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

Three distinct hepatocyte nuclear factor 3 (HNF-3) proteins are known to regulate the transcription of liver-specific genes. The HNF-3 proteins bind to DNA as a monomer through a modified helix-turn-helix, known as the winged helix motif, which is also utilized by a number of developmental regulators, including the Drosophila homeotic forkhead (fkh) protein. We have previously described the isolation, from rodent tissue, of an extensive family of tissue-specific HNF-3/fkh homolog (HFH) genes sharing homology in their winged helix motifs. In this report, we have determined the preferred DNA-binding consensus sequence for the HNF-3P protein as well as for two divergent family members, HFH-1 and HFH-2.We show that these HNF-3/fkh proteins bind to distinct DNA sites and that the specificity of protein recognition is dependent on subtle nucleotide alterations in the site. The HNF-3, HFH-1, and HFH-2 consensus binding sequences were also used to search DNA regulatory regions to identify potential target genes.Furthermore, an analysis of the DNA-binding properties of a series of HFH-1/HNF-3p protein chimeras has allowed us to identify a 20-amino-acid region, located adjacent to the DNA recognition helix, which contributes to DNA-binding specificity. These sequences are not involved in base-specific contacts and include residues which diverge within the HNF-3/fkh family. Replacement of this 20-amino-acid region in HNF-3P with corresponding residues from HFH-1 enabled the HNF-3I recognition helix to bind only HFH-1-specific DNA-binding sites. We propose a model in which this 20-amino-acid flanking region influences the DNA-binding properties of the recognition helix.Deciphering mechanisms which lead to transcriptional regulation of a distinct array of genes in a particular cell type is critical for understanding cellular commitment during mammalian embryogenesis. Differential expression of protein-encoding genes occurs at the point of transcriptional initiation and involves the assembly of several well-characterized basal factors with TATA-binding protein and RNA polymerase II at the initiation site of the promoter region (17). Promoter and enhancer regions are also composed of multiple DNA sites that interact with sequence-specific transcription factors which are believed to enhance the recruitment of basal factors to the initiation complex. Tissue-restricted gene expression thus relies upon the recognition of multiple cis-acting DNA sequences by cell-specific nuclear factors that potentiate or, in some instances, repress transcriptional initiation (23,28,32). Because transcription factors play a central role in regulating cellular differentiation, the analysis of their molecular structure and expression patterns has been fruitful in elucidating regulatory pathways involved in establishing tissue-specific gene transcription.The functional analysis of a number of transcription factors has demonstrated that they are modular in structure, consisting of independently functioning protein domains (11,(18)(19)(20).

Hepatocyte-specific gene expression requires the interaction of many proteins with multiple binding sites in the regulatory regions. HNF-3 is a site found to be important in the maximal hepatocyte-specific expression of several genes. We find that liver nuclear extracts contain three major binding activities for this site, which we call HNF-3A, HNF-3B, and HNF-3C. Purification from rat liver nuclear extracts of HNF-3A and HNF-3C reveals that each activity corresponds to a distinct polypeptide, as determined by SDS-PAGE. Peptide sequence derived from the most abundant species, HNF-3A, was used for synthesizing probes with which to isolate a cDNA clone of this protein. The encoded protein contains 466 amino acids (48.7 kD) and has binding properties identical to those of the purified protein. A 160-amino-acid region that does not resemble the binding domain of any known transcription factor is essential for DNA binding. The mRNA for HNF-3A is present in the rat liver but not in brain, kidney, intestine, or spleen, and the basis for this difference is cell-specific regulation of HNF-3A gene transcription.

Previous liver regeneration studies demonstrated that the mouse forkhead box M1B (FoxM1B) transcription factor regulates hepatocyte proliferation through expression of cell cycle genes that stimulate cyclin-dependent kinase 2 (Cdk2) and Cdk1 activity. In this study, we demonstrated that disruption of the FoxM1B Cdk1/2 phosphorylation site at Thr residue 596 significantly reduced both FoxM1B transcriptional activity and Cdk phosphorylation of the FoxM1B T596A mutant protein in vivo. Retention of this FoxM1B 596 Cdk phosphorylation site was found to be essential for recruiting the histone acetyltransferase CREB binding protein (CBP) to the FoxM1B transcriptional activation domain. Consistent with these findings, dominant negative Cdk1 protein significantly reduced FoxM1B transcriptional activity and inhibited FoxM1B recruitment of the CBP coactivator protein. Likewise, Cdc25B-mediated stimulation of Cdk activity together with elevated levels of the CBP coactivator protein provided a 6.2-fold synergistic increase in FoxM1B transcriptional activity. Furthermore, mutation of the FoxM1B Leu 641 residue within an LXL motif (residues 639 to 641) inhibited recruitment of Cdk-cyclin complexes and caused significant reduction in both FoxM1B transcriptional activity and in vivo Cdk phosphorylation of the FoxM1B Thr 596 residue. We demonstrated that FoxM1B transcriptional activity requires binding of either S-phase or M-phase Cdk-cyclin complexes to mediate efficient Cdk phosphorylation of the FoxM1B Thr 596 residue, which is essential for recruitment of p300/CBP coactivator proteins.Cellular proliferation involves stimulation of the mitogenactivated protein kinase (MAPK) pathway, consisting of the Ras/Raf1/MEK/MAPK cascade (19,47), and activation of the phosphoinositol 3-kinase (PI3K) pathway, consisting of the PI3K/phosphoinositide-dependent kinase 1 (PDK1)/Akt cascade (5). However, cell division is tightly regulated at the G 1 /S (DNA replication) and G 2 /M (mitosis) transitions of the cell cycle by temporal activation of multiple cyclin-dependent kinases (Cdks) complexed with their corresponding cyclin regulatory subunits. Cdk2-cyclin E or A and Cdk1-cyclin B kinase activity is essential for progression through the G 1 /S and G 2 /M transitions of the cell cycle, respectively. Furthermore, Cdk activity is negatively regulated by phosphorylation of Thr 14 and Tyr 15 by the dual specific Myt1 kinase (6, 30, 31, 37). Both MAPK and PDK1 phosphorylate and activate the downstream p90 ribosomal S6 kinase (Rsk) (4, 21, 51), which provides inhibitory phosphorylation to the dual Myt1 kinase (44, 45). Simulation of Cdk1 or Cdk2 activity involves removal of inhibitory phosphates at Thr 14 and Tyr 15 by either Cdc25A phosphatase (G 1 /S phase), Cdc25B (late S phase), or Cdc25C phosphatase (G 2 /M phase) (7,41,62). In addition, Cdk activity requires phosphorylation of Thr 160 by the Cdk-activating kinase (CAK) protein, which is also a direct target for MAPK phosphorylation (14,28,55).Cdk1 and Cdk2 proteins are proline-directed kinase...