OBJECTIVE Forkhead box O (FoxO) transcription factors represent evolutionarily conserved targets of insulin signaling, regulating metabolism and cellular differentiation in response to changes in nutrient availability. Although the FoxO1 isoform is known to play a key role in adipogenesis, its physiological role in differentiated adipose tissue remains unclear. RESEARCH DESIGN AND METHODS In this study, we analyzed the phenotype of FoxO1 haploinsufficient mice to investigate the role of FoxO1 in high-fat diet–induced obesity and adipose tissue metabolism. RESULTS We showed that reduced FoxO1 expression protects mice against obesity-related insulin resistance with marked improvement not only in hepatic insulin sensitivity but also in skeletal muscle insulin action. FoxO1 haploinsufficiency also resulted in increased peroxisome proliferator–activated receptor (PPAR)γ gene expression in adipose tissue, with enhanced expression of PPARγ target genes known to influence metabolism. Moreover, treatment of mice with the PPARγ agonist rosiglitazone caused a greater improvement in in vivo insulin sensitivity in FoxO1 haploinsufficient animals, including reductions in circulating proinflammatory cytokines. CONCLUSIONS These findings indicate that FoxO1 proteins negatively regulate insulin action and that their effect may be explained, at least in part, by inhibition of PPARγ function.

Foxo transcription factors regulate cell cycle progression, cell survival and DNA-repair pathways. Here we demonstrate that deficiency in Foxo3 resulted in greater expansion of T cell populations after viral infection. This exaggerated expansion was not T cell intrinsic. Instead, it was caused by the enhanced capacity of Foxo3-deficient dendritic cells to sustain T cell viability by producing more interleukin 6. Stimulation of dendritic cells mediated by the coinhibitory molecule CTLA-4 induced nuclear localization of Foxo3, which in turn inhibited the production of interleukin 6 and tumor necrosis factor. Thus, Foxo3 acts to constrain the production of key inflammatory cytokines by dendritic cells and to control T cell survival.

The Foxo subfamily of FOX transcription factors plays a variety of roles in a broad assortment of diverse physiological processes including cellular differentiation, tumor suppression, metabolism, cell cycle arrest, cell death and protection from stress. Animal models have proved to be invaluable tools in furthering our understanding of the role of particular genes in complex organismal processes. Multiple animal models in diverse species, including Caenorhabditis elegans, Drosophila. melanogaster and the laboratory mouse, exist for the Foxo family of transcription factors. Foxo genes are highly conserved throughout the evolution and each of these model systems has provided valuable insight into the roles of Foxo factors. Many roles are conserved among the different model organisms. Several Foxo-related animal model systems are reviewed here along with the knowledge gleaned to date from each model system.

Human cancer cells frequently harbor chromosomal translocations that create chimeric fusion genes. The t( 2; 13) translocation is characteristic of the pediatric muscle tumor, alveolar rhabdomyosarcoma, and produces the chimeric transcription factor, PAX3-FOXO1, that contains the DNA binding elements of PAX3 and the transcriptional activation domain of FOXO1. Experiments designed to determine how PAX3-FOXO1 expression contributes to the development of muscle cell-derived tumors resulted in the discovery that the fusion protein misregulates gene expression and interrupts myogenic differentiation through a unique gain of function mechanism. These results yield new insight into how tumor-associated genetic alterations increase the likelihood of cancer formation and may lead to new therapeutic approaches.

In the paper, Guilt by association:PAX3-FOXO1 regulates gene expression through selective destabilization of the EGR1 transcription factor by Wendy Roeb, Antonia Boyer, Webster K. Cavenee and Karen C. Arden (Cell Cycle 2008; 7:837-41), Figure 1 and Figure 2 were mislabeled and published alongside the incorrect figure legends. Figures 1 and 2 appear with their correct figure legends below.

The transcription factors Foxo1, Foxo3 and Foxo4 modulate cell fate 'decisions' in diverse systems. Here we show that Foxo1-dependent gene expression was critical at many stages of B cell differentiation. Early deletion of Foxo1 caused a substantial block at the pro-B cell stage due to a failure to express interleukin 7 receptor-alpha. Foxo1 inactivation in late pro-B cells resulted in an arrest at the pre-B cell stage due to lower expression of the recombination-activating genes Rag1 and Rag2. Deletion of Foxo1 in peripheral B cells led to fewer lymph node B cells due to lower expression of L-selectin and failed class-switch recombination due to impaired upregulation of the gene encoding activation-induced cytidine deaminase. Thus, Foxo1 regulates a transcriptional program that is essential for early B cell development and peripheral B cell function.

By integrating genome-wide maps of RNA polymerase II (Polr2a) binding with gene expression data and H3ac and H3K4me3 profiles, we characterized promoters with enriched activity in mouse embryonic stem cells (mES) as well as adult brain, heart, kidney, and liver. We identified approximately 24,000 promoters across these samples, including 16,976 annotated mRNA 5' ends and 5153 additional sites validating cap-analysis of gene expression (CAGE) 5' end data. We showed that promoters with CpG islands are typically non-tissue specific, with the majority associated with Polr2a and the active chromatin modifications in nearly all the tissues examined. By contrast, the promoters without CpG islands are generally associated with Polr2a and the active chromatin marks in a tissue-dependent way. We defined 4396 tissue-specific promoters by adapting a quantitative index of tissue-specificity based on Polr2a occupancy. While there is a general correspondence between Polr2a occupancy and active chromatin modifications at the tissue-specific promoters, a subset of them appear to be persistently marked by active chromatin modifications in the absence of detectable Polr2a binding, highlighting the complexity of the functional relationship between chromatin modification and gene expression. Our results provide a resource for exploring promoter Polr2a binding and epigenetic states across pluripotent and differentiated cell types in mammals.

The FoxO transcription factors have been implicated in many processes including tumor suppression and cell death. In this issue, two groups now report on mice that conditionally lack the three predominant FoxO transcription factors. Tothova et al. (2007) demonstrate that FoxOs are critical for the long-term maintenance of hematopoietic stem cells, and Paik et al. (2007) show that FoxOs suppress the formation of hemangiomas and lymphomas in mice.

The chimeric protein PAX3-FOXO1, resulting from a translocation between chromosomes 2 and 13, is the most common genetic aberration in the alveolar subtype of the human skeletal muscle tumor, rhabdomyosarcoma. To understand how PAX3-FOXO1 contributes to tumor development, we isolated and characterized muscle cells from transgenic mice expressing PAX3-FOXO1 under control of the PAX3 promoter. We demonstrate that these myoblasts are unable to complete myogenic differentiation because of an inability to up-regulate p57Kip2 transcription. This defect is caused by reduced levels of the EGR1 transcriptional activator resulting from a direct, destabilizing interaction with PAX3-FOXO1. Neither PAX3 nor FOXO1 share the ability to regulate p57Kip2 transcription. Thus, the breakage and fusion of the genes encoding these transcription factors creates a unique chimeric protein that controls a key cell-cycle and -differentiation regulator.

The FOXO family of transcription factors has been implicated in several cellular processes including cell cycle arrest, cell death and protection from stress stimuli. FOXO function is influenced by multiple signaling pathways. Many of these pathways are known to be misregulated in cancer. Perturbation of FOXO function leads to uncontrolled cell proliferation and accumulation of DNA damage. It is becoming clear that active FOXO proteins play an important role in keeping cells in check and inactivation of FOXO proteins is associated with characteristics of cancer cells. FOXO proteins may represent new therapeutic targets for a broad spectrum of cancers.

The total mass of islets of Langerhans is reduced in individuals with type 2 diabetes 1 , possibly contributing to the pathogenesis of this condition. Although the regulation of islet mass is complex, recent studies have suggested the importance of a signaling pathway that includes the insulin or insulin-like growth factor–1 receptors, insulin receptor substrate and phosphatidylinositol (PI) 3-kinase 2 , 3 , 4 . 3-Phosphoinositide–dependent protein kinase 1 ( PDK1 ) is a serine-threonine kinase that mediates signaling downstream of PI 3-kinase. Here we show that mice that lack PDK1 specifically in pancreatic β cells (β Pdk1 −/− mice) develop progressive hyperglycemia as a result of a loss of islet mass. The mice show reductions in islet density as well as in the number and size of cells. Haploinsufficiency of the gene for the transcription factor Foxo1 resulted in a marked increase in the number, but not the size, of cells and resulted in the restoration of glucose homeostasis in β Pdk1 −/− mice. These results suggest that PDK1 is important in maintenance of pancreatic cell mass and glucose homeostasis.

The t(2;13) chromosomal translocation is found in the majority of human alveolar rhabdomyosarcomas (RMS). The resulting PAX3-FKHR fusion protein contains PAX3 DNA-binding domains fused to the potent transactivation domain of FKHR, suggesting that PAX3-FKHR functions to deregulate PAX3-specific target genes and signaling pathways. We previously developed transgenic mice expressing PAX3-FKHR under the control of mouse Pax3 regulatory sequences to test this hypothesis. We reported that PAX3-FKHR interferes with normal Pax3 developmental functions, with mice exhibiting neural tube and neural crest abnormalities that mimic those found in Pax3-deficient Splotch mice. Here we expanded those studies to show that developmental expression of PAX3-FKHR results in aberrant myogenesis in the developing somites and neural tube, leading to ectopic skeletal muscle formation in the mature spinal cord. Gene expression profiling indicated that PAX3-FKHR expression in the developing neural tube induces a myogenic pattern of gene expression at the expense of the normal neurogenic program. Somite defects in PAX3-FKHR transgenic animals resulted in skeletal malformations that included rib fusions and mis-attachments. As opposed to the neural tube defects, the severity of the rib phenotype was rescued by reducing Pax3 levels through mating with Splotch mice. Embryos from the transgenic line expressing the highest levels of PAX3-FKHR had severe neural tube defects, including exencephaly, and almost half of the embryos died between gestational ages E13.5-E15.5. Nearly all of the embryos that survived to term died after birth due to severe spina bifida, rather than the absence of a muscular diaphragm. These studies reveal a prominent role for PAX3-FKHR in disrupting Pax3 functions and in deregulating skeletal muscle development, suggesting that this fusion protein plays a critical role in the pathogenesis of␣alveolar RMS by influencing the commitment␣and differentiation of the myogenic cell lineage.

Prostate tumors are complex entities composed of malignant cells mixed and interacting with nonmalignant cells. However, molecular analyses by standard gene expression profiling are limited because spatial information and nontumor cell types are lost in sample preparation. We scored 88 prostate specimens for relative content of tumor, benign hyperplastic epithelium, stroma, and dilated cystic glands. The proportions of these cell types were then linked in silico to gene expression levels determined by microarray analysis, revealing unique cell-specific profiles. Gene expression differences for malignant and nonmalignant epithelial cells (tumor versus benign hyperplastic epithelium) could be identified without being confounded by contributions from stroma that dominate many samples or sacrificing possible paracrine influences. Cell-specific expression of selected genes was validated by immunohistochemistry and quantitative PCR. The results provide patterns of gene expression for these three lineages with relevance to pathogenetic, diagnostic, and therapeutic considerations.

Forkhead transcription factors of the FoxO subfamily are emerging as a shared component among pathways regulating diverse cellular functions, such as differentiation, metabolism, proliferation, and survival. Their transcriptional output is controlled via a two-tiered mechanism of phosphorylation and acetylation. Modest alterations of this balance can result in profound effects. The gamut of phenotypes runs from protection against diabetes and predisposition to neoplasia, conferred by FoxO loss of function, to increased cellular survival and a marked catabolic response associated with gain of function.

Two recent reports (Seoane et al., 2004; Hu et al., 2004) reveal new roles for FoxO proteins in cell proliferation and tumorigenesis. Seoane and colleagues show that FoxO proteins play key roles in the TGFbeta-dependent activation of p21Cip1 by partnering with Smad3 and Smad4. FoxG1, a protein from a distinct Fox subfamily, binds FoxO/Smad complexes and blocks p21Cip1 expression. These interactions establish a relationship between the PI3K pathway, FoxG1, and the TGFbeta/Smad pathways. The second report identifies IKB kinase as a negative regulator of FoxO proteins, suggesting a mechanism for relieving negative regulation of cell cycle and promoting tumor cell proliferation.