PowerPoint-Pr

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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
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.
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).

• Summary (Arial 36, bold, RGB 38/84/124)
• (Arial 30, bold, black) The transcriptional
  activity of the receptor protein depends on
• 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)

(Arial 20, bold, black) Fig. 1 Gene design of
the firefly luciferase (luc2) of pGL 4.19 (luc2CP)
Results (Arial 36, bold, RGB 38/84/124) 1) Transfe
ction efficiency is nearly the same according to
microscopical control of a fluorescent control
plasmid, but the concentration of EcR isoforms is
different.
DR1
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.
EcRE
DR12
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.
Fig. 2 Vector circle map of the constructs
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. 3 Scheme of EcR isoforms
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
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. 4 Scheme of Usp wt and the Usp variants
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.