Introduction (Arial 36, bold, RGB 38/84/124)
1
Literature and Acknowledgements Modulation of Transcriptional Activity of Ecdysone dependent Genes by Different Hormone Response Elements (Arial 60, bold, white) Introduction (Arial 36, bold, RGB 38/84/124) (Arial 30, bold, black) The responsiveness of ecdysteroids is mediated by the ecdysteroid receptor (EcR) and Ultraspiracle (Usp) [1, 2], which act as ligand-activated transcription factors. Thus the recognition of their cognate hormone response element (EcRE) is essentially. The heterodimer of EcR and Usp is able to bind to direct repeat sequences (like DR1) [3], to perfect inverted repeat sequences (like PAL1) and to imperfect palindromes (like hsp27) [4]. We used a rapid response luciferase reporter vector (pGL 4.19, Promega), including trimeric sequences of DR1 and DR12, to investigate their influence on EcR and EcR/Usp. S. Schauer, S. Braun, M. Spindler-Barth (Arial 48, bold, black) Institute of General Zoology and Endocrinology, Ulm University, Germany [1] Beato et al. (1995) Cell 83: 851-857 (Arial, 16, black) [2] Mangelsdorf et al. (1995). Cell 83: 835-839 [3] Horner et al. (1995) Dev Biol 168: 490-502 [4] Riddihough G, Pelham HRB (1987) EMBO J. 6: 3729-3734 [5] Pratt WB, Toft DO (1997) Endocrinol Rev 18: 306-360 [6] Promega (2006) Technical Manual, pGL4 Luciferase Reporter Vectors The gifts of Usp- and EcR-constructs by Dr. V.C. Henrich, (University of Greensboro, USA) and A. Ozyhar (Technical University of Wroclaw, Poland) are gratefully acknowledged. We thank N. Möbius, E. Arnold, S. Raith and M. Burret for skillful technical assistance. The work was supported by the Graduate College1041. Material and methods (Arial 36, bold, RGB 38/84/124) (Arial 30, bold, black) The firefly luciferase of pGL 4.19 (luc2CP) is destabilized by protein degradation sequences (fig. 1). Destabilized reporter proteins are more responsive and better suited to monitor rapid processes [6]. The used constructs are shown in fig. 2. CHO-cells were transfected with those constructs and with different isoforms of EcR (fig. 3) and different variants of Usp (fig. 4). Luciferase activity was normalized on receptor concentration determined by quantification of specific Western blot signals (see poster Ruff et al. [7]), because the receptor protein concentration varies between the EcR isoforms, the absence / presence of Ultraspiracle and between the absence / presence of hormone, despite identical transfection efficiency determined with a constitutively expressed reporter (ß-galactosidase). Fig. 4: Scheme of Usp wt and the Usp variants (Arial 20, bold, black) Fig. 1: Gene design of the firefly luciferase (luc2) of pGL 4.19 (luc2CP) Summary (Arial 36, bold, RGB 38/84/124) (Arial 30, bold, black) The transcriptional activity of the receptor protein depends on: 1)The type of hormone response element 2) The EcR isoform 3) The presence / absence of Usp and the Usp variant 4) The presence / absence of hormone (see also poster Ruff et al. [7]) Fig. 2: Vector circle map of the constructs DR1 EcRE DR12 0 5000 10000 15000 20000 25000 EcR -A EcR -B1 EcR -B2 rel.luc. act./ rec. prot.conc.[rel. Units] Fig. 6 – 9: Receptor protein concentration (6 and 7) and transcriptional activity (8 and 9) of EcR Isoforms in absence of its heterodimerization partner Usp (A) and in presence of Usp (B and C). □ : without hormone, ■ : with 1µM muristerone A. Fig. 6A: DR1 3x without Usp Fig. 7A: DR12 3x without Usp Fig. 8A: DR1 3x without Usp Fig. 9A: DR12 3x without Usp Fig. 6B: DR1 3x with Usp wt Fig. 7B: DR12 3x with Usp wt Fig. 8B: DR1 3x with Usp wt Fig. 9B: DR12 3x with Usp wt Fig. 6C: DR1 3x with Usp III Fig. 7C: DR12 3x with Usp III Fig. 8C: DR1 3x with Usp III Fig. 9C: DR12 3x without Usp III 0 10 20 30 40 50 60 70 EcR -A EcR -B1 EcR -B2 rec.prot.conc.[rel.Units] 0 10 20 30 40 50 60 70 EcR -A E cR -B1 EcR -B2 rec.prot.conc.[rel.Units] 0 10 20 30 40 50 60 70 EcR -A EcR -B1 EcR -B2 rec.prot.conc.[rel.Units] 0 10 20 30 40 50 60 70 EcR -A EcR -B1 EcR -B2 rec.prot.conc. [rel.Units] 0 10 20 30 40 50 60 70 EcR -A EcR -B1 EcR -B2 rec. prot.conc. [rel.Units] 0 10 20 30 40 50 60 70 EcR -A EcR -B1 EcR -B2 rec. prot.conc.[rel. Units] 0 5000 10000 15000 20000 25000 EcR -A EcR -B1 EcR -B2 rel.luc.act./rec. prot.conc.[rel.Units] 0 5000 10000 15000 20000 25000 EcR -A EcR -B1 EcR -B2 rel. luc. act. / rec. prot. conc. [rel. U nits] 0 5000 10000 15000 20000 25000 EcR -A EcR -B1 EcR -B2 rel. luc. act. / rec. prot. conc. [rel. U nits] 0 5000 10000 15000 20000 25000 EcR -A EcR -B1 EcR -B2 rel. luc. act./rec. prot. conc.[rel.Units] 0 5000 10000 15000 20000 25000 EcR -A EcR -B1 EcR -B2 rel. luc.act./ rec. prot. conc.[rel.Units] 2) Stabilization of the receptor protein and its transcriptional activity varies between the presence / absence of a hormone response element. 3) Stabilization of the receptor protein in presence of hormone and / or Usp varies depending on the isoform and type of hormone response element. Results (Arial 36, bold, RGB 38/84/124) 1) Transfection efficiency is nearly the same according to microscopical control of a fluorescent control plasmid, but the concentration of EcR isoforms is different. Fig. 3: Scheme of EcR isoforms Fig. 5: Receptor protein concentration and transcriptional activity of EcR isoforms in absence of its heterodimerization partner Usp and in presence / absence of hormone and HREs. □ : without HRE, ■ : with HRE, - : without hormone, + : with 1µM muristerone A. 0 50 100 150 200 250 E cR -A /- E cR -A / + E cR -B1 / - E cR -B1 / + EcR -B 2 / - EcR -B 2 / + rec.prot.conc.[rel.U nits] 0 5000 10000 15000 EcR -A / - E cR -A / + EcR -B1 /- EcR -B1 /+ E cR -B2 /- EcR -B2 /+ rel.luc.act./rec. prot.conc.[rel.U nits]
description
Modulation of Transcriptional Activity of Ecdysone dependent Genes by Different Hormone Response Elements (Arial 60, bold, white). S. Schauer, S. Braun, M. Spindler-Barth (Arial 48, bold, black) Institute of General Zoology and Endocrinology, Ulm University, Germany. - PowerPoint PPT Presentation
Transcript of Introduction (Arial 36, bold, RGB 38/84/124)
Literature and Acknowledgements
Modulation of Transcriptional Activity of Ecdysone dependent Genes by Different Hormone Response Elements (Arial 60, bold, white)
Introduction (Arial 36, bold, RGB 38/84/124)
(Arial 30, bold, black) The responsiveness of ecdysteroids is mediated by the ecdysteroid receptor (EcR) and Ultraspiracle (Usp) [1, 2], which act as ligand-activated transcription factors. Thus the recognition of their cognate hormone response element (EcRE) is essentially. The heterodimer of EcR and Usp is able to bind to direct repeat sequences (like DR1) [3], to perfect inverted repeat sequences (like PAL1) and to imperfect palindromes (like hsp27) [4]. We used a rapid response luciferase reporter vector (pGL 4.19, Promega), including trimeric sequences of DR1 and DR12, to investigate their influence on EcR and EcR/Usp.
S. Schauer, S. Braun, M. Spindler-Barth (Arial 48, bold, black)
Institute of General Zoology and Endocrinology, Ulm University, Germany
[1] Beato et al. (1995) Cell 83: 851-857 (Arial, 16, black) [2] Mangelsdorf et al. (1995). Cell 83: 835-839[3] Horner et al. (1995) Dev Biol 168: 490-502 [4] Riddihough G, Pelham HRB (1987) EMBO J. 6: 3729-3734[5] Pratt WB, Toft DO (1997) Endocrinol Rev 18: 306-360 [6] Promega (2006) Technical Manual, pGL4 Luciferase Reporter Vectors
The gifts of Usp- and EcR-constructs by Dr. V.C. Henrich, (University of Greensboro, USA) and A. Ozyhar (Technical University of Wroclaw, Poland) are gratefully acknowledged. We thank N. Möbius, E. Arnold, S. Raith and M. Burret for skillful technical assistance. The work was supported by the Graduate College1041.
Material and methods (Arial 36, bold, RGB 38/84/124)
(Arial 30, bold, black) The firefly luciferase of pGL 4.19 (luc2CP) is destabilized by protein degradation sequences (fig. 1). Destabilized reporter proteins are more responsive and better suited to monitor rapid processes [6]. The used constructs are shown in fig. 2. CHO-cells were transfected with those constructs and with different isoforms of EcR (fig. 3) and different variants of Usp (fig. 4). Luciferase activity was normalized on receptor concentration determined by quantification of specific Western blot signals (see poster Ruff et al. [7]), because the receptor protein concentration varies between the EcR isoforms, the absence / presence of Ultraspiracle and between the absence / presence of hormone, despite identical transfection efficiency determined with a constitutively expressed reporter (ß-galactosidase).
Fig. 4: Scheme of Usp wt and the Usp variants
(Arial 20, bold, black) Fig. 1: Gene design of the firefly luciferase (luc2) of pGL 4.19 (luc2CP)
Summary (Arial 36, bold, RGB 38/84/124)(Arial 30, bold, black) The transcriptional activity of the receptor protein
depends on:1) The type of hormone response element2) The EcR isoform3) The presence / absence of Usp and the Usp variant4) The presence / absence of hormone (see also poster Ruff et al. [7])
Fig. 2: Vector circle map of the constructs
DR1
EcRE
DR12
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EcR-A EcR-B1 EcR-B2
rel.
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. / r
ec.
prot
. con
c. [r
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nits
]
Fig. 6 – 9: Receptor protein concentration (6 and 7) and transcriptional activity (8 and 9) of EcR Isoforms in absence of its heterodimerization
partner Usp (A) and in presence of Usp (B and C). □ : without hormone, ■ : with 1µM muristerone A.
Fig. 6A: DR1 3x without Usp Fig. 7A: DR12 3x without Usp Fig. 8A: DR1 3x without Usp Fig. 9A: DR12 3x without Usp
Fig. 6B: DR1 3x with Usp wt Fig. 7B: DR12 3x with Usp wt Fig. 8B: DR1 3x with Usp wt Fig. 9B: DR12 3x with Usp wt
Fig. 6C: DR1 3x with Usp III Fig. 7C: DR12 3x with Usp III Fig. 8C: DR1 3x with Usp III Fig. 9C: DR12 3x without Usp III
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EcR-A EcR-B1 EcR-B2
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nc. [
rel.
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EcR-A EcR-B1 EcR-B2
rec.
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EcR-A EcR-B1 EcR-B2
rec.
pro
t. co
nc. [
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EcR-A EcR-B1 EcR-B2
rec.
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EcR-A EcR-B1 EcR-B2
rec.
pro
t. co
nc. [
rel.
Uni
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EcR-A EcR-B1 EcR-B2
rec.
pro
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ts]
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EcR-A EcR-B1 EcR-B2
rel.
luc.
act
. / r
ec.
prot
. con
c. [r
el. U
nits
]
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EcR-A EcR-B1 EcR-B2
rel.
luc.
act
. / r
ec.
pro
t. c
on
c. [
rel.
Un
its]
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EcR-A EcR-B1 EcR-B2
rel.
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act
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ec.
pro
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c. [
rel.
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its]
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EcR-A EcR-B1 EcR-B2
rel.
luc.
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ec.
prot
. con
c. [r
el. U
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]
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EcR-A EcR-B1 EcR-B2
rel.
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ec.
prot
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c. [r
el. U
nits
]
2) Stabilization of the receptor protein and its transcriptional activity varies between the presence / absence of a hormone response element.
3) Stabilization of the receptor protein in presence of hormone and / or Usp varies depending on the isoform and type of hormone response element.
Results (Arial 36, bold, RGB 38/84/124)
1) Transfection efficiency is nearly the same according to microscopical control of a fluorescent control plasmid, but the concentration of EcR isoforms is different.
Fig. 3: Scheme of EcR isoforms
Fig. 5: Receptor protein concentration and transcriptional activity of EcR isoforms in absence of its heterodimerization partner Usp and in presence / absence of hormone and HREs.
□ : without HRE, ■ : with HRE,
- : without hormone, + : with 1µM muristerone A.
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EcR-A / - EcR-A /+
EcR-B1 /-
EcR-B1 /+
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EcR-B2 /+
rec
. p
rot.
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. [r
el.
Un
its
]
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c.
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c.
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s]
