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FoxK mediates TGF-beta signalling during midgut differentiation in flies.

Casas-Tinto S, Gomez-Velazquez M, Granadino B, Fernandez-Funez P - J. Cell Biol. (2008)

Bottom Line: Genet.This regulatory activity does not require direct labial activation by the TGF-beta effector Mad.Thus, we propose that the combined activity of the TGF-beta target genes FoxK and Dfos is critical for the direct activation of lab in the endoderm.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Texas Medical Branch, Galveston, TX 77555, USA. scasas@cnio.es

ABSTRACT
Inductive signals across germ layers are important for the development of the endoderm in vertebrates and invertebrates (Tam, P.P., M. Kanai-Azuma, and Y. Kanai. 2003. Curr. Opin. Genet. Dev. 13:393-400; Nakagoshi, H. 2005. Dev. Growth Differ. 47:383-392). In flies, the visceral mesoderm secretes signaling molecules that diffuse into the underlying midgut endoderm, where conserved signaling cascades activate the Hox gene labial, which is important for the differentiation of copper cells (Bienz, M. 1997. Curr. Opin. Genet. Dev. 7:683-688). We present here a Drosophila melanogaster gene of the Fox family of transcription factors, FoxK, that mediates transforming growth factor beta (TGF-beta) signaling in the embryonic midgut endoderm. FoxK mutant embryos fail to generate midgut constrictions and lack Labial in the endoderm. Our observations suggest that TGF-beta signaling directly regulates FoxK through functional Smad/Mad-binding sites, whereas FoxK, in turn, regulates labial expression. We also describe a new cooperative activity of the transcription factors FoxK and Dfos/AP-1 that regulates labial expression in the midgut endoderm. This regulatory activity does not require direct labial activation by the TGF-beta effector Mad. Thus, we propose that the combined activity of the TGF-beta target genes FoxK and Dfos is critical for the direct activation of lab in the endoderm.

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FoxK binds to specific DNA sequences and regulates transcription. (A) EMSA performed with GST-FoxK(414–654) or GST alone (protein [P]) and the radiolabeled double-stranded oligonucleotide Oligo-FH containing an optimum FH-binding site (red). Cold Oligo-FH probe (FH), a suboptimum FH-binding site (Sub), and an unrelated sequence (GAS) were used at 100-fold molar excess (competitors [C]). The higher bands (arrows) indicate specific binding of FoxK to Oligo-FH. Cold Oligo-FH efficiently competes for FoxK, whereas a suboptimum FH-binding site is a less efficient competitor and GAS does not compete for FoxK. GST alone did not bind to Oligo-FH. Free oligonucleotides complexes accumulate in the bottom (arrowhead). (B and C) A plasmid driving luciferase under the control of six consecutive Oligo-FH sequences (6xFH) was cotransfected with pAc5C-FoxK-L-V5, pAc5C-FoxK-S-V5, or empty vector in S2 cells. As expected, both FoxK-L and FoxK-S isoforms exhibit nuclear localization in transfected S2 cells (green, FoxK-L-V5). (D) Both FoxK-L and FoxK-S induce a fourfold activation of the 6xFH target sequence. The error bars correspond to the standard deviation of three independent experiments. (E) The FoxK-L and FoxK-S proteins migrate in two distinct bands in Western blot, suggesting posttranslational modification. β-Galactosidase was used for normalization.
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fig2: FoxK binds to specific DNA sequences and regulates transcription. (A) EMSA performed with GST-FoxK(414–654) or GST alone (protein [P]) and the radiolabeled double-stranded oligonucleotide Oligo-FH containing an optimum FH-binding site (red). Cold Oligo-FH probe (FH), a suboptimum FH-binding site (Sub), and an unrelated sequence (GAS) were used at 100-fold molar excess (competitors [C]). The higher bands (arrows) indicate specific binding of FoxK to Oligo-FH. Cold Oligo-FH efficiently competes for FoxK, whereas a suboptimum FH-binding site is a less efficient competitor and GAS does not compete for FoxK. GST alone did not bind to Oligo-FH. Free oligonucleotides complexes accumulate in the bottom (arrowhead). (B and C) A plasmid driving luciferase under the control of six consecutive Oligo-FH sequences (6xFH) was cotransfected with pAc5C-FoxK-L-V5, pAc5C-FoxK-S-V5, or empty vector in S2 cells. As expected, both FoxK-L and FoxK-S isoforms exhibit nuclear localization in transfected S2 cells (green, FoxK-L-V5). (D) Both FoxK-L and FoxK-S induce a fourfold activation of the 6xFH target sequence. The error bars correspond to the standard deviation of three independent experiments. (E) The FoxK-L and FoxK-S proteins migrate in two distinct bands in Western blot, suggesting posttranslational modification. β-Galactosidase was used for normalization.

Mentions: To determine the transcriptional activity of this putative transcription factor, we first assayed its ability to bind specific DNA sequences. Mouse MNF/FOXK1 binds both strands of the consensus FH-binding site composed of the heptanucleotide core 5′-(A/G)TAAA(C/T)A-3′ (Weigel and Jackle, 1990; Granadino et al., 2000). Electrophoretic mobility shift assays (EMSA) performed with a recombinant fusion protein including the FH domain of FoxK (GST-FoxK[414–654]) and a radiolabeled oligonucleotide probe containing a consensus FH-binding site (Oligo-FH) produced high molecular mass complexes (Fig. 2 A, arrows). The addition of cold Oligo-FH efficiently displaced the labeled probe, whereas a suboptimal probe (Fig. 2 A, Sub) was less efficient. Conversely, an unrelated oligonucleotide (Fig. 2 A, GAS) did not interfere with Oligo-FH binding. Together, these results showed that the FH domain of FoxK specifically recognized a DNA sequence carrying a consensus FH-binding site.


FoxK mediates TGF-beta signalling during midgut differentiation in flies.

Casas-Tinto S, Gomez-Velazquez M, Granadino B, Fernandez-Funez P - J. Cell Biol. (2008)

FoxK binds to specific DNA sequences and regulates transcription. (A) EMSA performed with GST-FoxK(414–654) or GST alone (protein [P]) and the radiolabeled double-stranded oligonucleotide Oligo-FH containing an optimum FH-binding site (red). Cold Oligo-FH probe (FH), a suboptimum FH-binding site (Sub), and an unrelated sequence (GAS) were used at 100-fold molar excess (competitors [C]). The higher bands (arrows) indicate specific binding of FoxK to Oligo-FH. Cold Oligo-FH efficiently competes for FoxK, whereas a suboptimum FH-binding site is a less efficient competitor and GAS does not compete for FoxK. GST alone did not bind to Oligo-FH. Free oligonucleotides complexes accumulate in the bottom (arrowhead). (B and C) A plasmid driving luciferase under the control of six consecutive Oligo-FH sequences (6xFH) was cotransfected with pAc5C-FoxK-L-V5, pAc5C-FoxK-S-V5, or empty vector in S2 cells. As expected, both FoxK-L and FoxK-S isoforms exhibit nuclear localization in transfected S2 cells (green, FoxK-L-V5). (D) Both FoxK-L and FoxK-S induce a fourfold activation of the 6xFH target sequence. The error bars correspond to the standard deviation of three independent experiments. (E) The FoxK-L and FoxK-S proteins migrate in two distinct bands in Western blot, suggesting posttranslational modification. β-Galactosidase was used for normalization.
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Related In: Results  -  Collection

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fig2: FoxK binds to specific DNA sequences and regulates transcription. (A) EMSA performed with GST-FoxK(414–654) or GST alone (protein [P]) and the radiolabeled double-stranded oligonucleotide Oligo-FH containing an optimum FH-binding site (red). Cold Oligo-FH probe (FH), a suboptimum FH-binding site (Sub), and an unrelated sequence (GAS) were used at 100-fold molar excess (competitors [C]). The higher bands (arrows) indicate specific binding of FoxK to Oligo-FH. Cold Oligo-FH efficiently competes for FoxK, whereas a suboptimum FH-binding site is a less efficient competitor and GAS does not compete for FoxK. GST alone did not bind to Oligo-FH. Free oligonucleotides complexes accumulate in the bottom (arrowhead). (B and C) A plasmid driving luciferase under the control of six consecutive Oligo-FH sequences (6xFH) was cotransfected with pAc5C-FoxK-L-V5, pAc5C-FoxK-S-V5, or empty vector in S2 cells. As expected, both FoxK-L and FoxK-S isoforms exhibit nuclear localization in transfected S2 cells (green, FoxK-L-V5). (D) Both FoxK-L and FoxK-S induce a fourfold activation of the 6xFH target sequence. The error bars correspond to the standard deviation of three independent experiments. (E) The FoxK-L and FoxK-S proteins migrate in two distinct bands in Western blot, suggesting posttranslational modification. β-Galactosidase was used for normalization.
Mentions: To determine the transcriptional activity of this putative transcription factor, we first assayed its ability to bind specific DNA sequences. Mouse MNF/FOXK1 binds both strands of the consensus FH-binding site composed of the heptanucleotide core 5′-(A/G)TAAA(C/T)A-3′ (Weigel and Jackle, 1990; Granadino et al., 2000). Electrophoretic mobility shift assays (EMSA) performed with a recombinant fusion protein including the FH domain of FoxK (GST-FoxK[414–654]) and a radiolabeled oligonucleotide probe containing a consensus FH-binding site (Oligo-FH) produced high molecular mass complexes (Fig. 2 A, arrows). The addition of cold Oligo-FH efficiently displaced the labeled probe, whereas a suboptimal probe (Fig. 2 A, Sub) was less efficient. Conversely, an unrelated oligonucleotide (Fig. 2 A, GAS) did not interfere with Oligo-FH binding. Together, these results showed that the FH domain of FoxK specifically recognized a DNA sequence carrying a consensus FH-binding site.

Bottom Line: Genet.This regulatory activity does not require direct labial activation by the TGF-beta effector Mad.Thus, we propose that the combined activity of the TGF-beta target genes FoxK and Dfos is critical for the direct activation of lab in the endoderm.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Texas Medical Branch, Galveston, TX 77555, USA. scasas@cnio.es

ABSTRACT
Inductive signals across germ layers are important for the development of the endoderm in vertebrates and invertebrates (Tam, P.P., M. Kanai-Azuma, and Y. Kanai. 2003. Curr. Opin. Genet. Dev. 13:393-400; Nakagoshi, H. 2005. Dev. Growth Differ. 47:383-392). In flies, the visceral mesoderm secretes signaling molecules that diffuse into the underlying midgut endoderm, where conserved signaling cascades activate the Hox gene labial, which is important for the differentiation of copper cells (Bienz, M. 1997. Curr. Opin. Genet. Dev. 7:683-688). We present here a Drosophila melanogaster gene of the Fox family of transcription factors, FoxK, that mediates transforming growth factor beta (TGF-beta) signaling in the embryonic midgut endoderm. FoxK mutant embryos fail to generate midgut constrictions and lack Labial in the endoderm. Our observations suggest that TGF-beta signaling directly regulates FoxK through functional Smad/Mad-binding sites, whereas FoxK, in turn, regulates labial expression. We also describe a new cooperative activity of the transcription factors FoxK and Dfos/AP-1 that regulates labial expression in the midgut endoderm. This regulatory activity does not require direct labial activation by the TGF-beta effector Mad. Thus, we propose that the combined activity of the TGF-beta target genes FoxK and Dfos is critical for the direct activation of lab in the endoderm.

Show MeSH
Related in: MedlinePlus