Fig. 7. HNF6 and Foxm1 regulate transcription of the HNF6 promoter as determined by cotransfection and ChIP assays.

(A) Infection of HepG2 cells with AdHNF6 stimulated expression of the endogenous human HNF6 gene. HepG2 cells were infected with AdHNF6 and RNA was isolated at different times after AdHNF6 infection and analyzed for HNF6 expression by RPA. (B) HNF6 protein binds to its own promoter region. Electrophoretic mobility shift assays with AdHNF6 infected HepG2 cell extracts and radioactively labeled double stranded oligonucleotide synthesized to the −1368 to −1329 bp human HNF6 promoter region forms a specific HNF6 protein-DNA complex that was inhibited by including in the binding reaction either a 100-fold excess of cold homologous oligonucleotide or HNF6 antisera. (C) HepG2 cell cotransfection assays shows that HNF6 and FoxM1b stimulates transcription of the −1.4 KB human HNF6 promoter region. HepG2 cells were cotransfected with either −1.4 KB or −1.3 KB human HNF6 promoter Luciferase plasmid and CMV-HNF6 and CMV-FoxM1b expression vectors either alone or together. At 24 hours after transfection, the cells were used to prepare cell extracts, which were then analyzed for dual Luciferase enzyme activity as described in Materials and Methods. (D–E) Quantitative ChIP assays revealed that Foxm1 and HNF6 proteins bind to the endogenous human HNF6 promoter region. HepG2 cells were mock infected or infected with either AdHNF6 or AdGFP and 24 hours after infection the chromatin was cross-linked and processed for ChIP assays as described in the Materials and Methods. The cross-linked and sonicated mouse chromatin was immunoprecipitated (IP) with antibodies specific to either HNF6 (+, D) or FoxM1 (+, E) or with rabbit antisera (−) and the amount of promoter DNA associated with the IP chromatin was quantitated by Real-Time PCR with primers specific to −1499 to −1337 bp of the human HNF6 promoter region. The asterisks indicate statistically significant changes: *P ≤ 0.05, **P ≤ 0.01 and P ≤ 0.001.