Fig. 5.

Characterization of a functional IR1 in the proximal promoter of ADH1A and ADH1B. A: Schematic representation of human ADH1 gene cluster and localization of the IR1 elements identified by NUBIScan in each proximal promoter. Alignment of the three IR1 and the FXRE consensus is shown below. The RGGTCA half-sites are indicated by arrows. B: The promoters of ADH1A and ADH1B, but not of ADH1C, respond to activated FXR. Huh7 cells were transfected with a plasmid containing luciferase reporter constructs driven by ∼2–2.9 kb fragments corresponding to ADH1 gene promoters (pGL3-ADH1A, pGL3-ADH1B, and pGL3-ADH1C, respectively), or the empty pGL3-basic vector (bv) as negative control, along with a plasmid expressing FXR, or the empty expression vector pSG5 as control, and then treated for 24 h with vehicle (Control) or 1 μM GW4064 and luciferase activities were measured. C: IR1 elements in ADH1A and ADH1B, but not ADH1C, confer FXR responsiveness to a heterologous promoter. Experiments were performed as in (B) with reporter constructs containing four copies of the IR1 site identified in the proximal promoter of ADH1 genes cloned in front of a heterologous thymidine kinase (TK) promoter-driven luciferase gene. pGL3-TK reporter vector was used as negative control. D: Disruption of ADH1A and ADH1B IR1 elements by site-directed mutagenesis abrogates the response to FXR. Experiments were performed as in (B) with the indicated reporter constructs containing wild-type or IR1 mutated sequence. E: Conversion of ADH1C IR1 to ADH1A IR1 element by site-directed mutagenesis confers FXR responsiveness to ADH1C promoter. Experiments were performed as in (B) with the constructs containing wild-type ADH1C promoter or a modified version containing the IR1 converted to ADH1A IR1 element. Results are expressed as -fold induction over control. ***P < 0.001 versus untreated controls. The results are representative of three independent experiments from triplicate dishes.