embryos. The luciferase activity was measured in pools of four animal caps after four hours cultivation in Steinberg's solution. To calculate the increase of luciferase activity in HNF1B injected animal caps the ratio of firefly luciferase (FL) to renilla luciferase (RL) was used (FL/RL). The vertical line in the middle represents the mean and the small bars the standard deviation. Since experiments with different pools of animal caps were not comparable in quantitative terms, we used the Mann-Whitney-test to score significant differences. * and ** refer to p-value of ≤ 0.05 and ≤ 0.01, respectively and n.s. means not significant. N is the number of animal cap pools tested. (C) The upper panel shows the sequence logo of HNF1 binding site given in JASPAR [44] and the lower panel the sequences of the HNF1 binding site (capital letters) in Ex1(-120/+3) and Ex1(-117/+3) constructs. The vector sequence is indicated by italics.