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Angie Rizzino - One of the best experts on this subject based on the ideXlab platform.

  • Transcription Factor Sox-2 inhibits co-activator stimulated Transcription.
    Molecular reproduction and development, 2004
    Co-Authors: Cory T. Bernadt, Tamara K. Nowling, Angie Rizzino
    Abstract:

    Previous studies have shown that Transcription of the fibroblast growth Factor-4 (FGF-4) gene by early embryonic cells is dependent upon a powerful distal enhancer located 3 kb downstream of the Transcription start site within the untranslated region of the last exon. The Transcription Factors Sox-2 and Oct-3 cooperatively bind to critical cis-regulatory elements within the enhancer to synergistically activate Transcription. Moreover, the co-activator p300 can mediate the synergistic activity of Sox-2 and Oct-3, and p300 associates with the FGF-4 enhancer in vivo. Embryonal carcinoma (EC) cells have been used extensively as a model system to study the regulation of the FGF-4 gene during early development. Recently, it has been suggested that suboptimal levels of Sox-2 expression in F9 EC cells limit the Transcription of the FGF-4 gene. The studies presented in this report argue that Sox-2 levels are not limiting in F9 EC cells. Moreover, overexpression of Sox-2 in F9 EC cells decreases FGF-4 promoter activity. In addition, overexpression of Sox-2 in these cells inhibits activation by the co-activators p300, CBP, and OCA-B in a manner that requires the transactivation domain of Sox-2. These findings suggest that Sox-2 levels in F9 EC cells are regulated carefully to avoid interference with the Transcription of critical genes.

  • Regulation of the FGF-4 gene by a complex distal enhancer that functions in part as an enhanceosome.
    Gene, 2003
    Co-Authors: Troy A. Luster, Angie Rizzino
    Abstract:

    Abstract The exact mechanisms by which enhancers regulate Transcription are currently under investigation. For some genes, activation is accomplished by an intricate array of enhancer cis -regulatory elements that direct the assembly of a gene-specific activation complex known as an “enhanceosome”. Transcription of the fibroblast growth Factor-4 (FGF-4) gene during early development is controlled by a powerful distal enhancer located 3 kb downstream of the Transcription start site within the 3' untranslated region of the gene. Previous studies have shown that FGF-4 enhancer function is mediated by at least three critical positive cis -regulatory elements: an HMG, a POU, and a GT-box motif, which bind the Transcription Factors Sox-2, Oct-3, and Sp1/Sp3, respectively. In this study, we identify a second essential HMG motif within the FGF-4 enhancer that binds the Transcription Factor Sox-2. Moreover, we demonstrate that spatial alignment of the new HMG motif, relative the other enhancer cis -regulatory elements, is important. Based on findings presented in this report, and work published earlier, we propose that the previously identified core HMG and POU cis -regulatory elements of the FGF-4 enhancer are dependent on one another and function in an enhanceosome-like manner. In contrast, the HMG motif identified in this study is only partially dependent on the other enhancer cis -regulatory elements for its function.

  • Identification of the transactivation domain of the Transcription Factor Sox-2 and an associated co-activator.
    The Journal of biological chemistry, 2000
    Co-Authors: Tamara K. Nowling, Lance R. Johnson, Matthew S. Wiebe, Angie Rizzino
    Abstract:

    The importance of interactions between Sox and POU Transcription Factors in the regulation of gene expression is becoming increasingly apparent. Recently, many examples of the involvement of Sox-POU partnerships in Transcription have been discovered, including a partnership between Sox-2 and Oct-3. Little is known about the mechanisms by which these Factors modulate Transcription. To better understand the molecular interactions involved, we mapped the location of the transactivation domain of Sox-2. This was done in the context of its interaction with Oct-3, as well as its ability to transactivate as a fusion protein linked to the DNA-binding domain of Gal4. Both approaches demonstrated that Sox-2 contains a transactivation domain in its C-terminal half, containing a serine-rich region and the C terminus. We also determined that the viral oncoprotein E1a inhibits the ability of the Gal4/Sox-2 fusion protein to transactivate, as well as the Transcriptional activation mediated by the combined action of Sox-2 and Oct-3. In contrast, a mutant form of E1a, unable to bind p300, lacks both of these effects. Importantly, we determined that p300 overcomes the inhibitory effects of E1a in both assays. Together, these findings suggest that Sox-2 mediates its effects, at least in part, through the co-activator p300.

  • Role of the Transcription Factor Sox-2 in the expression of the FGF-4 gene in embryonal carcinoma cells.
    Molecular reproduction and development, 1998
    Co-Authors: Lance R. Johnson, Kimberly A. Lamb, Qingsheng Gao, Tamara K. Nowling, Angie Rizzino
    Abstract:

    It has been shown previously that the FGF-4 gene is regulated by a powerful downstream enhancer in embryonal carcinoma (EC) cells. This enhancer contains an essential HMG motif; however, the Transcription Factor that binds to the HMG motif in EC cells has not been determined definitively. In earlier studies, this HMG motif was shown to bind a heat-stable, redox-insensitive Factor expressed by F9 EC cells. Others have proposed that the Transcription Factor Sox-2 binds to the FGF-4 enhancer HMG motif. In this study, we demonstrate that the N-terminal half of Sox-2, which contains the DNA binding domain, binds to the FGF-4 enhancer HMG motif and we show that this binding is unaffected by heat and oxidation. In addition, we employed two experimental approaches to demonstrate that Sox-2 regulates the Transcription of the FGF-4 gene in EC cells. As part of these studies, an expression plasmid that codes for a dominant-negative form of Sox-2 was used in transient expression assays. In other experiments, a Sox-2 antisense expression plasmid was used. When co-transfected into F9 EC cells along with an FGF-4 promoter/reporter gene construct, each expression plasmid caused a significant reduction in reporter activity. Our studies also demonstrate that Sox-2 affects the expression of the FGF-4 gene in the multipotent EC cell line, P19. Taken together, these studies argue strongly that Sox-2 plays an important role in the expression of the FGF-4 gene in vivo.

Timothy L. Dunn - One of the best experts on this subject based on the ideXlab platform.

  • Trans-activation and DNA-binding properties of the Transcription Factor, Sox-18
    Nucleic acids research, 1995
    Co-Authors: Brett M. Hosking, Peter Koopman, George E. O. Muscat, Dennis H. Dowhan, Timothy L. Dunn
    Abstract:

    Sox-18 is a member of the Sox multi-gene family (Sry-related HMG-box gene). We have bacterially expressed this 378 amino acid protein and demonstrated sequence-specific binding to the Sox DNA-binding motif AACAAAG. A distinct 95 amino acid activation domain was mapped in Sox-18 using GAL4-Sox-18 fusions (amino acids 160-225). Furthermore, Sox-18 was capable of trans-activating gene expression through the AACAAA motif. Our results suggest that Sox-18 functions as a classical trans-activator of gene expression.

  • Trans-activation andDNA-binding properties ofthe Transcription Factor, Sox-18
    1995
    Co-Authors: Brett M. Hosking, Peter Koopman, Dennis H. Dowhanand, Timothy L. Dunn
    Abstract:

    Sox-18 isa memberoftheSoxmulti-gene family (Sry-related HMG-boxgene). We havebacterially expressed this 378aminoacidprotein anddemonstrated sequence-specifi binding totheSoxDNA-binding motif AACAAAG.A distinct 95aminoacidactivation domain wasmappedinSox-18 using GAL4-Sox-18 fusions (amino acids160-225). Furthermore, Sox-18 was capable oftrans-activating geneexpression through theAACAAAGmotif. Ourresults suggest that Sox-1 8functions asaclassical trans-activator ofgene expression.

Tamara K. Nowling - One of the best experts on this subject based on the ideXlab platform.

  • Transcription Factor Sox-2 inhibits co-activator stimulated Transcription.
    Molecular reproduction and development, 2004
    Co-Authors: Cory T. Bernadt, Tamara K. Nowling, Angie Rizzino
    Abstract:

    Previous studies have shown that Transcription of the fibroblast growth Factor-4 (FGF-4) gene by early embryonic cells is dependent upon a powerful distal enhancer located 3 kb downstream of the Transcription start site within the untranslated region of the last exon. The Transcription Factors Sox-2 and Oct-3 cooperatively bind to critical cis-regulatory elements within the enhancer to synergistically activate Transcription. Moreover, the co-activator p300 can mediate the synergistic activity of Sox-2 and Oct-3, and p300 associates with the FGF-4 enhancer in vivo. Embryonal carcinoma (EC) cells have been used extensively as a model system to study the regulation of the FGF-4 gene during early development. Recently, it has been suggested that suboptimal levels of Sox-2 expression in F9 EC cells limit the Transcription of the FGF-4 gene. The studies presented in this report argue that Sox-2 levels are not limiting in F9 EC cells. Moreover, overexpression of Sox-2 in F9 EC cells decreases FGF-4 promoter activity. In addition, overexpression of Sox-2 in these cells inhibits activation by the co-activators p300, CBP, and OCA-B in a manner that requires the transactivation domain of Sox-2. These findings suggest that Sox-2 levels in F9 EC cells are regulated carefully to avoid interference with the Transcription of critical genes.

  • Identification of the transactivation domain of the Transcription Factor Sox-2 and an associated co-activator.
    The Journal of biological chemistry, 2000
    Co-Authors: Tamara K. Nowling, Lance R. Johnson, Matthew S. Wiebe, Angie Rizzino
    Abstract:

    The importance of interactions between Sox and POU Transcription Factors in the regulation of gene expression is becoming increasingly apparent. Recently, many examples of the involvement of Sox-POU partnerships in Transcription have been discovered, including a partnership between Sox-2 and Oct-3. Little is known about the mechanisms by which these Factors modulate Transcription. To better understand the molecular interactions involved, we mapped the location of the transactivation domain of Sox-2. This was done in the context of its interaction with Oct-3, as well as its ability to transactivate as a fusion protein linked to the DNA-binding domain of Gal4. Both approaches demonstrated that Sox-2 contains a transactivation domain in its C-terminal half, containing a serine-rich region and the C terminus. We also determined that the viral oncoprotein E1a inhibits the ability of the Gal4/Sox-2 fusion protein to transactivate, as well as the Transcriptional activation mediated by the combined action of Sox-2 and Oct-3. In contrast, a mutant form of E1a, unable to bind p300, lacks both of these effects. Importantly, we determined that p300 overcomes the inhibitory effects of E1a in both assays. Together, these findings suggest that Sox-2 mediates its effects, at least in part, through the co-activator p300.

  • Role of the Transcription Factor Sox-2 in the expression of the FGF-4 gene in embryonal carcinoma cells.
    Molecular reproduction and development, 1998
    Co-Authors: Lance R. Johnson, Kimberly A. Lamb, Qingsheng Gao, Tamara K. Nowling, Angie Rizzino
    Abstract:

    It has been shown previously that the FGF-4 gene is regulated by a powerful downstream enhancer in embryonal carcinoma (EC) cells. This enhancer contains an essential HMG motif; however, the Transcription Factor that binds to the HMG motif in EC cells has not been determined definitively. In earlier studies, this HMG motif was shown to bind a heat-stable, redox-insensitive Factor expressed by F9 EC cells. Others have proposed that the Transcription Factor Sox-2 binds to the FGF-4 enhancer HMG motif. In this study, we demonstrate that the N-terminal half of Sox-2, which contains the DNA binding domain, binds to the FGF-4 enhancer HMG motif and we show that this binding is unaffected by heat and oxidation. In addition, we employed two experimental approaches to demonstrate that Sox-2 regulates the Transcription of the FGF-4 gene in EC cells. As part of these studies, an expression plasmid that codes for a dominant-negative form of Sox-2 was used in transient expression assays. In other experiments, a Sox-2 antisense expression plasmid was used. When co-transfected into F9 EC cells along with an FGF-4 promoter/reporter gene construct, each expression plasmid caused a significant reduction in reporter activity. Our studies also demonstrate that Sox-2 affects the expression of the FGF-4 gene in the multipotent EC cell line, P19. Taken together, these studies argue strongly that Sox-2 plays an important role in the expression of the FGF-4 gene in vivo.

Brett M. Hosking - One of the best experts on this subject based on the ideXlab platform.

  • Trans-activation and DNA-binding properties of the Transcription Factor, Sox-18
    Nucleic acids research, 1995
    Co-Authors: Brett M. Hosking, Peter Koopman, George E. O. Muscat, Dennis H. Dowhan, Timothy L. Dunn
    Abstract:

    Sox-18 is a member of the Sox multi-gene family (Sry-related HMG-box gene). We have bacterially expressed this 378 amino acid protein and demonstrated sequence-specific binding to the Sox DNA-binding motif AACAAAG. A distinct 95 amino acid activation domain was mapped in Sox-18 using GAL4-Sox-18 fusions (amino acids 160-225). Furthermore, Sox-18 was capable of trans-activating gene expression through the AACAAA motif. Our results suggest that Sox-18 functions as a classical trans-activator of gene expression.

  • Trans-activation andDNA-binding properties ofthe Transcription Factor, Sox-18
    1995
    Co-Authors: Brett M. Hosking, Peter Koopman, Dennis H. Dowhanand, Timothy L. Dunn
    Abstract:

    Sox-18 isa memberoftheSoxmulti-gene family (Sry-related HMG-boxgene). We havebacterially expressed this 378aminoacidprotein anddemonstrated sequence-specifi binding totheSoxDNA-binding motif AACAAAG.A distinct 95aminoacidactivation domain wasmappedinSox-18 using GAL4-Sox-18 fusions (amino acids160-225). Furthermore, Sox-18 was capable oftrans-activating geneexpression through theAACAAAGmotif. Ourresults suggest that Sox-1 8functions asaclassical trans-activator ofgene expression.

Chiou-fen Chuang - One of the best experts on this subject based on the ideXlab platform.

  • a universal transportin protein drives stochastic choice of olFactory neurons via specific nuclear import of a Sox 2 activating Factor
    Proceedings of the National Academy of Sciences of the United States of America, 2019
    Co-Authors: Amel Alqadah, Yi-wen Hsieh, Chieh Chang, Rui Xiong, Bluma J Lesch, Chiou-fen Chuang
    Abstract:

    Stochastic neuronal cell fate choice involving notch-independent mechanisms is a poorly understood biological process. The Caenorhabditis elegans AWC olFactory neuron pair asymmetrically differentiates into the default AWCOFF and induced AWCON subtypes in a stochastic manner. Stochastic choice of the AWCON subtype is established using gap junctions and SLO BK potassium channels to repress a calcium-activated protein kinase pathway. However, it is unknown how the potassium channel-repressed calcium signaling is translated into the induction of the AWCON subtype. Here, we identify a detailed working mechanism of how the homeodomain-like Transcription Factor NSY-7, previously described as a repressor in the maintenance of AWC asymmetry, couples SLO BK potassium channels to transactivation of Sox-2 expression for the induction of the AWCON subtype through the identification of a unique imb-2 (transportin 1) allele. imb-2 loss-of-function mutants are not viable; however, we identify a viable imb-2 allele from an unbiased forward genetic screen that reveals a specific role of imb-2 in AWC olFactory neuron asymmetry. IMB-2 specifically drives nuclear import of NSY-7 within AWC neurons to transactivate the expression of the high mobility group (HMG)-box Transcription Factor Sox-2 for the specification of the AWCON subtype. This study provides mechanistic insight into how NSY-7 couples SLO BK potassium channels to transactivation of Sox-2 expression for the induction of the AWCON subtype. Our findings also provide structure-function insight into a conserved amino acid residue of transportins in brain development and suggest its dysfunction may lead to human neurological disorders.

  • An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides.
    Journal of visualized experiments : JoVE, 2016
    Co-Authors: Yi-wen Hsieh, Amel Alqadah, Chiou-fen Chuang
    Abstract:

    Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG Transcription Factor Sox-2 on olFactory neuron type diversification. We found that mutant Sox-2G73E protein alters specific DNA binding activity, thereby causing olFactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/Sox-2 adjacent target sites and purified Sox-2 proteins (WT and mutant Sox-2G73E proteins) as a biological example.

  • Postmitotic diversification of olFactory neuron types is mediated by differential activities of the HMG-box Transcription Factor Sox-2.
    The EMBO journal, 2015
    Co-Authors: Amel Alqadah, Yi-wen Hsieh, Berta Vidal, Chieh Chang, Oliver Hobert, Chiou-fen Chuang
    Abstract:

    Abstract Diversification of neuron classes is essential for functions of the olFactory system, but the underlying mechanisms that generate individual olFactory neuron types are only beginning to be understood. Here we describe a role of the highly conserved HMG‐box Transcription Factor Sox‐2 in postmitotic specification and alternative differentiation of the Caenorhabditis elegans AWC and AWB olFactory neurons. We show that Sox‐2 partners with different Transcription Factors to diversify postmitotic olFactory cell types. Sox‐2 functions cooperatively with the OTX/OTD Transcription Factor CEH‐36 to specify an AWC “ground state,” and functions with the LIM homeodomain Factor LIM‐4 to suppress this ground state and drive an AWB identity instead. Our findings provide novel insights into combinatorial codes that drive terminal differentiation programs in the nervous system and reveal a biological function of the deeply conserved Sox2 protein that goes beyond its well‐known role in stem cell biology.