Limits...
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.

Show MeSH

Related in: MedlinePlus

Hsf alters the DNA-binding property of GAF. (A) EMSA was performed using a 25-bp 32P-labeled probe with various amounts of proteins. The volume of the control was 2 μl (lanes 1–7). Various volumes of Hsf (1.25, 2.5 and 5 μl in lanes 5–7; 5 μl in lanes 8–10) and GAF (1, 2 and 4 μl in lanes 2–4 and 8–10) were used. The DNA–protein complexes were separated in a 4% polyacrylamide gel and detected using a PhosphoImager (GE/Amersham Biosciences). An arrow head and arrow indicate the tor-RE bound by a monomer and dimer, respectively. (B) Shift-western blotting of the complexes formed with GAF (4 μl in lanes 1, 3, 4, 6 and 7), Hsf (5 μl in lanes 2, 3, 5, 6 and 7) and the labeled probe. Quantity of the probe was 5-fold higher than that used in EMSA. GAF and Hsf on the nitrocellulose membrane were detected by immunoblotting with anti-GAF and Hsf antibodies, respectively. The ‘auto’ represents an autoradiogram that shows position of the radiolabeled probe on the nylon membrane. The DNA–protein complexes indicated by an arrow and arrow head are the same as those shown in (A). (C) DNaseI footprinting was used to show where Hsf, GAF or GAF/Hsf binds in the tll-MRR. The control is proteins extracted from bacteria carrying the pET28a vector. The protein concentrations of the control, GAF and Hsf proteins determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Inc.) are 2.24, 1.75 and 0.34 mg/ml, respectively. Various amounts of proteins were incubated with the probe in a final volume of 50 μl. The reaction mixture was incubated at room temperature for 10 min. A constant volume of control (2 μl in lanes 2 and 4–6) and Hsf (10 μl in lanes 3 and 7–9) were used, whereas various volumes of GAF (1, 2 and 4 μl; represented by triangles) were used (lanes 4–6 and 7–9). Brightness of lane 5 is reduced by 15%. Regions protected by GAF alone are highlighted by open rectangles (a–d) at the right of lane 6. Comparing intensities of bands in lanes 9 with those in lane 6, bands with decreased and increased intensity are marked by asterisks and close circles at the right of lane 9, respectively. Numbers at the left indicate distance in nucleotide from the BstNI site (Supplementary Figure S2).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2651784&req=5

Figure 3: Hsf alters the DNA-binding property of GAF. (A) EMSA was performed using a 25-bp 32P-labeled probe with various amounts of proteins. The volume of the control was 2 μl (lanes 1–7). Various volumes of Hsf (1.25, 2.5 and 5 μl in lanes 5–7; 5 μl in lanes 8–10) and GAF (1, 2 and 4 μl in lanes 2–4 and 8–10) were used. The DNA–protein complexes were separated in a 4% polyacrylamide gel and detected using a PhosphoImager (GE/Amersham Biosciences). An arrow head and arrow indicate the tor-RE bound by a monomer and dimer, respectively. (B) Shift-western blotting of the complexes formed with GAF (4 μl in lanes 1, 3, 4, 6 and 7), Hsf (5 μl in lanes 2, 3, 5, 6 and 7) and the labeled probe. Quantity of the probe was 5-fold higher than that used in EMSA. GAF and Hsf on the nitrocellulose membrane were detected by immunoblotting with anti-GAF and Hsf antibodies, respectively. The ‘auto’ represents an autoradiogram that shows position of the radiolabeled probe on the nylon membrane. The DNA–protein complexes indicated by an arrow and arrow head are the same as those shown in (A). (C) DNaseI footprinting was used to show where Hsf, GAF or GAF/Hsf binds in the tll-MRR. The control is proteins extracted from bacteria carrying the pET28a vector. The protein concentrations of the control, GAF and Hsf proteins determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Inc.) are 2.24, 1.75 and 0.34 mg/ml, respectively. Various amounts of proteins were incubated with the probe in a final volume of 50 μl. The reaction mixture was incubated at room temperature for 10 min. A constant volume of control (2 μl in lanes 2 and 4–6) and Hsf (10 μl in lanes 3 and 7–9) were used, whereas various volumes of GAF (1, 2 and 4 μl; represented by triangles) were used (lanes 4–6 and 7–9). Brightness of lane 5 is reduced by 15%. Regions protected by GAF alone are highlighted by open rectangles (a–d) at the right of lane 6. Comparing intensities of bands in lanes 9 with those in lane 6, bands with decreased and increased intensity are marked by asterisks and close circles at the right of lane 9, respectively. Numbers at the left indicate distance in nucleotide from the BstNI site (Supplementary Figure S2).

Mentions: Volumes of various proteins were the same as those used in EMSA, except quantity of the [32P]-labeled probe and incubation temperature, as indicated in the legend of Figure 3 and Supplementary Figure S4. Shift-western blotting was performed as described previously (46). In brief, DNA–protein complexes were separated in 6% polyacrylamide gels and transferred onto stacked nitrocellulose (Schleicher & Schuell) and nylon membranes (Hybond-XL, GE/Amersham Biosciences). The radiolabeled probe on nylon membrane was detected by autoradiography, whereas the proteins in the complexes on the nitrocellulose membrane were detected by immunoblotting with the chemiluminescence assay kit (47; Western lightingTM, Blossom Biotechnologies, Inc, Taiwan).


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)

Hsf alters the DNA-binding property of GAF. (A) EMSA was performed using a 25-bp 32P-labeled probe with various amounts of proteins. The volume of the control was 2 μl (lanes 1–7). Various volumes of Hsf (1.25, 2.5 and 5 μl in lanes 5–7; 5 μl in lanes 8–10) and GAF (1, 2 and 4 μl in lanes 2–4 and 8–10) were used. The DNA–protein complexes were separated in a 4% polyacrylamide gel and detected using a PhosphoImager (GE/Amersham Biosciences). An arrow head and arrow indicate the tor-RE bound by a monomer and dimer, respectively. (B) Shift-western blotting of the complexes formed with GAF (4 μl in lanes 1, 3, 4, 6 and 7), Hsf (5 μl in lanes 2, 3, 5, 6 and 7) and the labeled probe. Quantity of the probe was 5-fold higher than that used in EMSA. GAF and Hsf on the nitrocellulose membrane were detected by immunoblotting with anti-GAF and Hsf antibodies, respectively. The ‘auto’ represents an autoradiogram that shows position of the radiolabeled probe on the nylon membrane. The DNA–protein complexes indicated by an arrow and arrow head are the same as those shown in (A). (C) DNaseI footprinting was used to show where Hsf, GAF or GAF/Hsf binds in the tll-MRR. The control is proteins extracted from bacteria carrying the pET28a vector. The protein concentrations of the control, GAF and Hsf proteins determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Inc.) are 2.24, 1.75 and 0.34 mg/ml, respectively. Various amounts of proteins were incubated with the probe in a final volume of 50 μl. The reaction mixture was incubated at room temperature for 10 min. A constant volume of control (2 μl in lanes 2 and 4–6) and Hsf (10 μl in lanes 3 and 7–9) were used, whereas various volumes of GAF (1, 2 and 4 μl; represented by triangles) were used (lanes 4–6 and 7–9). Brightness of lane 5 is reduced by 15%. Regions protected by GAF alone are highlighted by open rectangles (a–d) at the right of lane 6. Comparing intensities of bands in lanes 9 with those in lane 6, bands with decreased and increased intensity are marked by asterisks and close circles at the right of lane 9, respectively. Numbers at the left indicate distance in nucleotide from the BstNI site (Supplementary Figure S2).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651784&req=5

Figure 3: Hsf alters the DNA-binding property of GAF. (A) EMSA was performed using a 25-bp 32P-labeled probe with various amounts of proteins. The volume of the control was 2 μl (lanes 1–7). Various volumes of Hsf (1.25, 2.5 and 5 μl in lanes 5–7; 5 μl in lanes 8–10) and GAF (1, 2 and 4 μl in lanes 2–4 and 8–10) were used. The DNA–protein complexes were separated in a 4% polyacrylamide gel and detected using a PhosphoImager (GE/Amersham Biosciences). An arrow head and arrow indicate the tor-RE bound by a monomer and dimer, respectively. (B) Shift-western blotting of the complexes formed with GAF (4 μl in lanes 1, 3, 4, 6 and 7), Hsf (5 μl in lanes 2, 3, 5, 6 and 7) and the labeled probe. Quantity of the probe was 5-fold higher than that used in EMSA. GAF and Hsf on the nitrocellulose membrane were detected by immunoblotting with anti-GAF and Hsf antibodies, respectively. The ‘auto’ represents an autoradiogram that shows position of the radiolabeled probe on the nylon membrane. The DNA–protein complexes indicated by an arrow and arrow head are the same as those shown in (A). (C) DNaseI footprinting was used to show where Hsf, GAF or GAF/Hsf binds in the tll-MRR. The control is proteins extracted from bacteria carrying the pET28a vector. The protein concentrations of the control, GAF and Hsf proteins determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Inc.) are 2.24, 1.75 and 0.34 mg/ml, respectively. Various amounts of proteins were incubated with the probe in a final volume of 50 μl. The reaction mixture was incubated at room temperature for 10 min. A constant volume of control (2 μl in lanes 2 and 4–6) and Hsf (10 μl in lanes 3 and 7–9) were used, whereas various volumes of GAF (1, 2 and 4 μl; represented by triangles) were used (lanes 4–6 and 7–9). Brightness of lane 5 is reduced by 15%. Regions protected by GAF alone are highlighted by open rectangles (a–d) at the right of lane 6. Comparing intensities of bands in lanes 9 with those in lane 6, bands with decreased and increased intensity are marked by asterisks and close circles at the right of lane 9, respectively. Numbers at the left indicate distance in nucleotide from the BstNI site (Supplementary Figure S2).
Mentions: Volumes of various proteins were the same as those used in EMSA, except quantity of the [32P]-labeled probe and incubation temperature, as indicated in the legend of Figure 3 and Supplementary Figure S4. Shift-western blotting was performed as described previously (46). In brief, DNA–protein complexes were separated in 6% polyacrylamide gels and transferred onto stacked nitrocellulose (Schleicher & Schuell) and nylon membranes (Hybond-XL, GE/Amersham Biosciences). The radiolabeled probe on nylon membrane was detected by autoradiography, whereas the proteins in the complexes on the nitrocellulose membrane were detected by immunoblotting with the chemiluminescence assay kit (47; Western lightingTM, Blossom Biotechnologies, Inc, Taiwan).

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