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Gerhard H. Braus - One of the best experts on this subject based on the ideXlab platform.
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SCF Ubiquitin Ligase F-box Protein Fbx15 Controls Nuclear Co-repressor Localization, Stress Response and Virulence of the Human Pathogen Aspergillus fumigatus.
PLoS pathogens, 2016Co-Authors: Bastian Jöhnk, Özgür Bayram, Anja Abelmann, Thorsten Heinekamp, Derek J. Mattern, Axel A. Brakhage, Ilse D. Jacobsen, Oliver Valerius, Gerhard H. BrausAbstract:F-box proteins share the F-box domain to connect substrates of E3 SCF ubiquitin RING ligases through the adaptor Skp1/A to Cul1/A scaffolds. F-box protein Fbx15 is part of the general stress response of the human pathogenic mold Aspergillus fumigatus. Oxidative stress induces a transient peak of Fbx15 expression, resulting in 3x elevated Fbx15 protein levels. During non-stress conditions Fbx15 is phosphorylated and F-box mediated interaction with SkpA preferentially happens in smaller subpopulations in the cytoplasm. The F-box of Fbx15 is required for an appropriate oxidative stress response, which results in rapid dephosphorylation of Fbx15 and a shift of the cellular interaction with SkpA to the nucleus. Fbx15 binds SsnF/Ssn6 as part of the RcoA/Tup1-SsnF/Ssn6 co-repressor and is required for its correct nuclear localization. Dephosphorylated Fbx15 prevents SsnF/Ssn6 nuclear localization and results in the derepression of gliotoxin gene expression. Fbx15 deletion mutants are unable to infect immunocompromised mice in a model for invasive aspergillosis. Fbx15 has a novel dual molecular function by controlling transcriptional repression and being part of SCF E3 ubiquitin ligases, which is essential for stress response, gliotoxin production and virulence in the opportunistic human pathogen A. fumigatus.
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Fbx15 is required for nuclear localization of SsnF.
2016Co-Authors: Bastian Jöhnk, Özgür Bayram, Anja Abelmann, Thorsten Heinekamp, Derek J. Mattern, Axel A. Brakhage, Ilse D. Jacobsen, Oliver Valerius, Gerhard H. BrausAbstract:(A) Fluorescence microscopy of SsnF-GFP in wild type or ΔFbx15 background revealed that SsnF-GFP was localized to the periphery of the nuclei in ΔFbx15, whereas it was inside the nuclei in wild type. (B) SsnF-GFP localization in wild type Fbx15-RFP, unphosphorylated Fbx15[S468|9A]-RFP or phosphorylated Fbx15[S469D]-RFP in backgrounds before and after H2O2-treatment. Under normal growth conditions SsnF is nuclear in the presence of either the wild type or the phosphate mimicking Fbx15[S469D] variant, whereas SsnF-GFP accumulates at the nuclear envelope in presence of the non-phosphorylatable Fbx15[S468|9A]. Upon oxidative stress, the dephosphorylation of wild type Fbx15 leads to similar subpopulations of SsnF like the unphosphorylated Fbx15[S468|9A]. In phosphorylated Fbx15[S469D] SsnF enters the nucleus even after H2O2-treatment. (C) Growth tests of A. fumigatus strains expressing either wild type Fbx15::rfp (AfGB98), rfp tagged phosphovariants of Fbx15 (AfGB101 and AfGB102) or corresponding rfp-tagged versions of Fbx15 that lack the F-box domain (AfGB125, AfGB126 and AfGB127). 5 x 103 conidia were spotted on minimal medium (MM) plate, supplemented with 2 mM H2O2 for providing oxidative stress, and grown for four days at 37°C.
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Fbx15 interacts with the transcriptional repressor subunit SsnF without affecting its stability.
2016Co-Authors: Bastian Jöhnk, Özgür Bayram, Anja Abelmann, Thorsten Heinekamp, Derek J. Mattern, Axel A. Brakhage, Ilse D. Jacobsen, Oliver Valerius, Gerhard H. BrausAbstract:(A) Scheme of a subset of interacting proteins of Fbx15 and SconB based on LC-MS/MS identifications. Shown are presumably nuclear proteins, which are either exclusively found for each F-box protein or which were found for both. Further Fbx15-TAP co-purified proteins included three CSN subunits and the cyclin-dependent kinase NimX. (B) BiFC of Fbx15 and SsnF showed a predominantly cytoplasmic YFP-signal, whereas unphosphorylated Fbx15[S468|9A] interacted with SsnF primarily in the nucleus. (C) Protein stability assays of SsnF-GFP in wild type, ΔFbx15 or Fbx15 overexpression backgrounds with cultures exposed to cycloheximide (CHX) showed no Fbx15-dependent SsnF stability changes.
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Aspergillus fumigatus F-box domain protein Fbx15 is indispensible for oxidative stress response.
2016Co-Authors: Bastian Jöhnk, Özgür Bayram, Anja Abelmann, Thorsten Heinekamp, Derek J. Mattern, Axel A. Brakhage, Ilse D. Jacobsen, Oliver Valerius, Gerhard H. BrausAbstract:(A) Fbx15 genomic locus, structure and transcripts. (B) The primary sequence of A. fumigatus Fbx15 F-box domain was compared with fungal homologues by ClustalW alignment. Highly conserved amino acids are marked in white; an arrow indicates the characteristic proline residue at position seven of the F-box domain. (C) fbx deletion mutants ΔFbx15 and to less extent Δfbx23 and ΔgrrA showed increased oxidative stress sensitivity, which could be complemented by reintroduction of the wild type fbx-genes. (D) ΔFbx15 mutant showed hyphal defects at low H2O2 concentrations. (E) Expression levels of cat1 encoding a mycelial catalase were increased in the ΔFbx15 mutant, whereas wild type and complemented strain showed only slightly elevated cat1 mRNA levels after H2O2 exposure. Mean values ± s.d. of N = 3 independent experiments are shown. P-values were calculated using two-sample t-test (**P
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Phosphorylated or unphosphorylated Fbx15 can interact with SkpA and CulA.
2016Co-Authors: Bastian Jöhnk, Özgür Bayram, Anja Abelmann, Thorsten Heinekamp, Derek J. Mattern, Axel A. Brakhage, Ilse D. Jacobsen, Oliver Valerius, Gerhard H. BrausAbstract:(A) Immunohybridization of Fbx15-RFP and phosphomutant versions of Fbx15, which mimic either an unphosphorylated Fbx15[S468|9A]-RFP or a constant phosphorylation Fbx15[S469D]-RFP before and after H2O2-mediated oxidative stress. (B) Scheme of an active SCF-complex. (C) Heatmap of co-purified SCF subunits for RFP-tagged Fbx15 and phosphomutant versions after oxidative stress.
Shinya Yamanaka - One of the best experts on this subject based on the ideXlab platform.
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generation of germline competent induced pluripotent stem cells
Nature, 2007Co-Authors: Keisuke Okita, Tomoko Ichisaka, Shinya YamanakaAbstract:We have previously shown that pluripotent stem cells can be induced from mouse fibroblasts by retroviral introduction of Oct3/4 (also called Pou5f1), Sox2, c-Myc and Klf4, and subsequent selection for Fbx15 (also called Fbxo15) expression. These induced pluripotent stem (iPS) cells (hereafter called Fbx15 iPS cells) are similar to embryonic stem (ES) cells in morphology, proliferation and teratoma formation; however, they are different with regards to gene expression and DNA methylation patterns, and fail to produce adult chimaeras. Here we show that selection for Nanog expression results in germline-competent iPS cells with increased ES-cell-like gene expression and DNA methylation patterns compared with Fbx15 iPS cells. The four transgenes (Oct3/4, Sox2, c-myc and Klf4) were strongly silenced in Nanog iPS cells. We obtained adult chimaeras from seven Nanog iPS cell clones, with one clone being transmitted through the germ line to the next generation. Approximately 20% of the offspring developed tumours attributable to reactivation of the c-myc transgene. Thus, iPS cells competent for germline chimaeras can be obtained from fibroblasts, but retroviral introduction of c-Myc should be avoided for clinical application.
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Generation of germline-competent induced pluripotent stem cells
Nature, 2007Co-Authors: Keisuke Okita, Tomoko Ichisaka, Shinya YamanakaAbstract:The search for new ways of coaxing readily available cells to show the pluripotent activity of embryonic stem cells — the potential to differentiate into virtually any cell type — continues. The stakes are high, since if it can be achieved safely for human cells, cell transplantation therapy, even patient-specific therapy, will have come a step closer. Two groups now report an important advance in this direction: the creation of pluripotent stem cells from mouse fibroblasts. The epigenetic reprogramming requires the expression of four transcription factors, Oct3/4, Sox2, c-Myc and Klf4. The resulting cells resemble embryonic stem cells in both biological potency and epigenetic state. We have previously shown that pluripotent stem cells can be induced from mouse fibroblasts by retroviral introduction of Oct3/4 (also called Pou5f1), Sox2, c-Myc and Klf4, and subsequent selection for Fbx15 (also called Fbxo15 ) expression. These induced pluripotent stem (iPS) cells (hereafter called Fbx15 iPS cells) are similar to embryonic stem (ES) cells in morphology, proliferation and teratoma formation; however, they are different with regards to gene expression and DNA methylation patterns, and fail to produce adult chimaeras. Here we show that selection for Nanog expression results in germline-competent iPS cells with increased ES-cell-like gene expression and DNA methylation patterns compared with Fbx15 iPS cells. The four transgenes ( Oct3/4 , Sox2 , c-myc and Klf4 ) were strongly silenced in Nanog iPS cells. We obtained adult chimaeras from seven Nanog iPS cell clones, with one clone being transmitted through the germ line to the next generation. Approximately 20% of the offspring developed tumours attributable to reactivation of the c-myc transgene. Thus, iPS cells competent for germline chimaeras can be obtained from fibroblasts, but retroviral introduction of c-Myc should be avoided for clinical application. Four transcription factors Oct4, Sox2, c-Myc and Klf4 are known to convert fibroblasts to pluripotent stem cells, if Fbx 15 expression is also selected. But the induced stem cells were shown to be distinct from normal embryonic stem cells. However, if cells expressing Nanog and Oct4 are selected, then the reprogrammed fibroblasts are similar to embryonic stem cells in both biological potency and epigenetic state.
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Fbx15 is a novel target of oct3 4 but is dispensable for embryonic stem cell self renewal and mouse development
Molecular and Cellular Biology, 2003Co-Authors: Yoshimi Tokuzawa, Eiko Kaiho, Masayoshi Maruyama, Kazutoshi Takahashi, Kaoru Mitsui, Mitsuyo Maeda, Hitoshi Niwa, Shinya YamanakaAbstract:Embryonic stem (ES) cells are immortal and pluripotent cells derived from early mammalian embryos. Transcription factor Oct3/4 is essential for self-renewal of ES cells and early mouse development. However, only a few Oct3/4 target genes have been identified. In this study, we found that F-box-containing protein Fbx15 was expressed predominantly in mouse undifferentiated ES cells. Inactivation of Oct3/4 in ES cells led to rapid extinction of Fbx15 expression. Reporter gene analyses demonstrated that this ES cell-specific expression required an 18-bp enhancer element located approximately 500 nucleotides upstream from the transcription initiation site. The enhancer contained an octamer-like motif and an adjacent Sox-binding motif. Deletion or point mutation of either motif abolished the enhancer activity. The 18-bp fragment became active in NIH 3T3 cells when Oct3/4 and Sox2 were coexpressed. A gel mobility shift assay demonstrated cooperative binding of Oct3/4 and Sox2 to the enhancer sequence. In mice having a β-galactosidase gene knocked into the Fbx15 locus, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside staining was detected in ES cells, early embryos (two-cell to blastocyst stages), and testis tissue. Despite such specific expression of Fbx15, homozygous mutant mice showed no gross developmental defects and were fertile. Fbx15-null ES cells were normal in morphology, proliferation, and differentiation. These data demonstrate that Fbx15 is a novel target of Oct3/4 but is dispensable for ES cell self-renewal, development, and fertility.
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Fbx15 is a novel target of Oct3/4 but is dispensable for embryonic stem cell self-renewal and mouse development.
Molecular and cellular biology, 2003Co-Authors: Yoshimi Tokuzawa, Eiko Kaiho, Masayoshi Maruyama, Kazutoshi Takahashi, Kaoru Mitsui, Mitsuyo Maeda, Hitoshi Niwa, Shinya YamanakaAbstract:Embryonic stem (ES) cells are derived from mammalian blastocysts and maintain pluripotency, an ability to differentiate into all types of somatic and germ cells (32). Another important property of ES cells is their robust and infinite growth equivalent to tumor cells despite their normal karyotype. ES cells were developed from mouse blastocysts in 1981 (8, 15) and have been extensively used to generate knockout mice. Human ES cells were established in 1998 (33) and are considered promising sources for cell transplantation therapy. POU transcription factor Oct3/4 is expressed specifically in pluripotent cells, including ES cells, early embryos, and germ cells (27, 31). Targeted disruption of the Oct3/4 gene in the mouse results in early embryonic lethality (21). The inner cellular mass of Oct3/4-null blastocysts differentiates exclusively into trophoblasts. Furthermore, conditional deletion of Oct3/4 in ES cells leads to spontaneous differentiation into trophectoderm (25), demonstrating that Oct3/4 is essential for self-renewal of ES cells and mouse early development. Only a few Oct3/4 target genes have been identified. These include FGF-4 (4) and Rex-1 (2), in which Oct3/4 binds to an octamer motif, ATT(T/A)GCAT, located in regulatory elements. In FGF-4, SRY-related transcription factor Sox2 binds to a motif adjacent to the octamer sequence and synergistically activates transcription (5). In Rex-1, hypothetical factor ROX1 functions in a similar manner (2). It is not clear whether synergetic interaction with other transcription factors is common among target genes. Even consensus nucleotide sequences of Oct3/4-binding sites have not been fully determined. For example, the Oct3/4-binding site in UTF1 is one nucleotide different from the octamer sequence (22). Furthermore, it remains largely unknown how Oct3/4 maintains self-renewal of ES cells. Identification of novel Oct3/4 target genes is crucial to answering these questions. In this study, we utilized expression analyses, reporter gene analyses, and a gel mobility shift assay to demonstrate that Fbx15, which encodes an F-box-containing protein (35), is a novel target of Oct3/4. We also performed gene-targeting experiments to study physiological functions of Fbx15 in self-renewal of ES cells, mouse development, and fertility.
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Fbx15 is a novel target of oct3 4 but is dispensable for embryonic stem cell self renewal and mouse development
Molecular and Cellular Biology, 2003Co-Authors: Yoshimi Tokuzawa, Eiko Kaiho, Masayoshi Maruyama, Kazutoshi Takahashi, Kaoru Mitsui, Mitsuyo Maeda, Hitoshi Niwa, Shinya YamanakaAbstract:Embryonic stem (ES) cells are derived from mammalian blastocysts and maintain pluripotency, an ability to differentiate into all types of somatic and germ cells (32). Another important property of ES cells is their robust and infinite growth equivalent to tumor cells despite their normal karyotype. ES cells were developed from mouse blastocysts in 1981 (8, 15) and have been extensively used to generate knockout mice. Human ES cells were established in 1998 (33) and are considered promising sources for cell transplantation therapy. POU transcription factor Oct3/4 is expressed specifically in pluripotent cells, including ES cells, early embryos, and germ cells (27, 31). Targeted disruption of the Oct3/4 gene in the mouse results in early embryonic lethality (21). The inner cellular mass of Oct3/4-null blastocysts differentiates exclusively into trophoblasts. Furthermore, conditional deletion of Oct3/4 in ES cells leads to spontaneous differentiation into trophectoderm (25), demonstrating that Oct3/4 is essential for self-renewal of ES cells and mouse early development. Only a few Oct3/4 target genes have been identified. These include FGF-4 (4) and Rex-1 (2), in which Oct3/4 binds to an octamer motif, ATT(T/A)GCAT, located in regulatory elements. In FGF-4, SRY-related transcription factor Sox2 binds to a motif adjacent to the octamer sequence and synergistically activates transcription (5). In Rex-1, hypothetical factor ROX1 functions in a similar manner (2). It is not clear whether synergetic interaction with other transcription factors is common among target genes. Even consensus nucleotide sequences of Oct3/4-binding sites have not been fully determined. For example, the Oct3/4-binding site in UTF1 is one nucleotide different from the octamer sequence (22). Furthermore, it remains largely unknown how Oct3/4 maintains self-renewal of ES cells. Identification of novel Oct3/4 target genes is crucial to answering these questions. In this study, we utilized expression analyses, reporter gene analyses, and a gel mobility shift assay to demonstrate that Fbx15, which encodes an F-box-containing protein (35), is a novel target of Oct3/4. We also performed gene-targeting experiments to study physiological functions of Fbx15 in self-renewal of ES cells, mouse development, and fertility.
Bastian Jöhnk - One of the best experts on this subject based on the ideXlab platform.
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SCF Ubiquitin Ligase F-box Protein Fbx15 Controls Nuclear Co-repressor Localization, Stress Response and Virulence of the Human Pathogen Aspergillus fumigatus.
PLoS pathogens, 2016Co-Authors: Bastian Jöhnk, Özgür Bayram, Anja Abelmann, Thorsten Heinekamp, Derek J. Mattern, Axel A. Brakhage, Ilse D. Jacobsen, Oliver Valerius, Gerhard H. BrausAbstract:F-box proteins share the F-box domain to connect substrates of E3 SCF ubiquitin RING ligases through the adaptor Skp1/A to Cul1/A scaffolds. F-box protein Fbx15 is part of the general stress response of the human pathogenic mold Aspergillus fumigatus. Oxidative stress induces a transient peak of Fbx15 expression, resulting in 3x elevated Fbx15 protein levels. During non-stress conditions Fbx15 is phosphorylated and F-box mediated interaction with SkpA preferentially happens in smaller subpopulations in the cytoplasm. The F-box of Fbx15 is required for an appropriate oxidative stress response, which results in rapid dephosphorylation of Fbx15 and a shift of the cellular interaction with SkpA to the nucleus. Fbx15 binds SsnF/Ssn6 as part of the RcoA/Tup1-SsnF/Ssn6 co-repressor and is required for its correct nuclear localization. Dephosphorylated Fbx15 prevents SsnF/Ssn6 nuclear localization and results in the derepression of gliotoxin gene expression. Fbx15 deletion mutants are unable to infect immunocompromised mice in a model for invasive aspergillosis. Fbx15 has a novel dual molecular function by controlling transcriptional repression and being part of SCF E3 ubiquitin ligases, which is essential for stress response, gliotoxin production and virulence in the opportunistic human pathogen A. fumigatus.
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Stress Response SCF Ubiquitin Ligase F box Protein Fbx15 Controls Nuclear Co repressor Localization and Virulence of the Opportunistic Human Fungal Pathogen Aspergillus fumigatus
2016Co-Authors: Bastian JöhnkAbstract:Aspergillus fumigatus is the most prevalent cause for pulmonary infections in immuno-compromised patients. Virulence factors are often linked to developmental control mechanisms, which are often identified in the closely related model organism Aspergillus nidulans. This work presents the characterization of the F-box protein Fbx15 in A. fumigatus, which had been shown to have a crucial impact on development in A. nidulans. Deletion of Fbx15 resulted in severe growth defects under various stress conditions, including classical virulence factors like increased temperature, oxidative stress and amino acid starvation, whereas growth under standard conditions was not affected. Oxidative stress induces a transient peak of Fbx15 transcript expression leading to three-fold increased protein levels after 40 min. Fbx15 is a stable F-box protein with a half-life of more than 90 minutes. F box proteins normally act as substrate adaptors for SCF E3 ubiquitin ligases. Fbx15 is phosphorylated during non-stress conditions and interacts with the Skp1/A linker subunit of SCF complexes, preferentially in smaller subpopulations in the cytoplasm. The phosphorylated form of Fbx15 preferentially incorporates into SCF complexes. Oxidative stress results in rapid dephosphorylation of Fbx15. Fbx15 variants, which are unable to be phosphorylated, interact with Skp1/A primarily in the nucleus. Fbx15 recruits three subunits of the COP9 signalosome and proteins involved in transcription, translation, signal transduction, morphology or metabolism. Fbx15 binds the Ssn6/SsnF subunit of the conserved Tup1/RcoA-Ssn6/SsnF co-repressor and is required for its nuclear localization. Dephosphorylated Fbx15 interacts with Ssn6/SsnF in the nucleus and Fbx15-SsnF mediated control of gene repression is required to reduce the biosynthesis of gliotoxin. Fbx15 deletion strains are unable to infect immunocompromised mice in a model of invasive aspergillosis, supporting that Fbx15 is essential for virulence. This work suggests that Fbx15 is not only part of SCF E3 ubiquitin ligases but carries a novel second molecular function, which includes the physical interaction with the co-repressor subunit Ssn6/SsnF and control of its localization. This dual function results in a crucial role for Fbx15 in the control of oxidative stress response, secondary metabolism and virulence in the opportunistic human pathogen A. fumigatus.
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Fbx15 is required for nuclear localization of SsnF.
2016Co-Authors: Bastian Jöhnk, Özgür Bayram, Anja Abelmann, Thorsten Heinekamp, Derek J. Mattern, Axel A. Brakhage, Ilse D. Jacobsen, Oliver Valerius, Gerhard H. BrausAbstract:(A) Fluorescence microscopy of SsnF-GFP in wild type or ΔFbx15 background revealed that SsnF-GFP was localized to the periphery of the nuclei in ΔFbx15, whereas it was inside the nuclei in wild type. (B) SsnF-GFP localization in wild type Fbx15-RFP, unphosphorylated Fbx15[S468|9A]-RFP or phosphorylated Fbx15[S469D]-RFP in backgrounds before and after H2O2-treatment. Under normal growth conditions SsnF is nuclear in the presence of either the wild type or the phosphate mimicking Fbx15[S469D] variant, whereas SsnF-GFP accumulates at the nuclear envelope in presence of the non-phosphorylatable Fbx15[S468|9A]. Upon oxidative stress, the dephosphorylation of wild type Fbx15 leads to similar subpopulations of SsnF like the unphosphorylated Fbx15[S468|9A]. In phosphorylated Fbx15[S469D] SsnF enters the nucleus even after H2O2-treatment. (C) Growth tests of A. fumigatus strains expressing either wild type Fbx15::rfp (AfGB98), rfp tagged phosphovariants of Fbx15 (AfGB101 and AfGB102) or corresponding rfp-tagged versions of Fbx15 that lack the F-box domain (AfGB125, AfGB126 and AfGB127). 5 x 103 conidia were spotted on minimal medium (MM) plate, supplemented with 2 mM H2O2 for providing oxidative stress, and grown for four days at 37°C.
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Fbx15 interacts with the transcriptional repressor subunit SsnF without affecting its stability.
2016Co-Authors: Bastian Jöhnk, Özgür Bayram, Anja Abelmann, Thorsten Heinekamp, Derek J. Mattern, Axel A. Brakhage, Ilse D. Jacobsen, Oliver Valerius, Gerhard H. BrausAbstract:(A) Scheme of a subset of interacting proteins of Fbx15 and SconB based on LC-MS/MS identifications. Shown are presumably nuclear proteins, which are either exclusively found for each F-box protein or which were found for both. Further Fbx15-TAP co-purified proteins included three CSN subunits and the cyclin-dependent kinase NimX. (B) BiFC of Fbx15 and SsnF showed a predominantly cytoplasmic YFP-signal, whereas unphosphorylated Fbx15[S468|9A] interacted with SsnF primarily in the nucleus. (C) Protein stability assays of SsnF-GFP in wild type, ΔFbx15 or Fbx15 overexpression backgrounds with cultures exposed to cycloheximide (CHX) showed no Fbx15-dependent SsnF stability changes.
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Aspergillus fumigatus F-box domain protein Fbx15 is indispensible for oxidative stress response.
2016Co-Authors: Bastian Jöhnk, Özgür Bayram, Anja Abelmann, Thorsten Heinekamp, Derek J. Mattern, Axel A. Brakhage, Ilse D. Jacobsen, Oliver Valerius, Gerhard H. BrausAbstract:(A) Fbx15 genomic locus, structure and transcripts. (B) The primary sequence of A. fumigatus Fbx15 F-box domain was compared with fungal homologues by ClustalW alignment. Highly conserved amino acids are marked in white; an arrow indicates the characteristic proline residue at position seven of the F-box domain. (C) fbx deletion mutants ΔFbx15 and to less extent Δfbx23 and ΔgrrA showed increased oxidative stress sensitivity, which could be complemented by reintroduction of the wild type fbx-genes. (D) ΔFbx15 mutant showed hyphal defects at low H2O2 concentrations. (E) Expression levels of cat1 encoding a mycelial catalase were increased in the ΔFbx15 mutant, whereas wild type and complemented strain showed only slightly elevated cat1 mRNA levels after H2O2 exposure. Mean values ± s.d. of N = 3 independent experiments are shown. P-values were calculated using two-sample t-test (**P
Fuyuki Ishikawa - One of the best experts on this subject based on the ideXlab platform.
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oct 3 4 and sox2 regulate oct 3 4 gene in embryonic stem cells
Journal of Biological Chemistry, 2005Co-Authors: Sayaka Okumuranakanishi, Hitoshi Niwa, Motoki Saito, Fuyuki IshikawaAbstract:Oct-3/4 is a key transcriptional factor whose expression level governs the fate of primitive inner cell mass and embryonic stem (ES) cells. Previously, an upstream 3.3-kb distal enhancer (DE) fragment was identified to be responsible for the specific expression of mouse Oct-3/4 in the inner cell mass and ES cells. However, little is known about the cis-elements and trans-factors required for DE activity. In this study, we identified a novel cis-element, called Site 2B here, located approximately 30 bp downstream from Site 2A, which was previously revealed in DE by an in vivo chemical modification experiment. Using the luciferase reporter assay, we demonstrated that both Site 2A and Site 2B are necessary and sufficient for activating DE in the contexts of both the native Oct-3/4 promoter and the heterologous thymidine kinase minimal promoter. In an electrophoretic mobility shift assay we showed that Site 2B specifically binds to Oct-3/4 and Sox2 when ES-derived cell extracts were used, whereas Site 2A binds to a factor(s) present in both ES and NIH 3T3 cells. Furthermore, we showed that the physiological level of Oct-3/4 in ES cells is required for Site 2B-mediated DE activity using the inducible knock-out system of Oct-3/4 in ES cells. These results indicate that Oct-3/4 is a member of the gene family regulated by Oct-3/4 and Sox2, as reported before for the FGF-4, UTF1, Sox2, and Fbx15 genes. Thus, Oct-3/4 and Sox2 comprise a regulatory complex that controls the expression of genes important for the maintenance of the primitive state, including themselves. This autoregulatory circuit of the Sox2.Oct-3/4 complex may contribute to maintaining robustly the precise expression level of Oct-3/4 in primitive cells.
Hitoshi Niwa - One of the best experts on this subject based on the ideXlab platform.
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oct 3 4 and sox2 regulate oct 3 4 gene in embryonic stem cells
Journal of Biological Chemistry, 2005Co-Authors: Sayaka Okumuranakanishi, Hitoshi Niwa, Motoki Saito, Fuyuki IshikawaAbstract:Oct-3/4 is a key transcriptional factor whose expression level governs the fate of primitive inner cell mass and embryonic stem (ES) cells. Previously, an upstream 3.3-kb distal enhancer (DE) fragment was identified to be responsible for the specific expression of mouse Oct-3/4 in the inner cell mass and ES cells. However, little is known about the cis-elements and trans-factors required for DE activity. In this study, we identified a novel cis-element, called Site 2B here, located approximately 30 bp downstream from Site 2A, which was previously revealed in DE by an in vivo chemical modification experiment. Using the luciferase reporter assay, we demonstrated that both Site 2A and Site 2B are necessary and sufficient for activating DE in the contexts of both the native Oct-3/4 promoter and the heterologous thymidine kinase minimal promoter. In an electrophoretic mobility shift assay we showed that Site 2B specifically binds to Oct-3/4 and Sox2 when ES-derived cell extracts were used, whereas Site 2A binds to a factor(s) present in both ES and NIH 3T3 cells. Furthermore, we showed that the physiological level of Oct-3/4 in ES cells is required for Site 2B-mediated DE activity using the inducible knock-out system of Oct-3/4 in ES cells. These results indicate that Oct-3/4 is a member of the gene family regulated by Oct-3/4 and Sox2, as reported before for the FGF-4, UTF1, Sox2, and Fbx15 genes. Thus, Oct-3/4 and Sox2 comprise a regulatory complex that controls the expression of genes important for the maintenance of the primitive state, including themselves. This autoregulatory circuit of the Sox2.Oct-3/4 complex may contribute to maintaining robustly the precise expression level of Oct-3/4 in primitive cells.
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Fbx15 is a novel target of oct3 4 but is dispensable for embryonic stem cell self renewal and mouse development
Molecular and Cellular Biology, 2003Co-Authors: Yoshimi Tokuzawa, Eiko Kaiho, Masayoshi Maruyama, Kazutoshi Takahashi, Kaoru Mitsui, Mitsuyo Maeda, Hitoshi Niwa, Shinya YamanakaAbstract:Embryonic stem (ES) cells are immortal and pluripotent cells derived from early mammalian embryos. Transcription factor Oct3/4 is essential for self-renewal of ES cells and early mouse development. However, only a few Oct3/4 target genes have been identified. In this study, we found that F-box-containing protein Fbx15 was expressed predominantly in mouse undifferentiated ES cells. Inactivation of Oct3/4 in ES cells led to rapid extinction of Fbx15 expression. Reporter gene analyses demonstrated that this ES cell-specific expression required an 18-bp enhancer element located approximately 500 nucleotides upstream from the transcription initiation site. The enhancer contained an octamer-like motif and an adjacent Sox-binding motif. Deletion or point mutation of either motif abolished the enhancer activity. The 18-bp fragment became active in NIH 3T3 cells when Oct3/4 and Sox2 were coexpressed. A gel mobility shift assay demonstrated cooperative binding of Oct3/4 and Sox2 to the enhancer sequence. In mice having a β-galactosidase gene knocked into the Fbx15 locus, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside staining was detected in ES cells, early embryos (two-cell to blastocyst stages), and testis tissue. Despite such specific expression of Fbx15, homozygous mutant mice showed no gross developmental defects and were fertile. Fbx15-null ES cells were normal in morphology, proliferation, and differentiation. These data demonstrate that Fbx15 is a novel target of Oct3/4 but is dispensable for ES cell self-renewal, development, and fertility.
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Fbx15 is a novel target of Oct3/4 but is dispensable for embryonic stem cell self-renewal and mouse development.
Molecular and cellular biology, 2003Co-Authors: Yoshimi Tokuzawa, Eiko Kaiho, Masayoshi Maruyama, Kazutoshi Takahashi, Kaoru Mitsui, Mitsuyo Maeda, Hitoshi Niwa, Shinya YamanakaAbstract:Embryonic stem (ES) cells are derived from mammalian blastocysts and maintain pluripotency, an ability to differentiate into all types of somatic and germ cells (32). Another important property of ES cells is their robust and infinite growth equivalent to tumor cells despite their normal karyotype. ES cells were developed from mouse blastocysts in 1981 (8, 15) and have been extensively used to generate knockout mice. Human ES cells were established in 1998 (33) and are considered promising sources for cell transplantation therapy. POU transcription factor Oct3/4 is expressed specifically in pluripotent cells, including ES cells, early embryos, and germ cells (27, 31). Targeted disruption of the Oct3/4 gene in the mouse results in early embryonic lethality (21). The inner cellular mass of Oct3/4-null blastocysts differentiates exclusively into trophoblasts. Furthermore, conditional deletion of Oct3/4 in ES cells leads to spontaneous differentiation into trophectoderm (25), demonstrating that Oct3/4 is essential for self-renewal of ES cells and mouse early development. Only a few Oct3/4 target genes have been identified. These include FGF-4 (4) and Rex-1 (2), in which Oct3/4 binds to an octamer motif, ATT(T/A)GCAT, located in regulatory elements. In FGF-4, SRY-related transcription factor Sox2 binds to a motif adjacent to the octamer sequence and synergistically activates transcription (5). In Rex-1, hypothetical factor ROX1 functions in a similar manner (2). It is not clear whether synergetic interaction with other transcription factors is common among target genes. Even consensus nucleotide sequences of Oct3/4-binding sites have not been fully determined. For example, the Oct3/4-binding site in UTF1 is one nucleotide different from the octamer sequence (22). Furthermore, it remains largely unknown how Oct3/4 maintains self-renewal of ES cells. Identification of novel Oct3/4 target genes is crucial to answering these questions. In this study, we utilized expression analyses, reporter gene analyses, and a gel mobility shift assay to demonstrate that Fbx15, which encodes an F-box-containing protein (35), is a novel target of Oct3/4. We also performed gene-targeting experiments to study physiological functions of Fbx15 in self-renewal of ES cells, mouse development, and fertility.
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Fbx15 is a novel target of oct3 4 but is dispensable for embryonic stem cell self renewal and mouse development
Molecular and Cellular Biology, 2003Co-Authors: Yoshimi Tokuzawa, Eiko Kaiho, Masayoshi Maruyama, Kazutoshi Takahashi, Kaoru Mitsui, Mitsuyo Maeda, Hitoshi Niwa, Shinya YamanakaAbstract:Embryonic stem (ES) cells are derived from mammalian blastocysts and maintain pluripotency, an ability to differentiate into all types of somatic and germ cells (32). Another important property of ES cells is their robust and infinite growth equivalent to tumor cells despite their normal karyotype. ES cells were developed from mouse blastocysts in 1981 (8, 15) and have been extensively used to generate knockout mice. Human ES cells were established in 1998 (33) and are considered promising sources for cell transplantation therapy. POU transcription factor Oct3/4 is expressed specifically in pluripotent cells, including ES cells, early embryos, and germ cells (27, 31). Targeted disruption of the Oct3/4 gene in the mouse results in early embryonic lethality (21). The inner cellular mass of Oct3/4-null blastocysts differentiates exclusively into trophoblasts. Furthermore, conditional deletion of Oct3/4 in ES cells leads to spontaneous differentiation into trophectoderm (25), demonstrating that Oct3/4 is essential for self-renewal of ES cells and mouse early development. Only a few Oct3/4 target genes have been identified. These include FGF-4 (4) and Rex-1 (2), in which Oct3/4 binds to an octamer motif, ATT(T/A)GCAT, located in regulatory elements. In FGF-4, SRY-related transcription factor Sox2 binds to a motif adjacent to the octamer sequence and synergistically activates transcription (5). In Rex-1, hypothetical factor ROX1 functions in a similar manner (2). It is not clear whether synergetic interaction with other transcription factors is common among target genes. Even consensus nucleotide sequences of Oct3/4-binding sites have not been fully determined. For example, the Oct3/4-binding site in UTF1 is one nucleotide different from the octamer sequence (22). Furthermore, it remains largely unknown how Oct3/4 maintains self-renewal of ES cells. Identification of novel Oct3/4 target genes is crucial to answering these questions. In this study, we utilized expression analyses, reporter gene analyses, and a gel mobility shift assay to demonstrate that Fbx15, which encodes an F-box-containing protein (35), is a novel target of Oct3/4. We also performed gene-targeting experiments to study physiological functions of Fbx15 in self-renewal of ES cells, mouse development, and fertility.
