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The Torso signaling pathway modulates a dual transcriptional switch to regulate tailless expression.

Chen YC, Lin SI, Chen YK, Chiang CS, Liaw GJ - Nucleic Acids Res. (2009)

Bottom Line: Hsf is a substrate of mitogen-activated protein kinase and S378 is the major phosphorylation site.Phosphorylation converts Hsf from a repressor to an activator that works with GAF to activate tll expression.In conclusion, the GAF/Hsf/Ttk69 complex binding to the tor-RE remodels local chromatin structure to repress tll expression and the Tor signaling pathway activate tll expression by modulating a dual transcriptional switch.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, 112 Taiwan, ROC.

ABSTRACT
The Torso (Tor) signaling pathway activates tailless (tll) expression by relieving tll repression. None of the repressors identified so far, such as Capicuo, Groucho and Tramtrack69 (Ttk69), bind to the tor response element (tor-RE) or fully elucidate tll repression. In this study, an expanded tll expression pattern was shown in embryos with reduced heat shock factor (hsf) and Trithorax-like (Trl) activities. The GAGA factor, GAF encoded by Trl, bound weakly to the tor-RE, and this binding was enhanced by both Hsf and Ttk69. A similar extent of expansion of tll expression was observed in embryos with simultaneous knockdown of hsf, Trl and ttk69 activities, and in embryos with constitutively active Tor. Hsf is a substrate of mitogen-activated protein kinase and S378 is the major phosphorylation site. Phosphorylation converts Hsf from a repressor to an activator that works with GAF to activate tll expression. In conclusion, the GAF/Hsf/Ttk69 complex binding to the tor-RE remodels local chromatin structure to repress tll expression and the Tor signaling pathway activate tll expression by modulating a dual transcriptional switch.

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Phosphorylation by Mapk converts Hsf from a repressor to an activator for tll expression. (A) Amino acid sequences of HSF1s in vertebrates were retrieved from Genbank and the regulatory domains of these HSF1s (HSF-RD) were aligned using the ClustalW program to reveal conserved amino acids. Putative Mapk recognition sequences are boxed and S307, a target site of Mapk in HSF1s, is highlighted. The same procedure was applied to Hsfs in Drosophila species including melanogaster (mel), yakuba (yak), erecta (ere), ananassae (ana), pseudoobscura (pse), mojavensis (moj) and virilis (vir). Amino acid sequences in boxes that contain two putative Mapk phosphorylation sites, S378 and T390, are highly conserved among Drosophila species. (B) In an in vitro phosphorylation assay, 100 pmol of GST-Hsf-RD and two mutant forms (GST-RD-S378A and T390A), as well as negative (GST) and positive (myelin basic protein, MBP) controls were used. After incubation with Mapk and γ[32P]-ATP, the proteins were separated in a 12% SDS gel and visualized using Coomassie blue staining, shown at the left. After the gel was dried, autoradiography was used to reveal the phosphorylated proteins, shown at the right. (C–F) tll expression patterns in late stage-4 embryos from mothers transheterozygous for hsf1 and TrlDHΔ34, determined by in situ hybridization, are shown and the anterior is arranged towards the left. Supplementation with UAS-hsf-wt (D), -S378A (E) and -S378D (F) driven by GAL4-GCN4 suppresses the expanded tll expression patterns. GFP expression was used as a negative control (C). (G) Hsf-S378D binding to the tor-RE is enhanced by GAF. The protein concentration of Hsf-S378D is 0.34 mg/ml. The Hsf, GAF and the probe were the same as those used in Figure 3A. GAF (4 μl) was mixed with various volumes of Hsf (wt) or Hsf-S378D (S378D) (2.5 μl in lanes 3 and 6; 5 μl in lanes 4 and 7; and 10 μl in lanes 5 and 8–10) and 32P-labeled probe. The DNA–protein complexes were separated in a 4% polyacrylamide gel and visualized using a PhosphoImager.
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Figure 6: Phosphorylation by Mapk converts Hsf from a repressor to an activator for tll expression. (A) Amino acid sequences of HSF1s in vertebrates were retrieved from Genbank and the regulatory domains of these HSF1s (HSF-RD) were aligned using the ClustalW program to reveal conserved amino acids. Putative Mapk recognition sequences are boxed and S307, a target site of Mapk in HSF1s, is highlighted. The same procedure was applied to Hsfs in Drosophila species including melanogaster (mel), yakuba (yak), erecta (ere), ananassae (ana), pseudoobscura (pse), mojavensis (moj) and virilis (vir). Amino acid sequences in boxes that contain two putative Mapk phosphorylation sites, S378 and T390, are highly conserved among Drosophila species. (B) In an in vitro phosphorylation assay, 100 pmol of GST-Hsf-RD and two mutant forms (GST-RD-S378A and T390A), as well as negative (GST) and positive (myelin basic protein, MBP) controls were used. After incubation with Mapk and γ[32P]-ATP, the proteins were separated in a 12% SDS gel and visualized using Coomassie blue staining, shown at the left. After the gel was dried, autoradiography was used to reveal the phosphorylated proteins, shown at the right. (C–F) tll expression patterns in late stage-4 embryos from mothers transheterozygous for hsf1 and TrlDHΔ34, determined by in situ hybridization, are shown and the anterior is arranged towards the left. Supplementation with UAS-hsf-wt (D), -S378A (E) and -S378D (F) driven by GAL4-GCN4 suppresses the expanded tll expression patterns. GFP expression was used as a negative control (C). (G) Hsf-S378D binding to the tor-RE is enhanced by GAF. The protein concentration of Hsf-S378D is 0.34 mg/ml. The Hsf, GAF and the probe were the same as those used in Figure 3A. GAF (4 μl) was mixed with various volumes of Hsf (wt) or Hsf-S378D (S378D) (2.5 μl in lanes 3 and 6; 5 μl in lanes 4 and 7; and 10 μl in lanes 5 and 8–10) and 32P-labeled probe. The DNA–protein complexes were separated in a 4% polyacrylamide gel and visualized using a PhosphoImager.

Mentions: tll expression is activated by the Tor pathway by relieving transcriptional repression (13). In vertebrates, the S307 MAPK phosphorylation site in the RD of HSF1s (59) is highly conserved (Figure 6A). In order to investigate whether Hsf is a substrate of Mapk in Drosophila, putative Mapk phosphorylation sites in the RD that are conserved among Drosophila species were identified using the ClustalW program. These sites were tested to determine whether they function in tll repression in vitro and in vivo.Figure 6.


The Torso signaling pathway modulates a dual transcriptional switch to regulate tailless expression.

Chen YC, Lin SI, Chen YK, Chiang CS, Liaw GJ - Nucleic Acids Res. (2009)

Phosphorylation by Mapk converts Hsf from a repressor to an activator for tll expression. (A) Amino acid sequences of HSF1s in vertebrates were retrieved from Genbank and the regulatory domains of these HSF1s (HSF-RD) were aligned using the ClustalW program to reveal conserved amino acids. Putative Mapk recognition sequences are boxed and S307, a target site of Mapk in HSF1s, is highlighted. The same procedure was applied to Hsfs in Drosophila species including melanogaster (mel), yakuba (yak), erecta (ere), ananassae (ana), pseudoobscura (pse), mojavensis (moj) and virilis (vir). Amino acid sequences in boxes that contain two putative Mapk phosphorylation sites, S378 and T390, are highly conserved among Drosophila species. (B) In an in vitro phosphorylation assay, 100 pmol of GST-Hsf-RD and two mutant forms (GST-RD-S378A and T390A), as well as negative (GST) and positive (myelin basic protein, MBP) controls were used. After incubation with Mapk and γ[32P]-ATP, the proteins were separated in a 12% SDS gel and visualized using Coomassie blue staining, shown at the left. After the gel was dried, autoradiography was used to reveal the phosphorylated proteins, shown at the right. (C–F) tll expression patterns in late stage-4 embryos from mothers transheterozygous for hsf1 and TrlDHΔ34, determined by in situ hybridization, are shown and the anterior is arranged towards the left. Supplementation with UAS-hsf-wt (D), -S378A (E) and -S378D (F) driven by GAL4-GCN4 suppresses the expanded tll expression patterns. GFP expression was used as a negative control (C). (G) Hsf-S378D binding to the tor-RE is enhanced by GAF. The protein concentration of Hsf-S378D is 0.34 mg/ml. The Hsf, GAF and the probe were the same as those used in Figure 3A. GAF (4 μl) was mixed with various volumes of Hsf (wt) or Hsf-S378D (S378D) (2.5 μl in lanes 3 and 6; 5 μl in lanes 4 and 7; and 10 μl in lanes 5 and 8–10) and 32P-labeled probe. The DNA–protein complexes were separated in a 4% polyacrylamide gel and visualized using a PhosphoImager.
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Related In: Results  -  Collection

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Figure 6: Phosphorylation by Mapk converts Hsf from a repressor to an activator for tll expression. (A) Amino acid sequences of HSF1s in vertebrates were retrieved from Genbank and the regulatory domains of these HSF1s (HSF-RD) were aligned using the ClustalW program to reveal conserved amino acids. Putative Mapk recognition sequences are boxed and S307, a target site of Mapk in HSF1s, is highlighted. The same procedure was applied to Hsfs in Drosophila species including melanogaster (mel), yakuba (yak), erecta (ere), ananassae (ana), pseudoobscura (pse), mojavensis (moj) and virilis (vir). Amino acid sequences in boxes that contain two putative Mapk phosphorylation sites, S378 and T390, are highly conserved among Drosophila species. (B) In an in vitro phosphorylation assay, 100 pmol of GST-Hsf-RD and two mutant forms (GST-RD-S378A and T390A), as well as negative (GST) and positive (myelin basic protein, MBP) controls were used. After incubation with Mapk and γ[32P]-ATP, the proteins were separated in a 12% SDS gel and visualized using Coomassie blue staining, shown at the left. After the gel was dried, autoradiography was used to reveal the phosphorylated proteins, shown at the right. (C–F) tll expression patterns in late stage-4 embryos from mothers transheterozygous for hsf1 and TrlDHΔ34, determined by in situ hybridization, are shown and the anterior is arranged towards the left. Supplementation with UAS-hsf-wt (D), -S378A (E) and -S378D (F) driven by GAL4-GCN4 suppresses the expanded tll expression patterns. GFP expression was used as a negative control (C). (G) Hsf-S378D binding to the tor-RE is enhanced by GAF. The protein concentration of Hsf-S378D is 0.34 mg/ml. The Hsf, GAF and the probe were the same as those used in Figure 3A. GAF (4 μl) was mixed with various volumes of Hsf (wt) or Hsf-S378D (S378D) (2.5 μl in lanes 3 and 6; 5 μl in lanes 4 and 7; and 10 μl in lanes 5 and 8–10) and 32P-labeled probe. The DNA–protein complexes were separated in a 4% polyacrylamide gel and visualized using a PhosphoImager.
Mentions: tll expression is activated by the Tor pathway by relieving transcriptional repression (13). In vertebrates, the S307 MAPK phosphorylation site in the RD of HSF1s (59) is highly conserved (Figure 6A). In order to investigate whether Hsf is a substrate of Mapk in Drosophila, putative Mapk phosphorylation sites in the RD that are conserved among Drosophila species were identified using the ClustalW program. These sites were tested to determine whether they function in tll repression in vitro and in vivo.Figure 6.

Bottom Line: Hsf is a substrate of mitogen-activated protein kinase and S378 is the major phosphorylation site.Phosphorylation converts Hsf from a repressor to an activator that works with GAF to activate tll expression.In conclusion, the GAF/Hsf/Ttk69 complex binding to the tor-RE remodels local chromatin structure to repress tll expression and the Tor signaling pathway activate tll expression by modulating a dual transcriptional switch.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, 112 Taiwan, ROC.

ABSTRACT
The Torso (Tor) signaling pathway activates tailless (tll) expression by relieving tll repression. None of the repressors identified so far, such as Capicuo, Groucho and Tramtrack69 (Ttk69), bind to the tor response element (tor-RE) or fully elucidate tll repression. In this study, an expanded tll expression pattern was shown in embryos with reduced heat shock factor (hsf) and Trithorax-like (Trl) activities. The GAGA factor, GAF encoded by Trl, bound weakly to the tor-RE, and this binding was enhanced by both Hsf and Ttk69. A similar extent of expansion of tll expression was observed in embryos with simultaneous knockdown of hsf, Trl and ttk69 activities, and in embryos with constitutively active Tor. Hsf is a substrate of mitogen-activated protein kinase and S378 is the major phosphorylation site. Phosphorylation converts Hsf from a repressor to an activator that works with GAF to activate tll expression. In conclusion, the GAF/Hsf/Ttk69 complex binding to the tor-RE remodels local chromatin structure to repress tll expression and the Tor signaling pathway activate tll expression by modulating a dual transcriptional switch.

Show MeSH
Related in: MedlinePlus