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PubMed2004

AcFKH1, a novel member of the forkhead family, associates with the RFX transcription factor CPCR1 in the cephalosporin C-producing fungus Acremonium chrysogenum.

In the filamentous fungus Acremonium chrysogenum, a complex regulatory network of transcription factors controls the expression of at least seven cephalosporin C biosynthesis genes. The RFX transcription factor CPCR1 binds to regulatory sequences in the promoter region of cephalosporin C biosynthesis genes, and is involved in the transcriptional regulation of the pcbC gene which encodes isopenicillin N synthase. In this study, we used CPCR1 in a yeast two-hybrid screen to identify potential protein interaction partners. A cDNA was identified, encoding the C-terminal part (pos. 438-665) of the novel forkhead protein, AcFKH1. The full-length AcFKH1 amino acid sequence is 665 residues and shares between 31% and 60% identity with forkhead protein sequences in the genomes of Aspergillus nidulans, Fusarium graminearum, and Neurospora crassa. AcFKH1 is characterized by two conserved domains, the N-terminal forkhead-associated domain (FHA), which might be involved in phospho-protein interactions, and the C-terminal DNA-binding domain (FKH) of the winged helix/forkhead type. The two-hybrid system was also used to map the protein domains required for the interaction of transcription factors CPCR1 and AcFKH1. The observed interaction between CPCR1 and the C-terminus of AcFKH1 in the yeast system was verified in vitro in a GST pulldown assay. Using gel retardation analysis, the DNA-binding properties of the fungal forkhead protein AcFKH1 were investigated. AcFKH1 recognizes two forkhead consensus binding sites within the 1.2 kb promoter region of the divergently oriented cephalosporin biosynthesis gene pair pcbAB-pcbC from A. chrysogenum. Additionally, AcFKH1 is able to bind with high affinity to the SWI5-binding site of the yeast FKH2 protein.

Acremonium↗
PubMed2006

Structure of the forkhead domain of FOXP2 bound to DNA.

FOXP (FOXP1-4) is a newly defined subfamily of the forkhead box (FOX) transcription factors. A mutation in the FOXP2 forkhead domain cosegregates with a severe speech disorder, whereas several mutations in the FOXP3 forkhead domain are linked to the IPEX syndrome in human and a similar autoimmune phenotype in mice. Here we report a 1.9 A crystal structure of the forkhead domain of human FOXP2 bound to DNA. This structure allows us to revise the previously proposed DNA recognition mechanism and provide a unifying model of DNA binding for the FOX family of proteins. Our studies also reveal that the FOXP2 forkhead domain can form a domain-swapped dimer, made possible by a strategic substitution of a highly conserved proline in conventional FOX proteins with alanine in the P subfamily. Disease-causing mutations in FOXP2 and FOXP3 map either to the DNA binding surface or the domain-swapping dimer interface, functionally corroborating the crystal structure.

Amino Acid Sequence↗
PubMed2004

Vascular endothelial growth factor-mediated induction of manganese superoxide dismutase occurs through redox-dependent regulation of forkhead and IkappaB/NF-kappaB.

The mitochondrial antioxidant manganese superoxide dismutase (Mn-SOD) plays a critical cytoprotective role against oxidative stress. Vascular endothelial growth factor (VEGF) was shown previously to induce expression of Mn-SOD in endothelial cells by a NADPH oxidase-dependent mechanism. The goal of the current study was to determine the transcriptional mechanisms underlying this phenomenon. VEGF resulted in protein kinase C-dependent phosphorylation of IkappaB and subsequent translocation of p65 NF-kappaB into the nucleus. Overexpression of constitutively active IkappaB blocked VEGF stimulation of Mn-SOD. In transient transfection assays, VEGF increased Mn-SOD promoter activity, an effect that was dependent on a second intronic NF-kappaB consensus motif. In contrast, VEGF-mediated induction of Mn-SOD was enhanced by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and by dominant negative Akt and was decreased by constitutively active Akt. Overexpression of a constitutively active (phosphorylation-resistant) form of FKHRL1 (TMFKHRL1) resulted in increased Mn-SOD expression, suggesting that the negative effect of PI3K-Akt involves attenuation of forkhead activity. In co-transfection assays, the Mn-SOD promoter was transactivated by TMFKHRL1. Flavoenzyme inhibitor, diphenyleneiodonium (DPI), and antisense oligonucleotides against p47phox (AS-p47phox) inhibited VEGF stimulation of IkappaB/NF-kappaB and forkhead phosphorylation, supporting a role for NADPH oxidase activity in both signaling pathways. Like VEGF, hepatocyte growth factor (HGF) activated the PI3K-Akt-forkhead pathway. However, HGF-PI3K-Akt-forkhead signaling was insensitive to diphenyleneiodonium and AS-p47phox. Moreover, HGF failed to induce phosphorylation of IkappaB/NF-kappaB or nuclear translocation of NF-kappaB and had no effect on Mn-SOD expression. Together, these data suggest that VEGF is uniquely coupled to Mn-SOD expression through growth factor-specific reactive oxygen species (ROS)-sensitive positive (protein kinase C-NF-kappaB) and negative (PI3K-Akt-forkhead) signaling pathways.

Active Transport, Cell Nucleus↗
PubMed2002

Common mechanism for oncogenic activation of MLL by forkhead family proteins.

The mixed lineage leukemia (MLL) gene undergoes fusions with a diverse set of genes as a consequence of chromosomal translocations in acute leukemias. Two of these partner genes code for members of the forkhead subfamily of transcription factors designated FKHRL1 and AFX. We demonstrate here that MLL-FKHRL1 enhances the self-renewal of murine myeloid progenitors in vitro and induces acute myeloid leukemias in syngeneic mice. The long latency (mean = 157 days), reduced penetrance, and hematologic features of the leukemias were very similar to those observed for the forkhead fusion protein MLL-AFX and contrasted with the more aggressive features of leukemias induced by MLL-AF10. Transformation mediated by MLL-forkhead fusion proteins required 2 conserved transcriptional effector domains (CR2 and CR3), each of which alone was not sufficient to activate MLL. A synthetic fusion of MLL with FKHR, a third mammalian forkhead family member that contains both effector domains, was also capable of transforming hematopoietic progenitors in vitro. A comparable requirement for 2 distinct transcriptional effector domains was also displayed by VP16, which required its proximal minimal transactivation domain (MTD/H1) and distal H2 domain to activate the oncogenic potential of MLL. The functional importance of CR2 was further demonstrated by its ability to substitute for H2 of VP16 in domain-swapping experiments to confer oncogenic activity on MLL. Our results, based on bona fide transcription factors as partners for MLL, unequivocally establish a transcriptional effector mechanism to activate its oncogenic potential and further support a role for fusion partners in determining pathologic features of the leukemia phenotype.

Animals↗
PubMed1993

Six members of the mouse forkhead gene family are developmentally regulated.

The 110-aa forkhead domain defines a class of transcription factors that have been shown to be developmentally regulated in Drosophila melanogaster and Xenopus laevis. The forkhead domain is necessary and sufficient for target DNA binding as shown for the rat hepatic nuclear factor 3 (HNF3) gene family. We have cloned six forkhead gene family members from a mouse genomic library in addition to the mouse equivalents of the genes for HNF3 alpha, -beta, and -gamma. The six genes, termed fkh-1 to fkh-6, share a high degree of similarity with the Drosophila forkhead gene, having 57-67% amino acid identity within the forkhead domain. fkh-1 seems to be the mammalian homologue of the Drosophila FD1 gene, as the sequences are 86% identical. fkh-1 to fkh-6 show distinct spatial patterns of expression in adult tissues and are expressed during embryogenesis.

Amino Acid Sequence↗
PubMed1994

Cloning and characterization of seven human forkhead proteins: binding site specificity and DNA bending.

The forkhead domain is a monomeric DNA binding motif that defines a rapidly growing family of eukaryotic transcriptional regulators. Genetic and biochemical data suggest a central role in embryonic development for genes encoding forkhead proteins. We have used PCR and low stringency hybridization to isolate clones from human cDNA and genomic libraries that represent seven novel forkhead genes, freac-1 to freac-7. The spatial patterns of expression for the seven freac genes range from specific for a single tissue to nearly ubiquitous. The DNA binding specificities of four of the FREAC proteins were determined by selection of binding sites from random sequence oligonucleotides. The binding sites for all four FREAC proteins share a core sequence, RTAAAYA, but differ in the positions flanking the core. Domain swaps between two FREAC proteins identified two subregions within the forkhead domain as responsible for creating differences in DNA binding specificity. Applying a circular permutation assay, we show that binding of FREAC proteins to their cognate sites results in bending of the DNA at an angle of 80-90 degrees.

Amino Acid Sequence↗
PubMed1995

Chromosomal localization of six human forkhead genes, freac-1 (FKHL5), -3 (FKHL7), -4 (FKHL8), -5 (FKHL9), -6 (FKHL10), and -8 (FKHL12).

The forkhead family of transcription factors has been shown to be involved in the regulation of embryonic development in organisms from Drosophila to humans. Genes encoding forkhead proteins are also emerging as a new class of oncogenes with relevance for human cancer. We have used a combination of fluorescence in situ hybridization and somatic cell hybrids to map the chromosomal localizations of five previously cloned human forkhead genes, freac-1, -3, -4, -5, and -6. (HGMW-approved symbols FKHL5, FKHL7, FKHL8, FKHL9 and FKHL10). We also report the sequence and the chromosomal position of a novel human forkhead gene, freac-8 (FKHL12). The freac genes have been localized to the following positions: freac-1, 16q24; freac-3, 6p25; freac-4, 5q12-q13; freac-5, 9 pericen; freac-6, 5q34, and freac-8, 1p32.

Amino Acid Sequence↗
PubMed1998

Mouse forkhead (winged helix) gene LUN encodes a transactivator that acts in the lung.

Many transcription factors involved in the determination and maintenance of cell fates share DNA-binding motifs to form gene families. The most recently discovered evolutionarily conserved DNA-binding domain of 100 amino acids, termed the forkhead domain, emerged from a sequence comparison of the rat transcription factor HNF-3 alpha and the homeotic gene fork head of Drosophila. Here we describe the isolation of a new mouse forkhead gene named LUN. Of a total of five forkhead genes in the lung, the LUN gene was uniquely expressed in the bronchiolar epithelium and in type II pneumocytes. We found that the LUN protein has forkhead domain that is identical to the HFH8 gene and a C-terminal region that is very similar to the HFH8 gene. Based on the structural similarity of the two proteins, we investigated the structure-function relationship of the LUN protein in two kinds of promoters and found that regions C and D work differently as activation domains, namely that region C acts autonomously but region D cannot work without region C. These results suggest that the LUN gene may play an important role as a transactivator in the determination and maintenance of some types of cells in the lung.

Animals↗
PubMed2005

Forkhead transcription factors in immunology.

The forkhead (Fox) gene family comprises a diverse group of "winged-helix" transcription factors that play important roles in development, metabolism, cancer and aging. Recently, several forkhead genes have been demonstrated to play critical roles in lymphocyte development and effector function, including Foxp3 in the development of regulatory T cells, Foxj1 and Foxo3a in the regulation of CD4+ T cell tolerance, and Foxn1 in thymic development. Roles for other forkhead genes have also been proposed, including Foxp1 in macrophage differentiation, Foxq1 in natural killer cell effector function and Foxd2 in T cell activation. Thus, forkhead genes promise insight into the mechanisms of immunoregulation in several immune cell lineages, and their dysregulation likely contributes to the pathogenesis of several immunological disorders, suggesting that their study will lead to the development of novel therapeutic agents.

Animals↗
PubMed2003

The forkhead gene family of Caenorhabditis elegans.

The forkhead genes encode a family of transcriptional regulators with important roles in the control of animal development. The genomic sequence has revealed Caenorhabditis elegans has 15 forkhead genes of which six had been experimentally characterised previously. The remaining nine have now been investigated by RNAi and reporter gene expression pattern analysis. Two (B0286.5 and F26B1.7) have key developmental roles, four (F26D12.1, K03C7.2, F40H3.4 and C25A1.2) are expressed in, and may function in, the nervous system and three (C29F7.4, C29F7.5 and F26A1.2), which are closely related to each other, do not appear to be essential. Conservation with forkhead genes in the closely related nematode Caenorhabditis briggsae does not appear to correspond completely with demonstrated functionality in C. elegans. Comparisons of expression patterns and RNAi/mutant phenotypes for all 15 C. elegans forkhead genes reveal the potential complexity of functional interactions for this gene family.

Animals↗
PubMed2001

Solution structure of the DNA binding domain of the human forkhead transcription factor AFX (FOXO4).

AFX is a human forkhead transcription factor. Based on results from studies of the orthologous transcription factor DAF-16 in Caenorhabditis elegans, it was suggested that some of the metabolic defects in both type I and type II diabetes may be due to unregulated activity of AFX. In the present study, we report the high-resolution NMR solution structure of the DNA binding domain of AFX. It is the first structure of the DNA binding domain from a small subfamily of forkhead transcription factors (i.e., AFX, FKHR, FKHRL1, FKHRL1P1, and FKHRP1). Despite rather low sequence identity for a protein within the forkhead family, the structure is remarkably similar to those of the DNA binding domains of HNF3-gamma and FREAC-11, and to a lesser extent the DNA binding domain of Genesis which displays a slightly altered orientation of the DNA recognition helix. The high degree of structural similarity between the DNA binding domains of different forkhead transcription factors implies that the repositioning of helix 3, observed for Genesis, cannot be a general feature for modulation of the DNA binding specificity. Other mechanisms that could influence the DNA binding specificity are discussed.

Amino Acid Motifs↗
PubMed2001

Regulation of collagenase expression during replicative senescence in human fibroblasts by Akt-forkhead signaling.

The expression of collagenase (matrix metalloproteinase 1) in human fibroblasts increases during aging both in vivo and in vitro. This age-associated increase in collagenase expression has been postulated to contribute to the age related decline in tissue function by increasing proteolysis of matrix components, but little is known regarding the regulation of collagenase expression. We examined the role that the serine/threonine kinase Akt plays in collagenase expression during in vitro senescence of WI-38 normal human lung fibroblast cells. Our results indicate that Akt-mediated signals, acting through the forkhead transcription factor FKHRL1, can regulate collagenase expression in WI-38 fibroblasts. Dominant negative forms of Akt increase collagenase promoter activity in early passage WI-38 fibroblasts, whereas an active form of Akt suppresses steady state levels of collagenase mRNA in senescent WI-38 fibroblasts. In addition, the activity of a synthetic promoter containing forkhead-specific binding sites, as measured by luciferase activity, is much higher in senescent cells compared with early passage WI-38 fibroblasts. These results indicate that members of the forkhead family of transcription factors play a role in the regulation of the collagenase promoter and that increased activity of forkhead transcription factors may underlie the increase in collagenase expression observed during replicative senescence.

Binding Sites↗
PubMed2002

Transforming growth factor-beta 2 is a transcriptional target for Akt/protein kinase B via forkhead transcription factor.

Tumors evade cell death by constitutively activating cell survival pathways and suppressing intrinsic death machinery. Activation of cell survival pathways leads to transcriptional repression of genes associated with cell death and activation of ones promoting anti-apoptosis. Akt/protein kinase B phosphorylates forkhead transcription factors and prevents their nuclear localization, leading to repression of genes involved in apoptosis, such as Fas ligand (FasL). Using bioinformatic approaches, we have identified three consensus sequences for forkhead transcription factor binding in transforming growth factor beta2 (TGF-beta2) promoter. TGF-beta inhibits cell proliferation and induces apoptosis in many cell types, and acquisition of TGF-beta resistance is linked to tumorigenesis. In this study, we show that activated Akt down-regulates TGF-beta2 promoter, and sequences within the promoter that are related to consensus forkhead binding sites are necessary for repression. Forkhead factor FKHRL1 binds in vitro to the three consensus sequences and can activate TGF-beta2 promoter in normal and Akt-transformed cell lines. In human breast and pancreatic tumors, activated Akt expression correlated with down-regulation of TGF-beta 2 mRNA levels. A number of tumor cells expressing activated Akt were responsive to TGF-beta addition, indicating the presence of an intact TGF-beta-signaling pathway. These results suggest that repression of TGF-beta 2 promoter activity in cells expressing activated Akt may play a role in promoting tumorigenesis and escape from the growth-inhibitory and/or apoptotic effects of TGF-beta.

Base Sequence↗
PubMed2006

A novel class of vascular endothelial growth factor-responsive genes that require forkhead activity for expression.

Recently, we have shown that transient phosphorylation and inhibition of the pro-apoptotic transcription factor, forkhead, by vascular endothelial growth factor (VEGF) is essential for endothelial cell (EC) survival and proliferation. The goal of the present study was to determine whether forkhead (FKHR) also plays a positive role in agonist-mediated gene induction. Human coronary artery ECs were transduced with adenovirus overexpressing constitutively active phosphorylation-resistant triple mutant FKHR or transfected with small interference RNA (siRNA) against FKHR. The cells were then treated in the absence or presence of VEGF and assayed for gene expression using quantitative real-time PCR and Northern blots analyses. The data revealed a novel set of VEGF-responsive genes that require FKHR activity for optimal expression in ECs, including bone morphogenic protein 2, cbp/p300-interacting transactivator 2, decay accelerating factor (DAF), vascular cell adhesion molecule-1 (VCAM-1), manganese superoxide dismutase, endothelial-specific molecule-1, RING1 and YY1-binding protein, and matrix metalloproteinase-10. Consistent with a positive role for FKHR in mediating VEGF induction of DAF and VCAM-1 mRNA, siRNA against FKHR attenuated the effect of VEGF on complement-mediated EC lysis and monocyte adhesion, respectively. VEGF induction of the forkhead-dependent genes was down-regulated by the NF-kappaB inhibitor, constitutively active Ad-IkappaB, and in some cases by the nuclear factor of activated T-cells (NF-AT) inhibitor, cyclosporin. Together, these findings suggest that the VEGF-forkhead signaling axis plays an important functional role in ECs beyond the regulation of cell survival/apoptosis and cell cycle.

Adenoviridae↗
PubMed2005

Phosphorylation of p66Shc and forkhead proteins mediates Abeta toxicity.

Excessive accumulation of amyloid beta-peptide (Abeta) plays an early and critical role in synapse and neuronal loss in Alzheimer's Disease (AD). Increased oxidative stress is one of the mechanisms whereby Abeta induces neuronal death. Given the lessened susceptibility to oxidative stress exhibited by mice lacking p66Shc, we investigated the role of p66Shc in Abeta toxicity. Treatment of cells and primary neuronal cultures with Abeta caused apoptotic death and induced p66Shc phosphorylation at Ser36. Ectopic expression of a dominant-negative SEK1 mutant or chemical JNK inhibition reduced Abeta-induced JNK activation and p66Shc phosphorylation (Ser36), suggesting that JNK phosphorylates p66Shc. Abeta induced the phosphorylation and hence inactivation of forkhead transcription factors in a p66Shc-dependent manner. Ectopic expression of p66ShcS36A or antioxidant treatment protected cells against Abeta-induced death and reduced forkhead phosphorylation, suggesting that p66Shc phosphorylation critically influences the redox regulation of forkhead proteins and underlies Abeta toxicity. These findings underscore the potential usefulness of JNK, p66Shc, and forkhead proteins as therapeutic targets for AD.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Structural and functional analyses of disease-causing missense mutations in the forkhead domain of FOXC1.

Five missense mutations (P79L, P79T, I91S, I91T and R127H) within the forkhead DNA-binding domain of the FOXC1 transcription factor, identified in patients with Axenfeld-Rieger (AR) malformations, were studied to identify the effects of these mutations on FOXC1 structure and function. Molecular modeling and threading analyses predict that the I91S and T mutations may generate local disruptions to the structure of the forkhead domain while the R127H mutation alters the electrostatic charge of the DNA binding surface of the forkhead domain. The P79L and T mutations are not predicted to grossly perturb the structure of the forkhead domain. Biological analyses indicate that all of these missense mutations cause a range of FOXC1 perturbations, including nuclear localization defects, reduced or abolished DNA binding capacity, and a reduction in the transactivation capacity of FOXC1. These experiments extend our previous hypothesis that reduced transactivation of appropriate target genes by FOXC1, underlie AR malformations mapping to human chromosome 6p25. Importantly, these results can also be applied to predict the consequences of the molecular effects of mutations of other FOX genes that have analogous missense mutations, including FOXP2, FOXE3 and FOXC2.

Amino Acid Sequence↗
PubMed2002

MLL-AFX requires the transcriptional effector domains of AFX to transform myeloid progenitors and transdominantly interfere with forkhead protein function.

MLL-AFX is a fusion gene created by t(X;11) chromosomal translocations in a subset of acute leukemias of either myeloid or lymphoid derivation. It codes for a chimeric protein consisting of MLL fused to AFX, a forkhead transcription factor that normally regulates genes involved in apoptosis and cell cycle progression. We demonstrate here that forced expression of MLL-AFX enhances the self-renewal of hematopoietic progenitors in vitro and induces acute myeloid leukemias after long latencies in syngeneic recipient mice. MLL-AFX interacts with the transcriptional coactivator CBP, which is also a fusion partner for MLL in human leukemias. A potent minimal transactivation domain (CR3) at the C terminus of AFX mediates interactions with the KIX domain of CBP and is necessary for transformation of myeloid progenitors by MLL-AFX. However, CR3 alone is not sufficient, suggesting that simple acquisition of a transactivation domain per se does not activate the oncogenic potential of MLL. Rather, two conserved transcriptional effector domains (CR2 and CR3) of AFX are required for full oncogenicity of MLL-AFX and also endow it with the potential to competitively interfere with transcription and apoptosis mediated by wild-type forkhead proteins. Furthermore, a dominant-negative mutant of AFX containing CR2 and CR3 enhances the growth of myeloid progenitors in vitro, although considerably less effectively than does MLL-AFX. Taken together, these data suggest that recruitment of transcriptional cofactors utilized by forkhead proteins is a critical requirement for oncogenic action of MLL-AFX, which may impact both MLL- and forkhead-dependent transcriptional pathways.

3T3 Cells↗
PubMed2006

Coordination of NF-kappaB and NFAT antagonism by the forkhead transcription factor Foxd1.

Forkhead transcription factors play critical roles in the maintenance of immune homeostasis. In this study, we demonstrate that this regulation most likely involves intricate interactions between the forkhead family members and inflammatory transcription factors: the forkhead member Foxd1 coordinates the regulation of the activity of two key inflammatory transcription factors, NF-AT and NF-kappaB, with Foxd1 deficiency resulting in multiorgan, systemic inflammation, exaggerated Th cell-derived cytokine production, and T cell proliferation in autologous MLRs. Foxd1-deficient T cells possess increased activity of both NF-AT and NF-kappaB: the former correlates with the ability of Foxd1 to regulate casein kinase 1, an NF-AT inhibitory kinase; the latter with the ability of Foxd1 to regulate Foxj1, which regulates the NF-kappaB inhibitory subunit IkappaB beta. Thus, Foxd1 modulates inflammatory reactions and prevents autoimmunity by directly regulating anti-inflammatory regulators of the NF-AT pathway, and by coordinating the suppression of the NF-kappaB pathway via Foxj1. These findings indicate the presence of a general network of forkhead proteins that enforce T cell quiescence.

Animals↗