Identification of HNF4α binding sites in hFBP1 promoter.

<p>(A) Transactivation of 5’-truncated hFBP1 promoter-luciferase reporter construct by HNF4α in HepG2 cells. The 886 nucleotides-hFBP1 promoter-luciferase reporter gene or its 5’-truncated mutants (500, 400, 300, 200 and 100) were transiently co-transfected with empty vector (pcDNA3; grey bar) or plasmid overexpressing HNF4α (pcDNA3-HNF4α; black bar) into HepG2. The luciferase activity of wild type or mutant construct was normalized with β-galactosidase activity and shown as relative luciferase activity. Relative luciferase activity obtained from cells co-transfected with the hFBP1-promoter-luciferase constructs and plasmid encoding HNF4α was presented as “fold change” relative to those transfected with hFBP1-promoter-luciferase construct and empty vector, which was arbitrarily set as 1. (B) Nucleotide sequences of consensus HNF4α binding sites including classical DR1 and H4-SBM and three HNF4α binding sites (HNF4α1 (-566/-554, antisense strand), HNF4α2 (-358/-346; sense strand) and HNF4α3 site (-212/-198; antisense strand)) in hFBP1 promoter also shown (C) Effect of mutations of three HNF4α binding sites on FBP1 promoter activity. Mutations of the HNF4α1, HNF4α2 and HNF4α3 sites were introduced in the 886 FBP1 promoter-reporter construct and co-transfected with empty vector (pcDNA3; grey bars) or vector containing human HNF4α (pcDNA3-HNF4α; black bars) into HepG2 cells. The luciferase activity of each construct was normalized to β-galactosidase activity and expressed as relative luciferase activity. The values obtained from mutated constructs are expressed relative to the corresponding parental or mutant construct which was arbitrarily set as 100%. The values shown are means ± standard deviation (n = 3). *p < 0.01, **p < 0.05, ***p <0.001.