The Forkhead box m1 (Foxm1) gene is critical for G 1 /S transition and essential for mitotic progression. However, the transcriptional mechanisms downstream of FoxM1 that control these cell cycle events remain to be determined. Here, we show that both early-passage Foxm1 ؊/؊ mouse embryonic fibroblasts (MEFs) and human osteosarcoma U2OS cells depleted of FoxM1 protein by small interfering RNA fail to grow in culture due to a mitotic block and accumulate nuclear levels of cyclin-dependent kinase inhibitor (CDKI) proteins p21Cip1 and p27 Kip1 . Using quantitative chromatin immunoprecipitation and expression assays, we show that FoxM1 is essential for transcription of the mitotic regulatory genes Cdc25B, Aurora B kinase, survivin, centromere protein A (CENPA), and CENPB. We also identify the mechanism by which FoxM1 deficiency causes elevated nuclear levels of the CDKI proteins p21Cip1 and p27 Kip1 . We provide evidence that FoxM1 is essential for transcription of Skp2 and Cks1, which are specificity subunits of the Skp1-Cullin 1-F-box (SCF) ubiquitin ligase complex that targets these CDKI proteins for degradation during the G 1 /S transition. Moreover, early-passage Foxm1 ؊/؊ MEFs display premature senescence as evidenced by high expression of the senescence-associated ␤-galactosidase, p19 ARF , and p16 INK4A proteins. Taken together, these results demonstrate that FoxM1 regulates transcription of cell cycle genes critical for progression into S-phase and mitosis.

Transthyretin (TTR) and al-antitrypsin (al-AT) are expressed at high levels in the liver and also in at least one other cell type. We report here a detailed analysis of the proximal regulatory region of the TTR gene, which has uncovered two new DNA-binding factors that are present mainly (or only) in hepatocytes. One of these new factors, hepatocyte nuclear factor 3 (HNF-3), binds to two sites that are crucial in TTR expression as well as to two additional sites in the al-AT proximal enhancer region. The second new factor, HNF-4, binds to two sites in TTR that are required for gene activity. We had previously identified binding sites for another hepatocyte-enriched DNA-binding protein (C/EBP or a relative thereof), and additional promoter-proximal sites for that protein in both TTR and al-AT are also reported here. From these results it seems clear that cell-specific expression is not simply the result of a single cell-specific factor for each gene but the result of a combination of such factors. The variation and distribution of such factors among different cell types could be an important basis for the coordinate expression of the TTR and al-AT genes in the liver or the discordant transcriptional activation of these genes in a few other cell types. The identification of such cell-enriched factors is a necessary prelude to understanding the basis for cell specificity.Hepatocytes express a large number of proteins that are not expressed at all in most other cell types (8,10,19,21,41). However, occasionally other cell types in adults express one or a few of the same proteins as the liver. For example, transthyretin (TTR [14]) and transferrin (4) are expressed in the choroid plexus, apolipoprotein CIII (43) is expressed in the intestine, al-antitrypsin (al-AT) is expressed in macrophages (42), and all are expressed in hepatocytes. In the cells of the yolk sac, an extraembryonic tissue, a large subset of proteins made in the adult liver are produced (39, 47) and secreted. Transcriptional control most likely underlies the regulation of the various genes in all of these cell types (13). It has long been suggested that cell-specific transcriptional factors might exist as at least part of the explanation of cell-specific regulation (for a review, see Maniatis et al. [36]). However, problems immediately arise when one tries to develop a simple scheme to explain this regulation. First, the invocation of negative-acting factors to suppress inappropriate expression, while not impossible, requires such factors to exist for every gene whose transcriptional activity must be restricted in a large number of noneypressing cell types. Second, with respect to positive-acting factors, for example, since TTR, al-AT, and apolipoproteins all are expressed in the liver but only TTR is expressed in the choroid plexus, only al-AT is expressed in macrophages, and only apolipoprotein is expressed in the gut, then the activation mechanism in the hepatocyte must not be the same as the activation mechanism in the other cell types. Obviously, ...

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The hepatocyte nuclear factor 3␣ (HNF-3␣) and 3␤ proteins have homology in the winged helix/fork head DNA binding domain and regulate cell-specific transcription in hepatocytes and in respiratory and intestinal epithelia. In this study, we describe two novel isoforms of the winged helix transcription factor family, HNF-3/fork head homolog 11A (HFH-11A) and HFH-11B, isolated from the human colon carcinoma HT-29 cell line. We show that these isoforms arise via differential splicing and are expressed in a number of epithelial cell lines derived from tumors (HT-29, Caco-2, HepG2, HeLa, A549, and H441). We demonstrate that differentiation of Caco-2 cells toward the enterocyte lineage results in decreased HFH-11 expression and reciprocal increases in HNF-3␣ and HNF-3␤ mRNA levels. In situ hybridization of 16 day postcoitus mouse embryos demonstrates that HFH-11 expression is found in the mesenchymal and epithelial cells of the liver, lung, intestine, renal cortex, and urinary tract. Although HFH-11 exhibits a wide cellular expression pattern in the embryo, its adult expression pattern is restricted to epithelial cells of Lieberkühn's crypts of the intestine, the spermatocytes and spermatids of the testis, and the thymus and colon. HFH-11 expression is absent in adult hepatocytes, but its expression is reactivated in proliferating hepatocytes at 4, 24, and 48 h after partial hepatectomy. Consistent with these findings, we demonstrate that HFH-11 mRNA levels are stimulated by intratracheal administration of keratinocyte growth factor in adult lung and its expression in an adult endothelial cell line is reactivated in response to oxidative stress. These experiments show that the HFH-11 transcription factor is expressed in embryonic mesenchymal and epithelial cells and its expression is reactivated in these adult cell types by proliferative signals or oxidative stress.Cell-specific transcription relies on the combinatorial recognition of multiple cis-acting elements by families of cell-restricted transcription factors (80). One of these regulatory families is represented by the hepatocyte nuclear factor 3␣ (HNF-3␣), HNF-3␤, and HNF-3␥ proteins (43), which have homology in the winged helix DNA binding domain (12) and function in combination with other liver-enriched transcription factors to mediate hepatocyte-enriched transcription (17). The HNF-3␣ and -3␤ proteins also activate the transcription of genes important for respiratory epithelial cell function (7,14,35,40,60,82). The HNF-3 proteins thus appear to play an important transcriptional regulatory role in epithelial cell typespecific gene expression in adult tissues derived from gut endoderm.In the adult intestine, multipotent proliferative stem cells in Lieberkühn's crypts in the mouse intestine give rise to four terminally differentiated cell types: digestive and absorptive columnar enterocytes (representing the most abundant cell type), mucus-producing goblet cells, enteroendocrine cells, and Paneth cells (53). As the postmitotic enterocytes, goblet cells, and ente...

The Forkhead Box (Fox) proteins are an extensive family of transcription factors that shares homology in the winged helix DNAbinding domain and whose members play essential roles in cellular proliferation, differentiation, transformation, longevity, and metabolic homeostasis. Liver regeneration studies with transgenic mice demonstrated that FoxM1B regulates the onset of hepatocyte DNA replication and mitosis by stimulating expression of cell cycle genes. Here, we demonstrate that albumin-promoter-driven Cre recombinase-mediated hepatocyte-specific deletion of the Foxm1b Floxed (fl) targeted allele resulted in significant reduction in hepatocyte DNA replication and inhibition of mitosis after partial hepatectomy. Reduced DNA replication in regenerating Foxm1b ؊/؊ hepatocytes was associated with sustained increase in nuclear staining of the cyclin-dependent kinase (Cdk) inhibitor p21 Cip1 (p21) protein between 24 and 40 h after partial hepatectomy. Furthermore, increased nuclear p21 levels and reduced expression of Cdc25A phosphatase coincided with decreases in Cdk2 activation and hepatocyte progression into S-phase. Moreover, the significant reduction in hepatocyte mitosis was associated with diminished mRNA levels and nuclear expression of Cdc25B phosphatase and delayed accumulation of cyclin B1 protein, which is required for Cdk1 activation and entry into mitosis. Cotransfection studies demonstrate that FoxM1B protein directly activated transcription of the Cdc25B promoter region. Our present study shows that the mammalian Foxm1b transcription factor regulates expression of cell cycle proteins essential for hepatocyte entry into DNA replication and mitosis.knock-out mouse ͉ Cdc25A ͉ Cdc25B ͉ cyclin-dependent kinase inhibitor p21 Cip1