Search

The forkhead box (Fox) family of transcription factors share homology in the winged helix/forkhead DNA-binding domain and play important roles in regulating cellular proliferation, differentiation, longevity, and cellular transformation. Forkhead box M1B (FoxM1B) is a ubiquitously expressed member of the Fox transcription factor family whose expression is restricted to proliferating cells and that mediates hepatocyte entry into DNA synthesis and mitosis during liver regeneration. Recent cDNA microarray studies indicated that age-related defects in cellular proliferation are associated with diminished expression of the FoxM1B transcription factor. Here, we show that increased levels of FoxM1B in regenerating liver of old transgenic mice restore the sharp peaks in hepatocyte DNA replication and mitosis that are the hallmarks of young regenerating mouse liver. Restoration of the young regenerating liver phenotype is associated with increased expression of numerous cell cycle regulatory genes that include cyclin D1, cyclin A2, cyclin F, cyclin B1, cyclin B2, Cdc25B, and p55cdc. Cotransfection assays in the human hepatoma HepG2 cell line demonstrated that FoxM1B protein stimulated expression of both the cyclin B1 and cyclin D1 promoters, suggesting that these cyclin genes are a direct FoxM1B transcriptional target. These results suggest that FoxM1B controls the transcriptional network of genes that are essential for cell division and exit from mitosis. Our results indicate that reduced expression of the FoxM1B transcription factor contributes to the decline in cellular proliferation observed in the aging process.

Mammalian hepatocyte nuclear factor-3 (HNF-3) and the Drosophila homeotic gene fork head proteins are prototypes of an extensive family of cell-specific transcription factors that share homology in the winged helix DNA-binding domain. One of these mammalian family members, HNF-3/fork head homolog-4 (HFH-4), was isolated by PCR amplification of rodent brain cDNA and exhibits abundant expression in the adult bronchiolar epithelium. In this study, we performed in situ hybridization of stage-specific mouse embryos and report on a novel expression pattern of the HFH-4 gene in both the presumptive and differentiated choroid plexus epithelium, which is responsible for the synthesis and secretion of cerebrospinal fluid (CSF) proteins. We also showed that HFH-4 is a potent transcriptional activator in cotransfection assays and defined several protein sequences important for HFH-4 transcriptional activity. We used in vitro DNA-binding site selection with recombinant HFH-4 protein and determined that the HFH-4 protein recognizes the DNA consensus sequences HWDTGTTTGTTTA or KTTTGTTGTTKTW (where H is not G, W is A or T, D is not C, and K is G or T). We used this HFH-4 consensus to identify potential HFH-4 target genes in the choroid plexus epithelium and demonstrated that these promoter sequences bind to recombinant HFH-4 protein in electrophoretic mobility shift assays. Recombinant HFH-4 formed specific protein-DNA complexes with the promoter regions of the human prothrombin, beta amyloid precursor protein, alpha1-antichymotrypsin, cystic fibrosis transmembrane conductance regulator and rodent alpha2-macroglobulin, growth hormone receptors, and insulin-like growth factor II genes. Furthermore, we identified putative HFH-4 target genes in the bronchiolar epithelium including the clara cell secretory protein gene and the HNF-3alpha gene, a winged helix family member involved in the transcriptional regulation of genes in the bronchiolar epithelium. In support of these binding studies, cotransfection assays show that HFH-4 potentiates expression of the HNF-3alpha and clara cell secretory protein promoter regions.