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Nuclear localization and DNA binding of ecdysone receptor and ultraspiracle.

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Archives of Insect Biochemistry and Physiology 65:125–133 (2007)
Nuclear Localization and DNA Binding of Ecdysone
Receptor and Ultraspiracle
M.V. Cronauer, S. Braun, Ch. Tremmel, K.-D. Kröncke, and M. Spindler-Barth*
The Ecdysone receptor (EcR) is distributed between cytoplasm and nucleus in CHO cells. Nuclear localization is increased by the
ligand Muristerone A. The most important heterodimerization partner Ultraspiracle (Usp) is localized predominantly in the
nucleus. We used the diethylentriamine nitric oxide adduct DETA/NO, which releases NO and destroys the zinc-finger structure
of nuclear receptors, to investigate whether nuclear EcR and Usp interact with DNA. If expressed separately, Usp and EcR in the
absence of hormone do not interact with DNA. The hormone-induced increase in nuclear EcR is due to enhanced DNA binding.
In the presence of Usp, EcR is shifted nearly quantitatively into the nucleus. Only a fraction (approximately 30%) of the
heterodimer is sensitive to DETA/NO. Interaction of the heterodimer with DNA is mediated mainly by the C-domain of EcR.
Deletion of the DNA-binding domain of Usp only slightly reduces nuclear localization of EcR/Usp, although the nuclear localization signal of Usp is not present anymore. The results indicate that EcR and Usp can enter the nucleus independently, but
cotransport of both receptors mediated by dimerization via the ligand binding domains is possible even in the absence of
hormone. Arch. Insect Biochem. Physiol. 65:125–133, 2007. © 2007 Wiley-Liss, Inc.
KEYWORDS: dimerization; insect; molting hormone; NO; nuclear receptors; Zn-finger
INTRODUCTION
The functional ecdysone receptor is considered
as a heterodimer of the ecdysteroid receptor EcR
and Ultraspiracle (Usp). A prerequisite for transcriptional regulation of ecdysone-dependent genes
is nuclear localization of EcR/Usp and interaction
with specific DNA sequences, the hormone response elements. DNA binding is mediated by the
C-domain of nuclear receptors encompassing two
zinc fingers, which interact with the major groove
of the DNA-helix. Intracellular localization is the
result of continuous nucleocytoplasmatic shuttling
(Shank et al., 2005), which may be shifted in favour
of the nucleus by interaction with DNA, since proteins bound to DNA may not be fully available for
the export machinery (Sackey et al., 1996).
To study the influence of DNA binding on
nuclear localization of EcR and Usp, we selectively
destroyed the 3-D structure of the C-domain with
nitric oxide (NO) known to remove zinc from the
cysteine bonds in a reversible manner, without affecting the amino acid sequence (Kröncke and
Carlberg, 2000; Garban et al., 2005; Hongo et al.,
2005). NO inhibits DNA-binding activity in a reversible manner in different members of the
nuclear steroid hormone receptor superfamily, e.g..
the vitamin D3 receptor (VDR), the retinoic X receptor (RXR), the glucocorticoid receptor (GR), the
estrogen receptor α (ERα), and the androgen receptor (AR), as well as the transcription factor YY1
(Kröncke and Carlberg, 2000; Galigniana et al.,
1999; Garban et al., 2005; Cronauer et al., 2006;
Hongo et al., 2005). Although NO inhibits DNAbinding activity of the nuclear steroid hormone receptors, it does not affect intracellular receptor
protein levels or its nuclear import as recently
shown for the AR (Cronauer et al., 2006).
Institute of General Zoology and Endocrinology, University of Ulm, Ulm, Germany
*Correspondence to: Prof. Dr. M. Spindler-Barth, Institute of General Zoology and Endocrinology, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm,
Germany. E-mail: [email protected]
© 2007 Wiley-Liss, Inc.
DOI: 10.1002/arch.20184
Published online in Wiley InterScience (www.interscience.wiley.com)
126
Cronauer et al.
MATERIAL AND METHODS
Plasmids
The following plasmids were used for transfection studies: pEYFP-EcR-B1 (obtained from Dr.
Ozyhar, Wrozlaw, Poland). The plasmid CFP-VP16Usp I and III was generated from DNA fragments
containing the coding sequences (cds) for wild type
sequences of Usp from Drosophila melanogaster. A
plasmid containing the cds for VP16-Usp I and III
(provided by Dr. VC Henrich, Greensboro, NC;
Beatty et al., 2006) was digested with BglII and
HindIII. The resulting DNA fragment was subcloned in the pECFP-C1 vector (Clontech, Takara
Bio Clontech, Saint-Germain-en-Laye, France). The
original AB domain of Usp was replaced by the
activation domain of VP16 to overcome the inhibitory effect of the AB domain of Usp on transactivation of reporter genes in vertebrate cells
(Henrich, 2005).
Heterologeous Expression of EcR and USP
CHO-K1 cells were cultured under standard conditions in Ham F12 medium. Cells were seeded in
six-well plates (Nunc, Wiesbaden, Germany) with
3 × 105 cells per well the day prior to transfection.
Transfection was performed with lipofectamine
2000 (Invitrogen, Karlsruhe, Germany) according to
the manufacturer’s instructions. Each well received
0.25 µg of expression plasmid. Empty vector was
added when only one expression plasmid was used.
The cells were incubated with plasmid DNA for 1
hr. Then the NO donor DETA/NO ((Z)-1-[N-(2aminoethyl)-N-(2-ammonioethyl)amino]diazen-1ium-1,2-diolate), which releases NO during several
hours (t½ = 8 h), or denitrosylated DETA (Sigma,
Deisenhofen, Germany,) which served as a control,
was added (final concentration 800 µM), dissolved
in DMSO. Seven hours after transfection, intracellular localization was evaluated.
Microscopy
Intracellular distribution of fluorescence was
evaluated with an inverted microscope (Zeiss Axio-
vert, Jena, Germany). To ensure a randomised approach, various samples from different regions were
analysed with a total of about 50 cells. Three independent transfection experiments were performed.
Western Blots
Cell extracts were prepared as described previously (Nieva et al.,
2005). After SDS gel electrophoresis (Laemmli,
1970) Western blots were performed according to
Khyse-Andersen (1984). Gels were electroblotted on
nitrocellulose membranes (BA 85, 45 µm pore size,
Schleicher and Schuell, Dassel, Germany). Membranes were stained with Ponceau as a loading control and to check transblotting efficiency (data not
shown). The membranes were soaked in blocking
buffer (Tris-HCl, 137 mM NaCl, 0.1% Tween 20,
pH 7.6, 0.02% Thimerosal) containing 3% milk
powder (low fat <1%) and probed with a monoclonal anti-GFP (Clontech, Heidelberg, Germany)
diluted in the same buffer 1:100. For detection of
specific Western signals, peroxidase conjugated secondary antibodies (Anti-mouse IgG; no. A-5906
Sigma, Deisenhofen, Germany) diluted 1:1,000 in
TBS (0.1% Tween 20) was used. After visualization
with an ECL detection kit (Amersham, Freiburg, Germany) according to the instructions of the supplier,
specific signals were scanned (Scanner JX-325,
Sharp, 600 dpi, software ViceVersa Scan 1.2. Krystec
EDV, Norderstedt, Germany) and analysed with an
image analysis system.
Toxicity Test
The activity of mitochondrial dehydrogenases
was used as a measure for toxicity (Mossman et
al., 1983) of DETA and DETA/NO. Cells were
seeded in 96-well plates (12,000 cells suspended
in 200 µl cell culture medium/well) and allowed
to adhere for 8 h. Subsequently, medium was
changed and cells were cultured for 24 h in 200 µl
of medium with increasing concentrations of
DETA/NO. Following DETA/NO treatment, 20 µl
of MTT (3-(4,5-Dimethylthiazol-2yl)-2,5 diphenylArchives of Insect Biochemistry and Physiology
July 2007
doi: 10.1002/arch.
Intracellular Localization of EcR and Usp
tetrazoliumbromid (Sigma, Deisenhofen, Germany; 5 mg/ml in phosphate buffered saline) was
added, mixed carefully and incubated for 60 min
at 37°C. The medium was removed and the cells
lysed with DMSO-SDS-solution (99.4 ml DMSO,
0.6 ml acetic acid, 10 g SDS). After 10 min OD560
was measured.
127
of NO (Figs. 2 and 3). Nuclear localization of YFPEcR–B1 is considerably lower and is also not affected by NO (Figs. 4 and 5). These results confirm
that both Usp and EcR can enter the nucleus independently and show that neither receptor interact
with DNA to a significant extent in the absence of
hormone.
RESULTS
DNA Binding Is Not Important for Nuclear Localization
of EcR and Usp
The concentration of DETA/NO used does not
cause significant toxicity problems during the incubation time chosen; 83 ± 3.2 % of the cells were
still viable after incubation with 800 µM DETA/
NO for 6 h as determined by mitochondrial hydroxylase activity (Mossman et al., 1983). No degradation products of EcR and Usp are observed by
Western blots in the presence of DETA/NO although receptor concentrations were diminished after incubation for 24 h (Fig. 1) presumably due to
toxic effects after prolonged incubation times.
Nuclear localization of CFP-Usp I, which is
present to about 80% in the nucleus 6 h after transfection, is not significantly reduced in the presence
Fig. 1. Influence of DETA/NO on EcR and Usp. Extracts
from CHO-K1 cells were separated on SDS-PAGE and
nuclear receptors detected with antibodies against YFP. * =
CFP-Usp (calculated MW = 83.6 KD); ** = YFP-EcR (calculated MW = 122 KD).
Archives of Insect Biochemistry and Physiology
July 2007
doi: 10.1002/arch.
Fig. 2. Intracellular localization of Usp fused with a fluorescent protein. DETA/NO or DETA were added 1 h after
transfection. Localization of Usp was evaluated 6 h after
addition of DETA/NO. a: Effect of DETA/NO on CFP-UspI.
b: Intracellular localization of CFP-UspIII, where the Cdomain was deleted. c: Influence EcR on intranuclear localization of CFP-UspIII. E = EcR-B1, UI = CFP-UspI, UIII
= CFP-UspIII; DNO = DETA/NO; H= 1 µM Muristerone A
(n = 3, mean ± SD).
128
Cronauer et al.
Fig. 3. Microphotographs of CHO cells showing the intracellular localization of Usp fused to a fluorescent protein. a: CFP-UspI. b: CFP-UspI+DETA/NO. c: CFP-UspI +
DETA. d: CFP-UspIII. e: CFP-UspIII in the presence of EcR.
f: CFP-UspIII with EcR and 1 µM Muristerone A. g: CFP-
UspIII + DETA/NO. h: CFP-UspIII with EcR and DETA/
NO. i: CFP-UspIII with EcR, DETA/NO, and 1 µM Muristerone A. Experimental conditions are the same as described in Figure 2.
Ligand and Heterodimerization With Usp Promote
Interaction of EcR With DNA
C-domain was deleted (Beatty et al., 2006). CFPUsp III is localized predominately in the cytoplasm
(Figs. 2 and 3). Coexpression with EcR increases
nuclear localization of CFP-Usp III (Figs. 2 and 3)
and also of EcR compared to separate expression of
each receptor protein (Figs. 4 and 5) as shown for
YFP-EcR and CFP-Usp III in the presence of the corresponding receptor without a fused YFP-tag. We,
therefore, conclude that EcR can heterodimerize
with Usp in the absence of the C-domain via the
ligand binding domains and that heterodimerization with EcR can compensate the missing nuclear
localization signal of Usp III. It shows also that
nuclear localization is not dependent on the DNAbinding ability of Usp. The decrease in nuclear localization of CFP-UspIII after addition of DETA/NO
of about 40% (Fig. 2) indicates that a considerable
Nuclear localization of EcR is increased in the
presence of ligand (Fig. 4). This shows that ligand
binding to EcR is of functional importance.
The enhanced nuclear localization of EcR in the
presence of Muristerone A is abolished by NO,
demonstrating that hormone binding is necessary
for interaction of EcR with DNA (Fig. 4). About
30% of the EcR is sensitive to DETA/NO (Fig. 4).
The C-Domain of Usp Is Not Essential for Nuclear
Localization of EcR.
To investigate further whether DNA-binding capacity of Usp is necessary for nuclear localization
of EcR, we used a Usp variant (Usp III), where the
Archives of Insect Biochemistry and Physiology
July 2007
doi: 10.1002/arch.
Intracellular Localization of EcR and Usp
129
nitrosative stress caused by NO. The inhibitory effect of NO on the DNA-binding of zinc-finger proteins is most likely due to a reversible S-nitrosation
of cysteine residues of the two cys4-type zinc fingers, which are essential for DNA-binding (Garban
et al., 2005). Although NO inhibits DNA-binding
activity of the nuclear steroid hormone receptors,
it does not affect intracellular receptor protein levels or its nuclear import as recently shown for the
AR (Cronauer et al., 2006). The nuclear localization signal of EcR located in the CD-domain is
functional in the absence of zinc-fingers (Gwozdz
et al., 2007), which confirms that NO does not
change nuclear import. In the following study, the
diethylenetriamine nitric oxide adduct Deta/NO,
which spontaneously liberates NO in aqueous solutions, was used as a tool to selectively inhibit
DNA binding activity of EcR and Usp.
Nuclear Localization of EcR and Usp
Fig. 4. Intracellular localization of YFP-EcR. DETA/NO
was added 1 h after transfection. Intracellular localization
was evaluated 6 h after addition of DETA/NO. a: YFPEcR. b: YFP-EcR + UspI. c: YFP-EcR + UspIII. UI = UspI;
UIII = UspIII; E= YFP-EcR-B1; DNO = DETA/NO; H= 1
µM Muristerone A (n = 3, mean ± SD).
fraction seems to be bound to DNA. although Usp
III cannot directly interact with DNA itself.
DISCUSSION
NO as a Tool to Study Intranuclear Localization of
Nuclear Receptors
Zinc finger transcription factors are redox-sensitive and, therefore, molecular targets during
Archives of Insect Biochemistry and Physiology
July 2007
doi: 10.1002/arch.
EcR is heterogeneously distributed between cytoplasm and nucleus (Nieva et al., 2005). The dynamic equilibrium between different intracellular
localizations is influenced by various import signals,
which are identified in the AB, C, and E domains
and an export signal present in the E-domain of
EcR (Nieva et al., 2005; Gwozdz et al., 2007), which
allows leptomycin B sensitive transport of EcR into
the cytoplasm (Betanska et al., 2007).
The effective nuclear export seems to be responsible for the low nuclear concentration of EcR, since
nuclear import is quite effective. The high nuclear
content of EcR decreases with time as shown by
Nieva et al. (unpublished observation). No export
signal could be identified for Usp so far (Gwozdz
et al., 2007), which may be responsible for the constantly high Usp concentration in nuclei.
A reasonable explanation for the enhanced
nuclear localization of EcR in the presence of Usp
is that heterodimerization renders the nuclear export signal in the ligand-binding domain of EcR
inaccessible (Betanska et al., 2007).
Although both receptors can enter the nuclei
independently, the heterodimerization partners
mutually enhance nuclear localization. This is most
130
Cronauer et al.
Fig. 5. Microphotographs of CHO cells showing the intracellular localization of YFP-EcR. a: YFP-EcR. b: YFPEcR with 1 µM Muristerone A. c: YFP-EcR with DETA/
NO. d: YFP-EcR with DETA/NO and 1 µM Muristerone
A. e: YFP-EcR with UspI. f: YFP-Ecr with UspI and 1 µM
Muristerone A. g: YFP-EcR with UspI and DETA/NO. h:
YFP-EcR with UspI, DETA/NO, and 1 µM Muristerone A.
i: YFP-EcR with UspIII (C-domain of Usp deleted). j: YFPEcR with UspIII and 1 µM Muristerone A. k: YFP-EcR
with UspIII and DETA/NO. l: YFP-EcR with UspIII, DETA/
NO, and 1 µM Muristerone A. Experimental conditions
are the same as described in Figure 2. [Color figure can
be viewed in the online issue, which is available at
www.interscience.wiley.com]
Archives of Insect Biochemistry and Physiology
July 2007
doi: 10.1002/arch.
Intracellular Localization of EcR and Usp
obvious for EcR and can be demonstrated for Usp
only, if the nuclear localization signal in the DNAbinding domain of Usp is deleted.
In the absence of DNA, dimerization of nuclear
receptors in cytoplasm is mediated by the ligandbinding domains, which apparently occurs already
in the absence of hormone to a sufficient degree,
although ligand binding increases dimerization of
LBDs of EcR and Usp significantly (Grebe et al.,
2003; Greb-Markiewicz et al., 2005).
Within the nucleus, receptors are present in different compartments. Interaction of nuclear receptors with DNA seems to regulate interaction with
comodulators, which depends also on the relative
concentrations of the proteins involved (Voss et
al., 2005). As pointed out by Voss et al. (2005),
subnuclear distribution of receptor proteins provides an additional mechanism for regulation of
gene expression. Generally, it is assumed that DNA
binding enhances nuclear localization of receptors
by reducing nuclear export as reported for several
vertebrate nuclear receptors, e.g., the glucocorticoid
receptor (Sackey et al., 1996).
Initially, both nuclear receptors do not interact
with DNA. As shown previously, ecdysteroids bind
to EcR already in the absence of a heterodimerization partner (Grebe et al., 2003, 2004) leading
to increased nuclear localization of EcR in the presence of ligand. Only the increased nuclear fraction
of EcR observed after hormone treatment is NO
sensitive, indicating association with DNA. Therefore, we conclude that liganded EcR can bind to
DNA even in the absence of a dimerization partner, since the concentration of RXR, the ortholog
of Usp, in CHO-K1 cells is extremely low and
cotransfection with RXR has only a very weak influence on nuclear localization of EcR (Nieva et
al., personal communication), This is confirmed
by EMSA with EcR containing cell extracts and reporter gene assays in the absence of Usp resp RXR
(Braun and Tremmel, unpublished results).
As reported by Nieva et al. (2005) and Gwozdz
et al. (2007), nuclear localization of EcR is greatly
enhanced in the presence of the heterodimerization
partner Usp, although Usp alone present predominantly in the nucleus does not interact with DNA.
Archives of Insect Biochemistry and Physiology
July 2007
doi: 10.1002/arch.
131
Heterodimerization with EcR seems to be required
for efficient interaction with DNA. Mutational
analysis of the ligand-binding domain (LBD) of
EcR (Grebe et al., 2003, 2004) revealed that
heterodimerization can rescue to a certain extent
the deleterious effects of some mutations in the
LBD of EcR, which indicates an altered three-dimensional structure of the heterodimer. An altered
conformation of the LBD of EcR may be required
for interaction with DNA. This view is confirmed
by Nieva et al. (unpublished results), who showed
that the LBD of EcR enhances DNA binding of the
heterodimer.
Since the only nuclear localization signal present
in Usp (Gwozdz et al., 2007) is situated in the Cdomain, UspIII is localized predominantly in the
cytoplasm. Interestingly, nuclear localization of EcR
and UspIII is only modestly reduced, although Usp
III lacks a DNA-binding domain and NiedzielaMaijka et al. (2000) report that the DNA-binding
domain of Usp plays an important role in directing
EcR to the hormone response element. From our
results, we conclude that EcR can heterdimerize with
Usp in the absence of the C-domain via the ligandbinding domains and that heterodimerization with
EcR can compensate for the missing nuclear localization signal of Usp III. The reduction in nuclear
localization of CFP-Usp III after addition of DETA/
NO of about 40% (Fig. 2) indicates that a considerable fraction seems to be bound to DNA mediated by the C-domain of EcR. It shows also that
dimerization via the ligand-binding domains of EcR
and Usp occurs already in the absence of ligand and
stimulates DNA binding of the heterodimer, although UspIII itself can not directly interact with
DNA. According to Gbheish et al. (2001), some actions of EcR/Usp require DNA binding of both transcription factors, whereas for other effects only DNA
binding of the heterodimeric complex mediated
only by the DBD of EcR is sufficient.
Is NO a Functional Regulator of Nuclear Receptor
Activity In Vivo?
NO was used in this study as an experimental
tool only. Its presence and functional importance,
132
Cronauer et al.
e.g., during development in Drosophila melanogaster,
raises the question of whether regulation of DNA
binding of Zn-finger proteins by NO is also a physiological mechanism. It is well known that NO mediates its effects by modulating the activity of
guanylyl cyclase, e.g., dilatation of blood vessels in
vertebrates, but NO plays essential roles as a second messenger also in insects (Regulski et al., 2004).
An influence on transcriptional regulation by the
nuclear receptor E75 was shown by Reinking et al.
(2005). However, in this case a heme group is
present in the ligand-binding domain, which can
bind NO in addition to S-nitrosylation of the
cyteines of the zinc-fingers. The tight control of NO
synthase activity in Drosophila underlines the importance of NO-controlled pathways. NO regulates cell
proliferation in Drosophila (Kuzin et al., 1996, 2000)
and in the optic lobe of Manduca sexta in an
ecdysteroid hormone–controlled manner (Champlin and Truman, 2000).
Nitric oxide (NO) has been shown to play an
important role in a variety of physiological functions in vertebrates and invertebrates. In vertebrates, genes for three isoformes of NO synthase
(NOS) have been cloned. In Drosophila, only one
NOS was identified so far. The reversible regulation of protein function by S-nitrosation has led
to the proposal that posttranslational modification
of proteins by NO might be of physiological importance, although the role of nitrosation in vivo
remains unclear. Kröncke and Carlberg (2000) proposed a functional role of NO for regulation of
Zn-finger containing transcription factors activity.
This would provide an additional level of complexity for regulation of gene induction by nuclear receptors. The effect of NO on DNA binding is most
pronounced on receptor molecules before binding
to DNA and is attenuated by hormones. This may
indicate that the impact of NO is part of a regulatory network used for fine tuning of the hormone
response.
ACKNOWLEDGMENTS
YFP-EcR was a kind gift from Dr. A. Ozyhar
(Wrozlaw, Poland). Usp I and Usp-III were pro-
vided by Dr. V.C. Henrich (University of Greensboro, NC).
The skilful technical assistance of N. Möbius
and M. Burret is gratefully acknowledged.
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Archives of Insect Biochemistry and Physiology
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doi: 10.1002/arch.
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