Fig. 6. Association of the HNF6 and Foxm1 proteins results in stimulation of Foxm1 transcriptional activity.

(A) Co-immunoprecipitation assays with regenerating liver extracts demonstrate functional association between the HNF6 and Foxm1 transcription factors. We performed co-immunoprecipitation (Co-IP) assays with protein extracts from quiescent mouse liver (0h) that lack detectable expression of Foxm1 protein or regenerating mouse liver isolated at 40 hours following PHx (40h), which express abundant levels of Foxm1 protein (Foxm1 Input). Quiescent or regenerating mouse liver extracts were Co-IP with the HNF6 antibody and then IP proteins were subjected to Western blot analysis with the FoxM1 C-terminal Antibody. (B) In vitro GST pull-down assays demonstrated that HNF6 Cut-Homeodomain protein interacts with the FoxM1b winged helix domain. Right panel shows the expression levels of the Glutathione-S-Transferase (GST), GST-FoxM1b N-terminal (N; amino acids 1-138) or GST-FoxM1b binding domain (DBD; amino acids 221 to 355) fusion proteins separated by SDS-PAGE and stained with Commassie Blue. The HNF6 Cut-Homeodomain DBD plasmid was used to synthesize radioactively labeled HNF6 DBD protein using in vitro transcription and translation (IVT) with ³⁵S Methionine and used for the in vitro GST-FoxM1b pull-down assays and bound labeled HNF6 protein was separated by SDS-PAGE and visualized by autoradiography (left panel). (C–D) Cotransfection studies demonstrate that HNF6 stimulates FoxM1b’s ability to activate Foxm1-dependent target promoters. Human hepatoma HepG2 (C) or osteosarcoma U2OS (D) cells were cotransfected with the Foxm1 reporter gene 6X Foxm1 TATA Luciferase reporter gene ⁵³ and increasing amounts of CMV HNF6 expression vector and constant amount of CMV FoxM1b expression vectors (numbers represent ng transfected). Cotransfection of CMV-HNF6 expression vector could not stimulate expression of the FoxM1 reporter gene in either HepG2 or U2OS cells. (E) Cotransfection assays demonstrated that HNF6 also stimulated FoxM1b ability to stimulate transcription of the −2.7 KB Cdc25B promoter region, which is a known transcriptional target of Foxm1 ²³. The asterisks indicate statistically significant changes: *P ≤ 0.05 and **P ≤ 0.01.