Figure 4

HNF4α activates transcription from Alu elements. A. Reporter gene assay with the HNF4α-Alu elements from the indicated human gene promoters fused to a minimal core promoter driving luciferase (pGL4.23). Shown is luciferase activity (relative light units, RLU) normalized to β-gal activity (normalized RLU) from HEK 293T cells transiently transfected with 1 μg of reporter and different amounts of either empty vector or human HNF4α2 expression vector (100, 200, 300 and 500 ng). Reporter constructs contain only the HNF4α-Alu element and immediately adjacent sequence; they do not contain any additional known HNF4α binding sites. Data are the mean normalized RLU of triplicate samples from one representative experiment from two or more that were performed. P-values of the HNF4α2 signal compared to the empty vector are indicated. Fold induction by HNF4α2 compared to the empty vector is indicated. B. As in (A) but with two different reporter constructs containing classical HNF4α response elements (RE-1 and RE-2). Shown is fold induction by 500 ng HNF4α2 compared to the parent construct (pGL4.23). C. As in (A) but of the native human APOA4 promoter (-1343 to +247) fused to luciferase (pGL4.10) without (WT) or with mutations (MUT) in either the HNF4α-Alu element (Alu) or a classical HNF4α site identified by a previous PBM analysis (HNF4α-PBM) (see Figure 3). Shown is the fold induction by 500 ng HNF4α2 compared to the empty expression vector from one experiment performed in six replicates. A second independent experiment performed in triplicate gave similar results. (B) and (C), sequence of the relevant HNF4α binding sites are given with the spacer nucleotide in lower case and mutations in red.