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Molecular and Cellular Biology, October 1999, p. 7106-7122, Vol. 19, No. 10
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Developmental Effects of Ectopic Expression of the Glucocorticoid
Receptor DNA Binding Domain Are Alleviated by an Amino Acid
Substitution That Interferes with Homeodomain Binding
Jun Ming
Wang,1
Gratien G.
Préfontaine,2
Madeleine E.
Lemieux,1
Louise
Pope,1
Marie-Andrée
Akimenko,3,* and
Robert J. G.
Haché1,3,4,*
Department of
Medicine,1 Graduate Program in
Biochemistry,2 Department of Cellular
and Molecular Medicine,3 and Department
of Biochemistry, Microbiology, and Immunology,4
The Loeb Health Research Institute at the Ottawa Hospital,
University of Ottawa, Ottawa, Ontario, Canada K1Y 4E9
Received 10 May 1999/Returned for modification 21 June
1999/Accepted 24 June 1999
 |
ABSTRACT |
Steroid hormone receptors are distinguished from other members of
the nuclear hormone receptor family through their association with heat
shock proteins and immunophilins in the absence of ligands. Heat shock
protein association represses steroid receptor DNA binding and
protein-protein interactions with other transcription factors and
facilitates hormone binding. In this study, we investigated the
hormone-dependent interaction between the DNA binding domain (DBD) of
the glucocorticoid receptor (GR) and the POU domains of octamer
transcription factors 1 and 2 (Oct-1 and Oct-2, respectively). Our
results indicate that the GR DBD binds directly, not only to the
homeodomains of Oct-1 and Oct-2 but also to the homeodomains of several
other homeodomain proteins. As these results suggest that the
determinants for binding to the GR DBD are conserved within the
homeodomain, we examined whether the ectopic expression of GR DBD
peptides affected early embryonic development. The expression of GR DBD
peptides in one-cell-stage zebra fish embryos severely affected their
development, beginning with a delay in the epibolic movement during the
blastula stage and followed by defects in convergence-extension
movements during gastrulation, as revealed by the abnormal patterns of
expression of several dorsal gene markers. In contrast, embryos
injected with mRNA encoding a GR peptide with a point mutation that
disrupted homeodomain binding or with mRNA encoding the DBD of the
closely related mineralocorticoid receptor, which does not bind octamer
factors, developed normally. Moreover, coinjection of mRNA encoding the
homeodomain of Oct-2 completely rescued embryos from the effects of the
GR DBD. These results highlight the potential of DNA-independent
effects of GR in a whole-animal model and suggest that at least some of
these effects may result from direct interactions with homeodomain proteins.
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INTRODUCTION |
Nuclear hormone receptors comprise a
broadly distributed class of transcriptional regulators implicated in
diverse physiological processes (4, 54). Many nuclear
receptors play major roles in development; these include the control of
patterning and tissue differentiation. Nuclear receptors are marked by
a highly conserved DNA binding domain (DBD) comprised of two Cys4 zinc
fingers (48, 54). They also contain a less well conserved
C-terminal region that confers ligand responsiveness to the
transcriptional regulatory functions of many receptors.
Most nuclear receptors are constitutive transcriptional regulators
whose transcriptional regulatory potential is altered by association
with ligands. Steroid hormone receptors, however, are distinguished by
their association into transcriptionally inactive heat shock protein
(hsp)- and immunophilin-containing complexes in the absence of
steroidal ligands (4, 63). Association into hsp complexes
prevents steroid receptor DNA binding by physically masking the DBD.
hsp association also alters the conformation of the steroid receptor
ligand binding domain in a manner that facilitates ligand binding
(62, 63). Indeed, hsp association appears to be required for
ligand binding by the glucocorticoid receptor (GR).
In addition to direct regulation of transcription through DNA response
elements, it is becoming increasingly apparent that many important
functions of nuclear receptors are mediated through protein-protein
interactions with other transcription factors in the absence of DNA
binding (30, 37). The physiological importance of these
activities has recently been highlighted by a report that mice with a
mutation in the GR that compromised DNA binding are viable
(69).
Interestingly, several protein-protein interactions between nuclear
receptors and other sequence-specific transcription factors are
mediated through receptor DBDs (13, 20, 55, 76). The best
characterized of these interactions occurs with AP-1 (29, 38,
53). Several nuclear hormone receptors, including GR
(29), androgen receptor (AR) (75), estrogen
receptor 
(ER) (100), thyroid hormone receptor
(108), and retinoic acid receptors and retinoid X receptors
(81), form complexes with AP-1 that block the interaction of
AP-1 with its normal response elements. At least for GR, the
receptor-AP-1 complex is redirected to composite response elements
specific for the GR-AP-1 complex. In addition, GR can bind to and
repress the DNA binding of NF-
B (76). For steroid hormone
receptors, these interactions are dependent upon the dissociation of
hsp complexes and thus are sensitive to both steroids and steroid
antagonists (29).
Recently, we determined that three steroid hormone receptors, GR, AR,
and progesterone receptor (PR), associated through their DBDs with the
POU DBDs of octamer transcription factors 1 and 2 (Oct-1 and Oct-2,
respectively) (64, 65). In contrast, ER, mineralocorticoid receptor (MR), and several other nuclear receptors failed to interact with Oct-1 and Oct-2 in transfected cells. These
results indicated that Oct-1 and Oct-2 binding was restricted within
the nuclear hormone receptor superfamily. For GR, AR, and PR, octamer
factor binding was strictly dependent upon the dissociation of free
receptors from hsp-immunophilin complexes that followed ligand binding.
Functionally, binding to GR, AR, and PR affected the DNA targeting of
the octamer factors in the cell by preferentially increasing their
binding to octamer motifs in the transcriptional regulatory regions of
steroid hormone-responsive reporter genes (11, 64, 65, 97).
There also has been a report for GR that this interaction may
simultaneously decrease the occupancy of octamer motifs in
transcriptional regulatory regions lacking steroid hormone response
elements (46). For rat GR, C500Y and L501P substitutions in
the second zinc finger of the DBD abrogated octamer factor binding and
their recruitment to DNA (65).
Like GR, POU transcription factors comprise a large family of
transcriptional regulators with a highly conserved DBD (73). The POU domain is bipartite, consisting of POU-specific and POU homeodomain motifs that bind cooperatively to the 5' and 3' halves of
DNA response elements (43). Homeobox genes encode master developmental control proteins involved in virtually all aspects of
pattern formation and tissue differentiation in the developing embryo
(70, 72, 73, 101).
Several homeobox genes involved in the first steps of the establishment
of the vertebrate body axis during gastrulation have been
characterized. Homeobox genes such as goosecoid (8, 9, 32, 92), floating head or Xnot (95,
98), GSX (51), Xlim-1 (94), Otx2 (61, 89), Xtwin
(49), and Siamois (22, 39, 49, 52) are
expressed in the organizer and show dorsalizing properties when
ectopically expressed in the embryo. The homeobox genes
Xvent-1 and Vox (also named Xvent-2)
have been shown to be involved in ventral fate specification (27,
59, 78). Most of these genes are activated between the
midblastula transition and the onset of gastrulation. However, some of
them, such as goosecoid (92), Otx2
(61, 89), and Xtwin (49), as well as a
number of POU factors (58, 60, 79, 80) are expressed as
maternal factors prior to being zygotically expressed. Despite the
important functions of these genes at the onset of gastrulation, little
is known about the role of maternal homeodomain proteins during
cleavage of the vertebrate embryo and the role of the maternal and
zygotic homeodomain proteins prior to gastrulation.
Glucocorticoids are teratogenic (1). The most frequent
correlate of embryonic exposure is cleft palate (26). In
mammals, the expression of GR has been detected beginning at a stage
equivalent to day 9.5 of mouse embryogenesis (18, 42).
Further, the down regulation of GR mRNA at the final differentiation
stages of tissues in which it is expressed is suggestive of a
morphogenetic role (42). Indeed, insertional inactivation of
the GR gene results in mice that die soon after birth due to a defect
in the final stages of lung development (17). Studies with
Xenopus also indicate a role for GR at the earliest stages
of embryogenesis. GR mRNA is abundant in Xenopus oocytes but
is rapidly degraded during the early cleavage stages of embryogenesis.
GR transcripts are reexpressed prior to the completion of gastrulation
and become localized to the dorsal ectoderm (25).
In this study, we report the Oct-1 homeodomain and the Oct-2
homeodomain (Oct-2HD) to be necessary and sufficient for
the direct binding of Oct-1 and Oct-2 to the GR DBD. L501P-sensitive GR
binding to the homeodomain was not limited to Oct-1 and Oct-2 but also
was observed for several other homeodomain proteins, including zebra
fish (Danio rerio) dlx2 and hoxd4. Intriguingly, the
expression of GR DBD but not MR DBD peptides in one- or two-cell-stage zebra fish embryos specifically affected embryonic development at or
before the time of blastoderm migration in a manner that correlated
with the L501P-sensitive binding of the GR DBD to maternal and
embryonic factors. Moreover, these developmental defects were rescued
by the coexpression of a peptide containing Oct-2HD. These results establish the GR DBD as a molecular probe for important developmental events occurring near the time when embryonic
transcription is initiated and predict an important role for
homeodomain proteins that bind to the GR DBD during these events.
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MATERIALS AND METHODS |
Plasmids.
pGEM7Z-Oct-1 was produced by insertion of the
HindIII/BamHI Oct fragment from pCGN-Oct-1
(96) into the corresponding sites of pGEM7Z (Promega).
pGEX2T-GR (amino acids [aa] 407 to 568) and pGEX2TGRC500Y
(aa 407 to 556) were constructed by insertion of the
BamHI/EcoRI fragment from pSP64X568
(71) and the BamHI/SmaI fragment from
pT7C500Y (77), respectively, in frame into the appropriate
restriction sites of pGEX2T (Pharmacia). pTL2HAOct-2 was produced by
first inserting the Oct-2 XbaI (blunted)/BamHI fragment from pCGN-Oct-2 (96) into pACT-2 (Clontech) to
acquire a hemagglutinin epitope and then recloning with
BglII/XhoI into the corresponding sites of pTL2.
pTL2OCT2
HD was created by removing the
EagI/PstI DNA fragment corresponding to the
homeodomain (aa 296 to 359) and religating it in frame. pTL2-OCT2POU
and pTL2-OCT2SP were created through a similar deletion and
religation strategy to remove aa 152 to 349 and aa 152 to 286, respectively. pGEX2TGR-PKA vectors (wild type [WT], C500Y, L501P,
C460Y, and C460Y-L501P; aa 407 to 550) were constructed by insertion of
an oligonucleotide encoding a protein kinase A (PKA) recognition motif,
LARRASYP, into the StyI/EcoRI sites of plasmid
pGEX2T-GR. pGEX2T-OCT2HD (aa 294 to 377) was constructed by insertion
of a 14-bp linker into the BamHI/EagI sites of
pGEX2T-OCT2POU (aa 195 to 377) (65). Expression plasmids for
dlx2 (pGEX3X-dlx2 and pTL2-dlx2), hoxd4 (pTL2-hoxd4), and
msxB (pGEX3X-msxB; aa 135 to 196) were as described previously
(107), as were the pGEX2T-PrdHD, pGEX2T-FtzHD, and pGEX2T-OtdHD constructs (104). pGEX2T-HoxC4 (aa 148 to 227)
was subcloned from a human Jurkat T-cell cDNA library. pCGNOCT-1, pCGNOCT-2, and GR plasmids pT7X556 (aa 407 to 556), pT7C460Y, pT7R489R,
pT7L501P, and pT7C500Y have been described previously (71,
96). The GR double-mutant plasmid pT7C460Y/L501P was constructed
by replacing the XhoI/SphI DNA fragment from
pT7L501P with the corresponding fragment from pT7C460Y. pET-11a-MR-DBD and pET-11a-OCT2HD were constructed by insertion of the fragments containing the MR DBD (aa 567 to 700) and Oct-2HD (aa 294 to 377) coding sequences created by PCR from pGALO-MR-DBD
(64) and pGEX2T-OCT2HD, respectively, in frame into pET-11a
(Novagen; the NheI/BclI and NheI/BamHI sites, respectively). The plasmids
used for making the in situ probes have been described previously as
pBluescript SK for ntl (82), shh
(44), flh (95), MyoD
(102), gsc (92), and bmp4
(57) and pBluescript KS for axial
(93).
GST pull-down and immunoprecipitation protein binding
assays.
Glutathione S-transferase (GST) fusion proteins
were expressed, purified, and tested in a GST pull-down assay as
previously described (65). The 35S-Met-labeled
proteins were produced by in vitro translation (Promega TNT coupled in
vitro transcription-translation kit) with either T7 or SP6 RNA
polymerase. To produce C-terminal Oct-1 protein truncations, plasmid
DNAs were restricted prior to in vitro translation. To assay for
binding, labeled proteins were incubated with 0.5 µg of GST fusion
protein attached to glutathione-Sepharose in binding buffer (20 mM
HEPES [pH 7.9], 60 mM KCl, 12% glycerol, 1.5 mM EDTA, 1 mM
dithiothreitol, 0.15 mM phenylmethylsulfonyl fluoride [PMSF], 0.1%
Nonidet P-40 [NP-40]) for 2 h at 4°C. Following extensive
washing with binding buffer, the samples were eluted by boiling for 5 minutes in sodium dodecyl sulfate (SDS) sample buffer prior to
SDS-polyacrylamide gel electrophoresis (PAGE) analysis. The gels were
dried and visualized by fluorography.
For coimmunoprecipitation assays, in vitro-translated Oct-2FL,
Oct-2SP, Oct-2HD, Oct-2POU, dlx2, and hoxd4 were incubated with c-myc-tagged GR immunoprecipitated with antibody 9E10
from nuclear extracts prepared from dexamethasone-treated, stably
expressing SF-7 fibroblasts or the parental cell line as previously
described (65). Following extensive washing with binding
buffer, the bound proteins were resolved by SDS-PAGE and visualized by
fluorography. Myc-GR loading in each experiment was confirmed by
Western blot analysis with antibody 9E10 and visualized by enhanced
chemiluminescence (Amersham). Phosphorimager quantification (Bio-Rad
model GS-525) of the SDS-polyacrylamide gels and the immunoblots was
done with background subtraction.
For the direct binding assay, GST-GR WT and mutants tagged with a PKA
phosphorylation site were expressed in bacteria. The
GST fusion
proteins were first immobilized on glutathione-Sepharose
in TEGz50
buffer (50 mM Tris [pH 7.5], 50 mM NaCl, 10% glycerol,
0.5 mM EDTA,
50 µM ZnCl
2, 0.5 mM PMSF) plus 1% Triton X-100 and
then
labeled with [
-
32P]ATP by a kinase reaction with the
catalytic subunit of PKA (Sigma)
in HMK buffer (20 mM Tris [pH 7.5],
100 mM NaCl, 12 mM MgCl
2,
1 mM dithiothreitol) for 30 min
at 30°C. The reaction was terminated
by the addition of 1 ml of stop
buffer (10 mM NaPO
4, 10 mM
Na
4O
7P
2,
10 mM EDTA, 2 mg of bovine
serum albumin [BSA] per ml). The labeled
proteins were eluted in
TEGz50 buffer (
23) plus 1% Triton X-100
by thrombin (Sigma)
cleavage. Approximately 5 ng of the
32P-labeled peptides
was incubated with immobilized GST fusion proteins
in binding buffer in
the presence of 2 mg of BSA per ml and 1
mM PMSF. Following extensive
washing, the bound proteins were
eluted in SDS sample buffer, resolved
by SDS-PAGE, and visualized
by autoradiography. The input
GST-homeodomain proteins and BSA
were resolved by SDS-PAGE and
visualized by Coomassie blue
staining.
Tissue cultures and transient transfections.
CHO-K1 cells
(American Type Culture Collection [ATCC]) were maintained in
-minimal essential medium supplemented with 10% fetal bovine serum
(Life Technologies). SF-7 and clonal cell lines (65) were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum.
For the mammalian two-hybrid assay, bait plasmids pCGN-Oct-2,
pTL2-hoxd4, pTL2-dlx2, and pSG5CREB were cotransfected with
pGAL4-GR-DBD, pGAL4-GR
L501P, or pGALO and pG5E1BCAT into
CHO cells
(ATCC) with Lipofectamine (10 µl per 60-mm dish; 50 ng of
the
chloramphenicol acetyltransferase [CAT] reporter, 50 ng of the
GAL4 expression plasmid, and 25 ng of the bait plasmid). Cells
were
harvested 48 h posttransfection. The CAT activities of cytoplasmic
extracts were determined by standard protocols and are expressed
as
fold induction of GAL4-GR DBD or GAL4-GR
L501P over GALO.
pRSV-
GAL
(50 ng) was cotransfected to provide an internal control
for transfection
efficiency. All transfections and CAT assays were
performed in
duplicate in at least three independent assays. Error bars
represent
the standard errors of the
mean.
Fish and embryos.
Zebra fish eggs were obtained by natural
spawning from a colony of fish derived from stock from a local fish
supplier. Embryos were raised at a constant 28°C temperature in
system water or in embryo medium by standard methods (103).
Developmental stages are given in hours postfertilization (p.f.).
In vitro transcription and microinjection.
mRNAs of the GR
DBD were produced in vitro by linearizing the pT7 plasmids with
EcoRV, pET-11a-OCT2HD with BamHI, and
pET-11a-MR-DBD with HindIII and transcribing with T7
polymerase by use of an mRNA capping kit (Stratagene, La Jolla,
Calif.). Following ethanol precipitation, the RNAs were resuspended in
filtered distilled water. The purified mRNAs were quantified on a
Bio-Rad Gel Doc system following agarose gel electrophoresis.
Injections of 0.2 to 0.4 nl of synthetic, capped RNA at concentrations
of 100 to 600 µg/ml were given to one- or two-cell-stage zebra fish
embryos by use of backfilled capillaries (Flaming/Brown micropipette
puller; Sutter Instrument Co., Novato, Calif.) and a pressure-pulsed
microinjector (PV830 Pneumatic Picopump; WPI, Sarasota, Fla.). Injected
embryos were raised in embryo medium and monitored at 2-h intervals and at other times as specificallly indicated. Death and developmental abnormalities were recorded to 24 h p.f. Embryos were photographed with a Leica stereomicroscope. Embryos stored for in situ analysis were
fixed in phosphate-buffered saline (pH 7.4) containing 4% paraformaldehyde overnight at 4°C, the chorion was manually removed, and the samples were stored in methanol at 
20°C.
In situ hybridizations.
In situ hybridization with
whole-mount zebra fish embryos was performed as described previously
(2, 3) by use of nonhydrolyzed antisense RNA probes. Enzymes
used for linearization and for transcription for probe synthesis were
as follows: ntl (82), XhoI and T7 RNA polymerase (T7); shh (44), BglII and
T7; flh (95), EcoRI and T7;
axial (93), DraI and T3;
MyoD (102), EcoRI and T7;
gsc (92), BamHI and T7; and
bmp4 (57), EcoRI and T7.
Far-Western analysis of proteins binding to the GR DBD in
embryonic extracts.
Embryo lysates were prepared from zebra fish
embryos collected between cell cycles 8 and 10 (early blastula stages)
and from embryos at 50% epiboly (early gastrula stage). The dissected
embryos were placed in cell lysis buffer (25 mM HEPES [pH 7.9], 100 mM KCl, 20% glycerol, 0.2 mM PMSF, 2 mM EDTA, 2 mM DTT, 0.01% NP-40) (1 µl per embryo) at 4°C, and the cells were dispersed by
vortexing. An equal volume of 2× gel loading buffer (100 mM Tris-HCl,
[pH 6.8], 200 mM DTT, 4% SDS, 0.2% bromophenol blue, 20% glycerol) was added. Lysate (25 µg) from the early blastula or the early gastrula stage was loaded and resolved by SDS-12% PAGE. Following electrophoresis, the proteins were transferred to polyvinylidene difluoride membranes (Millipore). Denaturation and renaturation of
filter-bound proteins were accomplished by immersing the membranes in 6 M guanidine HCl, gently agitating the mixture at room temperature for
10 min, and renaturing the samples by 1:1 serial dilutions with binding
buffer (5, 7). The membrane was washed in binding buffer for
10 min, and the filter was blocked with 3% BSA (wt/vol) for a minimum
1 h at 20°C. The filter was then incubated with 32P-labeled GRC460Y (GR carrying a C460Y
substitution) or GRC460Y/L501P (GR carrying C460Y and L501P
substitutions) (in binding buffer containing 0.3% BSA; the final
specific activity was about 106 cpm per ml) overnight at
4°C. After five 15-min washes with binding buffer, the filter was
exposed to X-AR film (Kodak).
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RESULTS |
The octamer factor homeodomain is sufficient for C500Y- and
L501P-sensitive GR DBD binding in vitro.
To begin to delimit the
requirements within full-length Oct-1 and Oct-2 for binding to steroid
receptors, we examined the binding of C-terminally truncated forms of
Oct-1 to the GR DBD (aa 407 to 568) by using a GST pull-down assay
(Fig. 1A). In vitro-translated full-length Oct-1 bound specifically to the WT GR DBD
fused to GST but did not interact with GST-GRC500Y (Fig.
1A, lanes 1 and 2). Deletion of the Oct-1 C terminus up to the
homeodomain had only a slight effect on the interaction with GR (Fig.
1A, lane 3). Further truncation into the homeodomain, however,
abrogated binding (Fig. 1A, lanes 4 and 5). In an additional
experiment, it was confirmed that binding of the GR DBD to the Oct-1
homeodomain is distinct from the binding previously reported for herpes
simplex virus protein 16 (VP16), as substitutions in the Oct-1
homeodomain that have been shown previously to disrupt VP16 binding
(47) had no effect on GR binding in this assay
(66).
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FIG. 1.
The association of Oct-1 and Oct-2 with GR is lost
upon deletion of the Oct homeodomains. (A) Binding of in
vitro-translated, 35S-Met-labeled Oct-1 peptides to the WT
GR DBD (aa 407 to 568; lanes 2 to 5) or the GR DBD with a C500Y
substitution (lane 1), expressed as GST fusion proteins. Lanes 6 to 9 show the signal obtained with 10% of the labeled proteins added to the
binding assay. (B) Binding of full-length, 35S-labeled, in
vitro-translated Oct-2 (lane 1), Oct-2 deletion constructs lacking the
POU-specific domain (SP) (lane 2), the POU homeodomain (HD) (lane
3), and the complete POU domain (POU) (lane 4), and firefly
luciferase (Luc; lane 5) to full-length GR with a c-myc
epitope tag. Samples were immunoprecipitated with c-myc
antibody 9E10 from whole-cell extracts prepared from
dexamethasone-treated, stably transfected murine SF-7 fibroblasts.
Lanes 6 to 10 show the signal obtained with 10% of the labeled
proteins added to the binding assay. In both panels A and B, bound
peptides were resolved by SDS-PAGE (12% gels) and visualized by
fluorography.
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Similarly, immunoprecipitation assays performed with in
vitro-translated Oct-2 constructs containing internal deletions and
full-length GR with an N-terminal c-
myc tag in crude nuclear
extracts
prepared from murine SF-7 cells indicated that deletion of
Oct-2
HD was sufficient to eliminate GR binding (Fig.
1B).
While full-length
Oct-2 bound strongly to immunoprecipitated GR (Fig.
1B, lane 1),
deletion of the entire POU domain or the POU homeodomain
alone
from full-length Oct-2 eliminated binding (Fig.
1B, lanes 3 and
4). Interestingly, deletion of the POU-specific domain alone decreased
binding to the GR DBD somewhat (Fig.
1B, lane 2). This result
may
suggest a role for the POU-specific domain in GR binding.
However, as
the result was not supported in subsequent experiments,
it would seem
more likely that it reflects a decrease in the accessibility
of the
homeodomain to GR when the adjacent POU-specific domain
was deleted
from Oct-2.
To resolve whether the octamer factor homeodomain was sufficient for GR
binding and to determine whether binding was direct,
we performed a
binding assay with purified components that had
been expressed in
bacteria. WT and L501P-substituted GR DBDs (aa
407 to 550) containing
PKA recognition sequences were expressed
as GST fusion proteins,
labeled with
32P by use of PKA, and then separated from the
GST moiety by cleavage
with thrombin. The purified GR DBD peptides were
tested for binding
to the complete Oct-2 POU domain (aa 194 to 377) or
to Oct-2
HD (aa 294 to 377) expressed as GST fusion proteins
and bound to
glutathione-Sepharose (Fig.
2). The WT GR DBD bound strongly to
both
the complete POU domain and the POU homeodomain alone (Fig.
2, lanes 2 and 3), but no binding was detected to GST alone (Fig.
2, lane 4).
Further, the L501P substitution in the GR DBD prevented
binding (Fig.
2, lanes 6 to 8). Thus, Oct-2
HD was sufficient for
direct
binding to the GR DBD in vitro.
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FIG. 2.
The GR DBD binds directly to Oct-2HD.
GST-tagged WT GR DBD and GRL501P DBD (aa 407 to 550)
containing PKA consensus phosphorylation sites were expressed in and
purified to homogeneity from Escherichia coli and labeled
with 32P by use of PKA. Signals obtained with 10% of the
32P-labeled GR and GRL501P DBDs added to the
binding assay are shown in lanes 1 and 5. The 32P-labeled
GR peptides were tested for binding to the POU domain and POU
homeodomain of Oct-2 or to GST alone (lanes 2 to 4 and 6 to 8, respectively). MW, molecular weight (in thousands).
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As the homeodomain is remarkably well conserved, we next became
interested in determining whether GR might also bind directly
to the
homeodomains of other proteins. To first test this possibility
in
vitro, we expressed the homeodomains of several proteins as
GST fusion
proteins (Fig.
3A) and tested them for
binding to
32P-labeled GR DBD peptides (Fig.
3B and C).
Remarkably, all of
the homeodomains tested bound to the WT GR DBD
peptides (Fig.
3B, lanes 2 to 8), but none interacted significantly
with the
GR DBD peptide containing the L501P substitution (Fig.
3C,
lanes
2 to 8). By comparison with the input GST-homeodomain proteins,
binding was strongest to the HoxC4 homeodomain (Fig.
3B, lane
4) and to
full-length dlx2 (Fig.
3B, lane 3) and was weakest to
the Prd
homeodomain.
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FIG. 3.
The GR DBD binds directly to the homeodomains of several
proteins. (A) Coomassie blue-stained SDS-polyacrylamide gel of 0.5 µg
of BSA (lane 1) and GST fusion proteins containing human
Oct-2HD (lane 2), full-length zebra fish dlx2 (lane 3), and
the homeodomains of human HoxC4 (lane 4), Drosophila paired
(Prd, lane 5), Drosophila orthodenticle (Otd, lane 6), zebra
fish msxB (lane 7), and Drosophila fushi tarazu (Ftz, lane
8). MW, molecular weight (in thousands). (B and C) Autoradiographs of
SDS-polyacrylamide gels showing the binding of 32P-labeled
GR DBD (B) and GRL501P DBD (C) peptides to the GST fusion
proteins shown in panel A. Lanes 1 show the signal from 10% of the
32P-labeled peptides added to each incubation, while lane 9 shows binding to a glutathione-Sepharose extract from mock-transformed
bacterial cells.
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L501P-sensitive binding of GR to homeodomain proteins in vivo.
To determine whether GR DBD binding to full-length homeodomain proteins
could be detected in the cell, we selected two zebra fish homeodomain
proteins, hoxd4 and dlx2, which are unrelated outside the homeodomain
and only distantly related to Oct-1 and Oct-2 within the homeodomain
family, for further study. hoxd4 is a close zebra fish relative of
human HoxC4, whose homeodomain bound to the GR DBD (see above). First,
we compared the interaction of the GR DBD with hoxd4 and dlx2 in a
one-hybrid assay performed with CHO cells (Fig.
4). In these experiments, the activity of the CAT reporter gene used was dependent upon a specific association of
the GAL4-GR DBD fusion proteins and the full-length homeodomain proteins. Transactivation was mediated by the natural transcriptional activation domains within Oct-2, hoxd4, and dlx2. Expression of the
GAL4 DBD alone or fused to the GR DBD peptides had no significant effect on transcription of the CAT reporter gene (66).
However, coexpression of full-length Oct-2 with the WT GAL4-GR DBD
fusion protein potentiated CAT activity six- to eightfold above the
level obtained with the GAL4 DBD alone. This induction in activity was completely sensitive to the L501P mutation in the GR DBD, as
coexpression of Oct-2 with the GAL4-GRL501P fusion protein
had no effect on reporter gene expression.
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FIG. 4.
One-hybrid analysis of the L501P-sensitive interaction
of the GR DBD with full-length Oct-2, hoxd4, and dlx2 in mammalian
cells. The GR DBD, the DBD with an L501P substitution fused to the GAL4
DBD, or the GAL4 DBD alone was coexpressed with Oct-2, hoxd4, dlx2, or
CREB in CHO cells. At 48 h after transfection, transcription from
a reporter gene with an E1B minimal promoter and five GAL4 binding
sites was assessed by a CAT assay. The data are expressed as the fold
induction of CAT activity in the presence of GAL-GR
DBDL501P (hatched bars) and GAL-GR DBD (solid bars) versus
in the presence of GAL4 DBD (GALO). The error bars represent the
standard error of the mean of three to five independent experiments
performed in duplicate (P < 0.02).
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Coexpression of full-length hoxd4 and dlx2 with the GAL4-GR DBD fusion
proteins resulted in similar GR
L501P-sensitive activation
of transcription from the E1B promoter. In contrast, coexpression
of
the bZip transcription factor CREB had no significant effect
on
reporter gene activity under these conditions. These results
indicated
that specific binding to the GR DBD in the cell occurred
for at least
two homeodomain proteins outside the octamer transcription
factor
subfamily.
To confirm further the potential for interactions between full-length
GR and full-length homeodomain proteins, we examined
the ability of in
vitro-translated dlx2 and hoxd4 to bind to full-length,
ligand-activated GRs extracted from the nucleus of murine fibroblasts
(Fig.
5). Previously, we had demonstrated
that full-length GR
and both Oct-1 and Oct-2 associated in this assay
in a manner
that was completely sensitive to the GR
L501P
mutation (
64).
Similarly, both dlx2 and hoxd4 were observed
to bind WT GR from
extracts prepared from dexamethasone-treated cells
(Fig.
5A, lanes
2 and 6). In contrast, no binding was observed with
extracts prepared
from cells expressing GR
L501P (Fig.
5A,
lanes 3 and 7) or with
extracts prepared from control parental cells
lacking a stably
expressed GR (Fig.
5A, lanes 4 and 8).
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FIG. 5.
The binding of full-length GR in nuclear extracts to
full-length zebrafish dlx2 and hoxd4 is sensitive to
GRL501P. (A) Binding of full-length,
35S-labeled, in vitro-translated dlx2 (lanes 2 and 3) and
hoxd4 (lanes 6 and 7) to GR and GRL501P immunoprecipitated
with c-myc antibody 9E10 from whole-cell extracts prepared
from dexamethasone-treated, stably transfected murine SF-7 fibroblasts.
Lanes 4 and 8 show binding to extracts prepared from untransfected SF-7
cells, while lanes 1 and 5 show 10% of the in vitro-translated
homeodomain proteins added to the binding assay. (B) Western blot of
the GRs immunoprecipitated from stably transfected SF-7 cells
expressing WT GR and GRL501P and from untransfected SF-7
cells (lanes 9 to 11). Below each lane in panels A and B are the counts
obtained following phosphorimager analysis.
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Together, these results indicated, through three independent assays,
that a peptide encompassing the GR DBD could bind directly
to the
homeodomains of distantly related homeodomain proteins.
Further, in all
these assays, binding was sensitive to an L501P
substitution in the
DNA-contacting
-helix of the second zinc
finger.
L501P-sensitive, DNA-independent effects of the GR DBD on early
embryogenesis in zebra fish.
The homeodomain mediates DNA binding
and protein-protein interactions that determine a large component of
homeodomain protein function in the cell (73). Recent gene
replacement experiments have suggested that DNA-independent functions
of GR are important components of the steroid response (69).
The GR DBD contains several interfaces for protein-protein interactions
that paradoxically may mediate at least some of these DNA-independent
functions of GR (37). The diverse nature of GR
DBD-homeodomain binding observed in our molecular assays suggested that
ectopic expression of the GR-DBD in whole animals might affect the
normal activities of at least some of the proteins that can be
contacted by the GR DBD. Further, parallel experiments with the L501P
substitution would be expected to reveal effects specific to the
surface involved in homeodomain binding.
To test our hypothesis, we examined the consequences for development of
introducing mRNAs encoding GR DBD peptides into one-
or two-cell-stage
zebra fish embryos. Zebra fish are particularly
well suited for studies
examining the developmental consequences
of the ectopic expression of
peptides during embryogenesis (
19,
41). Eggs are fertilized
externally, and proteins can be reliably
expressed by microinjection of
mRNA immediately following fertilization.
Analysis of developmental
abnormalities that may be induced is
facilitated by the optical clarity
and rapid development of the
embryos (
40). Further,
preliminary microinjection experiments
with mRNA encoding green
fluorescent protein (GFP) established
that the proteins encoded from
the microinjected mRNAs in our
experiments were expressed uniformly
through 24 h of development
in almost all cells of the embryos
(
99).
All of the GR constructs expressed in the embryos in our experiments
contained the primary GR nuclear localization sequence
to ensure that
the peptides expressed would be concentrated in
the embryonic nuclei.
Further, to exclude effects on development
that might result from the
binding of GR DBD to DNA, the GR DBD
peptides expressed also contained
amino acid substitutions C460Y,
K489R, and/or L501P, which compromise
the DNA binding of GR (
77).
Although C460Y and K489R
interfere with GR DNA binding, they were
not observed to affect binding
to the POU domain of Oct-1 (
65).
In the first experiments, the status of the microinjected embryos was
examined 24 h after injection, at the end of the segmentation
period. The results of these experiments are presented in Fig.
6 and
7 and
in Table
1.
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FIG. 6.
Microinjection of mRNA encoding a GR peptide with the
homeodomain binding surface into one- or two-cell-stage zebra fish
embryos interferes with embryogenesis in an L501P-sensitive manner. The
outcome at 24 h is displayed for one- or two-cell-stage embryos
injected with 0.2 to 0.4 nl of RNAs (600 µg/ml) encoding GR mutant
peptides with a GR DBD backbone (aa 407 to 556), an antisense
transcript of the WT GR, or the MR DBD (aa 567 to 700) or with saline
alone. The peptides encoded by the RNAs injected are listed at the
left, followed by the total number of embryos injected (n) and the
total number of independent injection series performed with each
sample. To the right, the percentage of embryos failing to survive for
24 h following injection is indicated by dark gray bars, while the
percentage of embryos with malformations visible under the
stereomicroscope is indicated by light gray bars. The error bars
indicate the standard deviations for independent trials (for
GRC460Y and GRK489R compared to the controls,
the P value was <0.001).
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FIG. 7.
Examples of the four major classes of visible defects in
zebra fish embryos 24 h following injection with
GRC460Y. From microinjected embryos, the chorion was
manually removed, and the samples were photographed under a
stereomicroscope at a magnification of ×63. Anterior is to the left;
dorsal is to the top. (A) A normally developed control embryo injected
with saline. (B to D) Examples of embryos lacking a distinct axis. (E)
Embryo with a curved tail. (F) Embryo lacking both a head and a tail.
(G) Embryo missing a head and with a truncated tail. The arrowheads
indicate the axial mesoderm (B to D), and the arrow indicates the
somites (C). Scale bar: A, 130 µm; B to G, 106 µm.
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Microinjection of mRNAs encoding aa 407 to 556 of GR with a C460Y or
K489R substitution severely affected embryonic survival
(Fig.
6). Fewer
than 50% of the embryos injected with either mRNA
survived to 24 h. In contrast, only approximately 10% of embryos
microinjected with
saline or an antisense GR DBD-encoding mRNA
failed to develop to
24 h. This value is well within the percentage
of growth arrest
that is expected to result from the mechanical
manipulation and
puncture of the embryos in this procedure (
6,
24). Moreover,
the mean time to arrest for the embryos injected
with the test mRNAs
was 8 to 10 h, during late gastrulation, while
the mean time to
arrest for the control embryos was approximately
12 h
(
99). Remarkably, the development of embryos microinjected
with mRNA encoding GR DBD with an L501P mutation or a combination
of
C460Y and L501P mutations was indistinguishable from that of
the
control embryos (Fig.
6). Moreover, the effects on development
were
highly specific to expression of the GR DBD. In contrast
to the severe
effects of the ectopic expression of the GR DBD
peptides,
microinjection of mRNA encoding comparable DBD peptide
from MR, which
differs from GR by only 4 aa within the core of
the DBD but which
does not interact with the octamer factor homeodomains
(
65), resulted in completely normal embryonic development
that
was indistinguishable from that of all of the controls (Fig.
6).
In addition, over 50% of the embryos that were microinjected with the
GR
C460Y- and GR
K489R-encoding mRNAs and that
survived
to 24 h were afflicted with axial abnormalities that were
readily
visible under the stereomicroscope. In contrast, malformations
of embryos microinjected with L501P-substituted and C460Y- and
L501P-substituted GR DBDs and with the MR DBD occurred at the
same low
frequency as that observed for embryos microinjected
with antisense
mRNA or
saline.
Several examples of the developmental abnormalities observed at 24 h p.f. for embryos injected with the GR
C460Y construct
are
shown in Fig.
7. The predominant phenotype observed (46% ±
7%) was
dramatic underdevelopment, with embryos lacking a unitary
body axis as
well as differentiated anteroposterior structures
(Fig.
7B to D). These
embryos generally exhibited distinct notochord-like
regions that were
separated by lobes of yolk. Many embryos (25%
± 5%) contained a
nearly normal axis in the trunk region but lacked
head and tail
structures (Fig.
7F and G), while the rest exhibited
defects primarily
in the development of the tail (29% ± 8%) (Fig.
7E).
The severity of the phenotypes observed correlated directly with the
quantity of GR DBD mRNA microinjected into the embryos
(Table
1). A
decrease in the concentration of the GR
C460Y peptide
injected from 600 to 100 µg/ml led to a 40% decrease in the number
of embryos whose development was arrested prior to 24 h. In
addition,
at 100 µg/ml there was a concomitant increase in the
percentage
of severely malformed embryos that survived to 24
h.
Together, these results provided a strong indication that the region of
the GR DBD including the homeodomain binding interface
specifically
interfered with some early events important for the
axial development
of the
embryos.
Expression of the GRC460Y peptide perturbs the
formation of the axial mesoderm.
The phenotype of the malformed
embryos uniformly suggested that the defects induced by the GR DBD
peptides most likely resulted from defects in the formation of the
axial mesoderm, in particular, notochord formation. To examine in
greater detail the nature of the effect of the GR peptides on the early
development of the embryo, we analyzed the expression of several
markers for the early development of the axial mesoderm and notochord
by in situ hybridization with microinjected whole-mount embryos.
The zebra fish
no tail (
ntl) gene is a member of
the T-box gene family (
82-84) and is likely the orthologue
of the mammalian
Brachyury gene, a developmental control
gene which encodes a transcription
factor directly implicated in the
formation of the primitive streak
(
68,
105). In zebra fish,
ntl is required for notochord and
tail formation (
28,
82).
In wild-type embryos,
ntl expression is first detected at
the late blastula stage (4 h p.f.) in a few dispersed cells at the
dorsal side of the blastula (
84). Later,
ntl is
expressed in
cells of the presumptive mesoderm of the germ ring (or
marginal
zone) and at the early gastrula stage (5.5 h p.f.). It is also
activated in cells of the embryonic shield at the dorsal midline
(Fig.
8A). The expression of
ntl is
specifically maintained in
the axial mesoderm as cells migrate away
from the blastoderm margin
(Fig.
8F). By the end of gastrulation,
ntl expression is confined
to the developing notochord cells
in the axial mesoderm and to
both axial and nonaxial cells in the
developing tail bud (Fig.
8K). After 24 h of development,
ntl expression is localized to
the notochord cells of the
tail (Fig.
8P).
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FIG. 8.
The expression of the GRC460Y peptide in
zebra fish embryos disrupts the pattern of no tail
expression during embryogenesis. In situ hybridization of whole-mount
zebra fish embryos with a no tail antisense RNA probe at the
indicated times after fertilization is shown. Embryos were injected at
the one- or two-cell stage with saline (mock) (A, F, K, and P) or mRNAs
encoding the GR DBD (aa 407 to 556) with C460Y (B to D, G to I, L to N,
and Q) or C460Y and L501P (E, J, and O) mutations. Three examples of
the hybridization patterns observed upon expression of the
GRC460Y peptide at each time point are shown. Dorsal views
are shown in panels A and C to O, while an animal pole view is shown in
panel B and a lateral view of the tail is shown in panels P and Q. The
arrow in panel Q indicates the secondary notochord axis. a, axial
mesoderm; gr, germ ring; es, embryonic shield; n, notochord. Scale bar:
A to O, 100 µm; P and Q, 70 µm.
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The pattern of expression of
ntl in embryos injected with
RNAs encoding GR DBDs with the C460Y and L501P mutations was similar
to
that observed in control embryos at all developmental stages
(Fig.
8E,
J, and O). In contrast, microinjection of RNA encoding
GR DBD with the
C460Y mutation severely affected
ntl expression
from the
earliest stages analyzed. For the majority of the embryos,
blastoderm
migration over the yolk cell had not yet reached 50%
epiboly, as
expected at 5.5 h p.f. (Fig.
8B to D). In a large
proportion of
the GR
C460Y-injected embryos,
ntl transcripts
were
found in a few cells at the dorsal margin of the blastoderm, a
pattern similar to that observed in normal embryos at 4 h p.f.
(
84), suggesting a developmental delay in these embryos
(Fig.
8B). Strikingly, in the rest of the embryos,
ntl
expression was
either interrupted in the marginal zone (Fig.
8C) or was
seen
as a small indentation (Fig.
8D) at the dorsal side of the
embryos.
These observations were suggestive of defects in the formation
of the embryonic
shield.
At 8.5 h p.f., while
ntl expression in
GR
C460Y-injected embryos appeared essentially normal in
cells of the germ ring, expression
in the notochord precursors was
affected to various degrees (Fig.
8G to I). In the less affected
embryos, the domain of
ntl-expressing
cells in the axial
mesoderm was wider and less anteriorly extended
than in the control
embryos, a finding which may have reflected
defects in the convergence
and extension movements of the cells
during gastrulation (compare Fig.
8F and G). In more affected
embryos, in addition to a delay in
development, it seemed that
the shield area was devoid of
ntl-expressing cells (Fig.
8H and
I). Nevertheless,
involution movement probably occurred, since
rudiments of stripes of
cells extending anteriorly were visible
starting from the extremities
of the germ ring surrounding the
normal position of the shield (Fig.
8H
and I). These kind of patterns
would have presumably led to embryos (if
viable) with partially
duplicated
axes.
At later stages (12 h),
ntl expression revealed that most
surviving embryos had a short and kinked notochord axis (Fig.
8L
and
M), suggesting that extension movements toward the anterior
part of the
embryos may have been affected. Some embryos at the
tail bud stage had
two distinct short duplicated axes as a possible
consequence of
convergence defects during the gastrula stage (Fig.
8N). Finally, among
the embryos surviving to 24 h p.f., we observed
the presence of a
short secondary notochord axis expressing
ntl and developing
from the tail region of the embryos, a finding
which may have been the
result of a late duplication of the notochord
axis during the
elongation period of the embryos (Fig.
8Q).
To confirm and expand on the observations obtained with
ntl,
we examined the expression of other markers of the axial mesoderm
in
the microinjected embryos (Fig.
9). These
markers included
sonic hedgehog (
shh), which
encodes a signaling protein involved
in the differentiation and
patterning of various embryonic tissues,
including the notochord and
the floor plate cells of the neural
tube (
44); the homeobox
gene
floating head (
flh), the zebra
fish ortholog
of the
Xenopus gene
Xnot, which is involved in
notochord
development (
95); and
axial, the
ortholog of the mouse
HNF-3 gene, which encodes a
transcription factor of the winged-helix
family and which has been
shown to be essential for the development
of the axial mesoderm
(
93) (Fig.
9A, E, and I). Injection of
zebra fish embryos
with RNAs encoding GR DBDs carrying the C460Y
and L501P mutations did
not significantly alter the expression
of these markers (Fig.
9D, H,
and L). In contrast, the expression
of the GR
C460Y peptide
severely affected the pattern of expression
of
shh,
flh, and
axial in the developing axial mesoderm
in ways
that were strikingly similar to those observed for
ntl (Fig.
9B,
C, F, G, J, and K). Together, these results
strongly support our
conclusion that the GR DBD peptides interfered
with early embryonic
developmental processes required for the normal
formation of the
axial mesoderm.
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FIG. 9.
Whole-mount in situ hybridization showing the expression
of developmental markers for axial mesoderm (a) (A to L) and paraxial
mesoderm (p) (M to P) formation in 12-h embryos. Embryos were injected
at the one- or two-cell stage with saline (mock) (A, E, I, and M) or
mRNAs encoding the GR DBD (aa 407 to 556) with C460Y (B, C, F, G, J, K,
N, and O) or C460Y and L501P (D, H, L, and P) mutations. Dorsal views
of the patterns of expression of sonic hedgehog
(shh, A to D), floating head (flh, E
to H), axial (I to L), and MyoD (M to P) in 12-h
embryos are shown. In GRC460Y/L501P-injected embryos (D, H,
L, and P), the patterns of expression of shh,
flh, and axial in the developing notochord and of
MyoD in the paraxial mesoderm do not differ significantly
from those observed in control embryos (A, E, I, and M). In contrast,
in GRC460Y-injected embryos, the shh,
flh, axial, and MyoD patterns of
expression are highly perturbed (B, C, F, G, J, K, N, and O). tb, tail
bud. Scale bar: A to P, 140 µm.
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To examine whether the GR DBD peptides also affected development beyond
the axial mesoderm, we analyzed the expression of
MyoD, a
gene encoding a basic helix-loop-helix protein expressed
in presomitic
mesoderm during gastrulation and then in somites
(Fig.
9M to P)
(
21,
102). We observed that the expression of
MyoD in 10-h zebra fish embryos injected with RNA encoding
the
GR
C460Y/L501P peptide did not significantly differ from
that in
control embryos (Fig.
9M and P). At this stage,
MyoD
transcripts
are normally found in two elongating rows of cells, the
adaxial
cells, adjacent to the axial mesoderm. These cells
differentiate
into the slow muscle fibers of the zebra fish myotome
(
21).
In contrast, the pattern of expression of
MyoD in GR
C460Y-injected
embryos showed various
degrees of disruption, ranging from more
widely separated stripes of
adaxial cells (Fig.
9N) to a complete
disruption of
MyoD
expression (Fig.
9O), reflecting the defects
observed in the patterns
of the axial
markers.
As our analysis of axial markers indicated defects in the formation of
the embryonic shield, we extended our analysis further
to examine the
expression of
goosecoid (
gsc), a homeobox gene
that has been proposed to participate in the establishment and
maintenance of the organizer and shield (
16,
84,
92).
Furthermore,
gsc is expressed very early during zebra fish
embryogenesis; it
is first maternally and ubiquitously expressed in the
embryo,
and then zygotic transcripts are detected at 4 h p.f.,
shortly
after the midblastula transition (
84,
92).
Therefore, it is
a valuable marker for analyzing the developmental
effects of GR
peptides prior to
gastrulation.
At the late blastula stage,
gsc is expressed as a patch of
cells at the margin of the blastoderm (
84,
92). At the onset
of gastrulation, the domain of
gsc expression is limited to
the
shield region (Fig.
10A). As was
observed with the other axial
markers, only embryos injected with RNA
encoding the GR
C460Y/L501P peptide reproduced the normal
pattern of expression of
gsc at
that stage (Fig.
10C).
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FIG. 10.
Whole-mount in situ hybridization showing the
expression of goosecoid in microinjected embryos. Embryos
were injected at the one- or two-cell stage with saline (mock) (A and
D) or mRNAs encoding the GR DBD (aa 407 to 556) with C460Y (B and E) or
C460Y and L501P (C and F) mutations. Patterns of expression of
goosecoid at 5.5 h (A to C) and 12 h (D to F) are
shown. Dorsal views are shown in panels A, B, and D to F, while an
animal pole view is shown in panel C. Injection of GR
DBDC460Y/L501P failed to modify gsc expression
at all developmental stages (compare panels A, D, C, and F). (A and C)
At the onset of gastrulation, gsc is expressed in the
embryonic shield on the dorsal part of the embryo (indicated by an
arrowhead in panel A). (B) Blastoderm migration in most
GRC460Y-injected embryos is delayed compared to that in
control embryos and GRC460Y/L501P-injected embryos, and
gsc expression is greatly reduced. (D and F) gsc
expression is confined to the prechordal plate (indicated by an arrow
in panel D) and cells of the anterior part of the dorsal midline
(indicated by an arrowhead in panel D). (E) In
GRC460Y-injected embryos, gsc is expressed in a
cluster of cells without any distinct pattern; in particular, no
rostral crescent corresponding to the prechordal mesoderm is visible.
Scale bar: A to F, 106 µm.
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Even taking into account the developmental delay of these embryos, the
pattern of expression of
gsc differed from the normal
expression of
gsc observed in noninjected embryos at the
late
blastula stage. Indeed, at 5.5 h p.f., a time at which
embryos
injected with the GR
C460Y-encoding RNA had an
appearance normally
associated with 4.7 h p.f. (30% epiboly),
gsc was expressed only
in a very small subset of cells in
the dorsal area of the embryo
but was not localized at the embryonic
margin (Fig.
10B), as expected
for WT embryos at 30% epiboly. This
observation lends further
credence to the suggestion that the
misexpression of the GR
C460Y peptide affected events
occurring very early during
development.
As gastrulation proceeds,
gsc transcripts are restricted to
the anterior edge of the progressing shield and start to be expressed
in a rostral crescent of cells of the prechordal plate and some
cells
of the anterior part of the dorsal midline (Fig.
10D). While
embryos
injected with GR
C460Y/L501P-encoding RNA had a
gsc expression
pattern similar to that of control embryos,
GR
C460Y-injected embryos
always had a wider
gsc
domain of expression in the dorsal midline
and rarely showed the
characteristic crescent-shaped expression
in the prechordal plate cells
(Fig.
10E and
F).
To determine whether, in addition to the observed dorsal defects, the
expression of the GR
C460Y peptide also generated ventral
defects in the embryos, we analyzed the expression of the bone
morphogenetic gene,
bmp4 (
41,
57), which is
involved in the
ventral patterning of zebra fish embryos (Fig.
11). At the beginning
of gastrulation
(shield stage, 6 h p.f.),
bmp4 is strongly expressed
on
the ventral region of embryos, especially in the marginal zone
(Fig.
11A). In addition,
bmp4 transcripts are also found on the
lateral areas and in the inner cells of the embryonic shield (
41,
57). We observed, at the shield stage, a lateral expansion of
the
domain of
bmp4 expression on the dorsal region of
GR
C460Y-injected
embryos (Fig.
11B). In contrast,
bmp4 expression on the ventral
part of these embryos
appeared unchanged. Embryos injected with
saline or with
GR
C460Y/L501P-encoding RNA showed the normal
bmp4 pattern of expression (Fig.
11C). Similarly, the
expression of
bmp2, another member of the bone morphogenetic
gene family, and
eve-1, a homeobox gene related to the
Drosophila even-skipped gene, whose patterns of expression
on the ventrolateral areas
of embryos at the early gastrula stage
overlap extensively with
that of
bmp4 (
33,
57),
remained unchanged in GR
C460Y-injected
embryos
(
99). These results suggest that the developmental effects
of GR DBD peptide expression may be restricted to the dorsal region
of
zebra fish embryos.
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FIG. 11.
Whole-mount in situ hybridization showing the
expression of bmp4 in microinjected embryos at the shield
stage. Embryos were injected at the one- or two-cell stage with saline
(mock) (A) or mRNAs encoding the GR DBD (aa 407 to 556) with C460Y (B)
or C460Y and L501P (C) mutations. Animal pole views of embryos are
oriented with their ventral area to the left and their dorsal area to
the right. Note that the bmp4 pattern of expression is
affected only in the dorsal region of GRC460Y-injected
embryos. The arrow indicates the position of the embryonic shield.
Scale bar: A to C, 105 µm.
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Developmental defects induced by ectopic expression of the GR DBD
are rescued by coexpression of Oct-2HD.
To begin to
assess the specificity of the developmental defects induced by the GR
DBD peptides, we tested whether the phenotypes induced by the
GRC460Y peptide could be influenced by the coexpression of
a homeodomain peptide. For this experiment, we injected the mRNA
encoding GRC460Y alone (180 pg) or together with a twofold excess of the mRNA encoding Oct-2HD (360 pg).
Oct-2HD was selected for these experiments as it binds
poorly to DNA in the absence of the POU-specific domain but binds
avidly to the GR DBD. The results were striking (Fig.
12). The coexpression of
Oct-2HD almost completely rescued the embryos from the
effects of GRC460Y. Fully 65% of the coinjected embryos
developed without visible defects to 24 h p.f., compared to the
nearly 75% of embryos injected with the GRC460Y-encoding
mRNA alone that failed to develop or displayed the severely malformed
phenotypes illustrated in Fig. 7.
View larger version (24K):
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|
FIG. 12.
Microinjection of mRNA encoding Oct-2HD
into one- or two-cell-stage zebra fish embryos rescues the
developmental defects induced by GRC460Y. The outcome at
24 h is displayed for one- or two-cell-stage embryos injected with
0.2 to 0.4 nl of RNA encoding GRC460Y (600 µg/ml), saline
alone, or RNAs encoding GRC460Y and Oct-2HD
(1,200 µg/ml). The peptides encoded by the RNAs injected are listed
at the left, followed by the total number of embryos injected (n) and
the total number of independent injection series performed with each
sample. To the right, the percentage of embryos failing to survive for
24 h following injection is indicated by dark gray bars (D), the
percentage of embryos with malformations visible under the
stereomicroscope is indicated by light gray bars (M), and the
percentage of normal embryos is indicated by white bars (N). The error
bars indicate the standard deviations for independent trials (for
GRC460Y and GRC460Y plus Oct-2 compared to the
controls, the P value was <0.001).
|
|
The effect of the Oct-2
HD-encoding mRNA appeared to occur
in direct opposition to the effect of the GR
C460Y peptide
rather
than by nonspecifically influencing GR
C460Y
production. Coinjection
of the same amounts of
Oct-2
HD-encoding mRNA and GFP-encoding
mRNA had no effect
on the level of expression of GFP compared
to injection of GFP-encoding
mRNA alone, and coinjection of GFP-encoding
mRNA and
GR
C460Y-encoding RNA had no effect on the phenotypes
obtained (
99). Further, as the concentration of the
Oct-2
HD-encoding
mRNA was decreased within the same total
mRNA concentration, the
effects of GR
C460Y reappeared
proportionally (
99). These results
do not in any way prove
that the developmental effects of the
GR peptides were mediated through
binding to embryonic homeodomain
proteins. However, they do strongly
suggest that the defects resulting
from the embryonic expression of the
GR DBD arise from GR DBD-mediated
protein-protein interactions that
overlap with the binding of
the GR to a
homeodomain.
Last, our molecular analysis suggested that specific binding to a
protein or proteins expressed in the early embryo interfered
with their
normal function at a time coincident with the onset
of embryonic
transcription. To obtain direct evidence in support
of this hypothesis,
cell lysates prepared from early-blastula-
and early-gastrula-stage
zebra fish embryos were examined for
specific binding to WT and
GR
L501P DBD peptides by a far-Western
approach (Fig.
13). Extracts prepared from
early-blastula-stage
embryos collected between zygotic cell cycles 8 and 11 (approximately
between 2.25 and 2.75 h p.f.) were composed
entirely of proteins
derived from maternal mRNAs (
35). In
contrast, in extracts prepared
from early-gastrula-stage embryos
(collected at 50% epiboly),
there was also a strong representation of
proteins derived from
mRNA transcribed from the embryo genome (
35,
36,
40).
View larger version (49K):
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|
FIG. 13.
Far-Western analysis of the binding of
GRC460Y and GRC460Y/L501P peptides to proteins
extracted from early zebra fish embryos. (A) Protein lysates were
prepared from early-blastula-stage and early-gastrula-stage dissected
zebra fish embryos from which the chorion had been removed. A Coomassie
blue-stained SDS-12% polyacrylamide gel analysis of 25 µg of each
extract is shown. (B) Following transfer to nylon membranes, the
extracts were denatured and renatured and the membranes were hybridized
with either GRC460Y (lanes 1 and 2) or
GRC460Y/L501P (lanes 3 and 4) peptides labeled with
32P to the same specific activity at a PKA phosphorylation
site added to the C termini of the peptides. Incubation, washing, and
exposure to autoradiographic film of the membranes were performed
identically for each probe.
|
|
Following electrophoresis in duplicate on a single gel, transfer to a
nylon membrane, and renaturation, duplicate samples
were incubated with
equal numbers of counts of
32P-labeled GR
C460Y
and GR
C460Y/L501P peptides labeled to the same
specific
activity. Exposure of the membrane revealed the presence
of several
nuclear factors that were preferentially recognized
by the
GR
C460Y peptide in several independent experiments. In
the
extract from the early blastula, two bands at 31 and 28 kDa
bound the
GR
C460Y peptide (Fig.
13B, lane 1). While binding to
the
31-kDa band was only modestly affected by the L501P mutation,
the
signal at 28 kDa decreased markedly (Fig.
13B, lane 3). Thus,
the GR
DBD bound specifically to at least one maternally expressed
factor in a
manner that was sensitive to the L501P
substitution.
In the extract from the gastrula, the intensity of the signals obtained
with the GR
C460Y peptide at 31 and 28 kDa increased
severalfold (Fig.
13B, lane 2). However, although equal
amounts
of proteins from blastula and gastrula lysates were examined,
we cannot rule out the possibility that the increase in signal
intensity at 31 and 28 kDa reflected an increase in the number
of cells
expressing these proteins at the gastrula stage rather
than an increase
in protein synthesis per cell. In addition to
the 28- and 31-kDa
signals, a third L501P-sensitive signal, at
45 kDa, was also detected.
Interestingly, in this extract, the
31-kDa signal was highly
sensitive to the L501P substitution in
the GR DBD peptide (Fig.
13B,
lane 4), suggesting that the signal
originated from a factor different
from that in the extract from
the early blastula. Thus, it appears that
the L501P-sensitive
developmental defects induced by the
expression of the GR DBD
may arise from specific interactions with
proteins encoded by
either or both maternal or zygotically transcribed
RNAs.
| |
DISCUSSION |
Many studies have demonstrated the ability of nuclear hormone
receptor DBDs to enter into productive protein-protein interactions with other transcription factors. Elsewhere, we have demonstrated that
three steroid hormone receptors, GR, AR, and PR, can interact productively with the Oct-1 and Oct-2 proteins through their DBDs to recruit them to DNA but that MR is unable to interact with Oct-1 and
Oct-2 under the same conditions (64). In the present work,
we determined that, when ectopically expressed following mRNA
microinjection into one- or two-cell-stage zebra fish embryos, GR but
not MR DBD peptides severely perturbed the development of the axial
mesoderm. These effects correlated directly with the L501P-sensitive
direct binding of GR to the homeodomains of several homeodomain
proteins in vitro and in transfected cells and were rescued by the
coexpression of Oct-2HD in embryos. While the full extent
of the influence of GR-homeodomain binding on homeodomain protein
activity in vivo remains to be proven, the sensitivity in our
experiments of the developmental defects to the same L501P substitution
that abrogated GR-homeodomain binding in tissue culture cells is highly
suggestive of a causal linkage to the developmental effects.
DBD-mediated nuclear receptor-homeodomain protein
binding influences homeodomain protein function.
The
homeodomain contains a highly conserved DNA binding domain. Homeodomain
proteins generally bind to DNA sequences containing a highly redundant
core motif. However, alone, most homeodomain proteins bind DNA
only with a relatively low affinity. In many instances, therefore,
homeodomain protein targeting to specific DNA response elements in the
cell has been shown to be dependent upon protein-protein interactions
with other transcription factors that promote the localization of the
homeodomain proteins to specific transcriptional regulatory regions.
For example, Hox homeodomain proteins from Hox gene
complexes gain DNA binding specificity and affinity through cooperative
binding with the divergent homeodomain protein Pbx1 (87),
and abdB-like Hox proteins stabilize DNA binding via the homeodomain
protein Meis1 (88). It has also been demonstrated that Pbx1
and Meis1 dimerize and display distinctive DNA binding specificities
(14). In another example, the human homeodomain protein
Phox1 has been shown to impart serum-responsive transcriptional
activity to the c-fos serum response element by interacting
with the serum response factor (90). Another homeodomain protein, the cardiogenic Nkx-2.5 factor, is recruited by serum response
factor to activate cardiac alpha-actin gene transcription in murine
fibroblasts (15). Nkx-2.5 also cooperates with GATA-4, a
zinc finger transcription factor, to activate the transcription of the
cardiac alpha-actin and atrial natriuretic factor genes (50,
85); in both cases, the protein interaction region has been
mapped to the Nkx-2.5 homeodomain (15, 50, 85).
Protein-protein interactions with factors other than GR have also been
shown to influence the DNA targeting of POU domain
proteins beyond the
increase in DNA binding specificity provided
by the POU-specific
domain. For example, the formation of a ternary
complex among Oct-1,
host cell factor, and herpes simplex virus
protein 16 redirects Oct-1
from octamer motifs to TAATGARAT sequences
(
45).
Additionally, the transcription factor Ets-1, a nuclear
phosphoprotein
involved in cell proliferation, functionally and
physically interacts
with GHF-1/Pit-1 to direct transcription
from the prolactin promoter
(
10).
While it is noteworthy that all of the homeodomain proteins tested in
vitro in our experiments bound directly to the GR DBD
in an
L501P-sensitive manner and that the phenotype derived from
the ectopic
expression of the GR DBD was rescued by the coexpression
of
Oct-2
HD, the extent of GR-homeodomain binding in vivo
remains
to be established. In particular, the degree to which the GR
DBD
binds productively to homeodomain proteins in the cell under
physiological
conditions will need to be evaluated for each potential
interaction.
While the high degree of conservation within the
homeodomain may
permit broad-based binding to the GR DBD in vitro and
in transient
overexpression assays, it would seem more probable that
productive
interactions in vivo would be tightly restricted within
glucocorticoid-responsive
tissues to the subset of homeodomain proteins
with a physiologically
relevant affinity for GR. The first example of
such a physiologically
relevant effect appears to be the interaction
among GR, Oct-1,
and Oct-2, which seems to be crucial for the
responsiveness of
mouse mammary tumor virus to steroid hormones
(
11,
64,
65).
AR and PR also bind to the POU domains of Oct-1 and Oct-2 and promote
their binding to the mouse mammary tumor virus promoter
in transfected
cells (
64). Therefore, it may seem tempting to
speculate
more broadly that nuclear receptors generally bind to
and affect
homeodomain protein function. However, other experiments
reinforce the
expectation that nuclear receptor-homeodomain protein
binding in situ
will be found to occur at the individual, rather
than at the family,
level. For example, although GR, AR, and PR
appear to interact
productively with Oct-1 and Oct-2, several
other nuclear receptors,
including ER
, MR, retinoid X receptor
retinoic acid receptor
, and FTZ-F1
, were unable to interact
with Oct-1 and Oct-2 in two
hybrid experiments with transfected
cells (
64).
Nevertheless, we suggest that the apparently general nature of the
potential for direct GR DBD-homeodomain binding highlighted
in our
experiments may reflect a broadly conserved ability of
nuclear hormone
receptors to enter, on an individual basis, into
productive
interactions with specific homeodomain proteins. Further,
a conserved
basic mechanism of protein-protein binding may underlie
specific
interactions between individual proteins from each family.
In this
regard, we note a recent report that the
Drosophila FTZ-F1
nuclear receptor binds through its DBD to the homeodomain of
fushi tarazu (FTZ) in a way that promotes the cooperative
binding of
FTZ to transcriptional regulatory regions also containing
DNA
binding sites for FTZ-F1 (
106). Similarly, the POU
domains of
Brn-3a and Brn-3b interact with the DBD of ER
to
differentially
modulate transcription from estrogen response elements
(
12).
Ectopic expression of the GR DBD disrupts critical events that
control the earliest stages of embryogenesis.
The expression of GR
DBD peptides compromised for DNA binding by C460Y or K489R point
mutations after microinjection of mRNA into one- or two-cell-stage
zebra fish embryos affected development prior to the completion of the
blastula stage in a manner that predominantly led to embryonic death
during gastrulation. Remarkably, these effects were completely
sensitive to the L501P substitution that abrogated GR-homeodomain
binding. Further, the coexpression of Oct-2HD counteracted
the effects of the GR DBD to allow normal embryonic development.
Our analysis of the affected embryos suggests that the phenotypes and
the defects in gene expression observed between 5.5
and 8.5 h p.f.
are most likely to have arisen from an L501P-sensitive,
GR DBD-mediated
defect in the normal cell movements that occur
through the blastula and
gastrula stages and that are at the origin
of the formation of the
embryonic axis. At the blastula stage,
during epibolic movement, cells
of the blastoderm migrate toward
the vegetal pole of the embryo. These
movements occurred normally
in embryos microinjected with control RNA
and RNAs encoding GR
DBDs with L501P and with C460Y and L501P
substitutions. In contrast,
the spreading of the blastoderm toward the
vegetal pole was clearly
delayed following the microinjection of a GR
DBD-encoding RNA
lacking the L501P
substitution.
This observation suggested that the defects were initiated at or before
the early phase of epiboly. The blastula stage is
a crucial
developmental stage during which, concomitant with the
acquisition of
cell motility, zygotic transcription is activated
during the
midblastula transition period that initiates at cell
cycle 10 of zebra
fish embryogenesis (
35). However, the molecular
events
underlying the onset of transcription in the zygote are
not presently
understood. Our results raise the interesting possibility
that
the GR
C460Y peptide affected the function of key maternal
proteins required for the initiation of zygotic transcription
and/or
interacted with and affected the developmental function
of one or more
of the earliest zygotic proteins. Further, they
also may reflect the
specific interference of the GR DBD with
the transcriptional regulatory
activity of homeodomain proteins
at the onset of zygotic
transcription.
However, it is also possible that the defects observed during epiboly
were mediated at a level other than transcription. They
may reflect the
consequences of GR DBD peptide effects on proteins
acting at an even
earlier developmental stage, such as during
the early phase of the
blastula stage or during the cleavage period
(0 to 2.25 h).
Indeed, far-Western analysis of potential GR DBD
binding factors
indicated the potential for both maternally and
zygotically expressed
proteins to be recognized by the GR DBD
in an L501P-sensitive manner.
Further experiments, including analyses
of cell death and cell
proliferation at these early times of development,
are being pursued to
more clearly localize the onset of the effects
of the GR DBD
peptides.
Interestingly, the GR
C460Y-injected embryos share some
early phenotypic characteristics with some zebra fish epiboly mutants
isolated in zebra fish mutagenesis screens (
34,
56) which
illustrated that both maternal and embryonic contributions are
essential for controlling early embryonic cell movements. Indeed,
Kane
et al. described the characterization of four recessive epiboly
mutations which, when homozygous, result in a slowdown and arrest
of
epiboly around midepiboly; these mutations are lethal during
or shortly
after gastrulation (
34). The similarity of the phenotypes
of
some of these mutants with the defects observed in the
GR
C460Y-injected
embryos suggests that the ectopic
expression of GR DBD peptides
may have interfered with proteins
involved in the same pathways
as those affected in these mutants. Thus,
GR DBD peptides may
offer a means to identify key components of the
machinery responsible
for early morphogenetic
movements.
The consequences of the early L501P-sensitive events induced by the GR
DBD peptides became more prominent as embryogenesis
progressed. In
zebra fish, gastrulation is characterized by several
morphogenetic
movements, including involution of the cells at
the embryonic margin
and convergence and extension movements that
reshape the blastula
embryo. Our present analysis does not allow
us to comment on potential
defects in the involution movement,
which marks the onset of
gastrulation. To obtain this information,
a detailed analysis of cell
movement at the margin of the embryo
by use of Nomarski optics and time
lapse will be
necessary.
However, the localization of the expression of axial mesodermal markers
surrounding but not within the normal field of the
embryonic shield is
suggestive of L501P-sensitive inhibition of
cell migration or
convergence to the organizer field in affected
embryos. Moreover, the
blunting of the anterior extension and
the widening of the axial
mesoderm in GR
C460Y-expressing embryos,
which in extreme
cases led to axial duplication, are also suggestive
of defects in
convergence and/or extension movements. Alternatively,
it is also
possible that the widening of the domains of expression
of the axial
markers observed in the GR
C460Y-injected embryos
originated
from an increased number of dorsal cells. However,
the induction of
axial duplication in many embryos is more likely
to be consistent with
the hypothesis of incomplete convergence
of laterally and ventrally
located cells toward the dorsal midline,
as an increase in cell number
would be expected to enlarge the
width of only a single
axis.
The combination of defects in convergence and extension movements
during gastrulation could account for the phenotype of the
surviving
24-h embryos lacking anterior and posterior structures.
Interestingly,
this phenotype resembles that which has been observed
for a number of
gastrulation mutants that remain to be molecularly
characterized,
including mutants showing defects in convergence
and extension
movements (
56,
91).
Finally, we note with interest that the defects that we have observed
in the development of zebra fish embryos upon expression
of the GR DBD
with a C460Y or K489R substitution resemble the
effects observed upon
the overexpression of full-length nuclear
receptors in
Xenopus embryos (
25,
26,
67,
86). In
preliminary
studies, the overexpression of full-length GR in
Xenopus embryos
resulted in developmental defects starting
during the early blastula
stage (
25,
26).
Interestingly, while these defects were also
lethal prior to the
completion of the gastrula stage, they were
entirely dependent upon
steroid
treatment.
The defects observed in our experiments also overlapped extensively
with the defects observed upon the overexpression of retinoid
X
receptor and thyroid hormone receptor in
Xenopus embryos
(
67).
In these experiments, the amounts of receptors
required to generate
the severe phenotypes decreased markedly when the
embryos were
treated with triiodothyronine (
67). Moreover,
the occurrence
of phenotypic defects in these experiments and other,
related
transgenic experiments in which the role of the DBD was
investigated
was dependent on the presence of an intact receptor DBD
(
31,
67,
74,
86). Our results suggest that, rather than
resulting
strictly from the DNA-dependent properties of the nuclear
receptors
used in these studies, these phenotypes also may be
determined
by the DNA-independent actions of the receptor
DBDs.
Our far-Western and one-hybrid analysis results suggest that the
identity of the key factors controlling early morphogenetic
events that
are targeted by GR may be expected to be revealed
by expression library
screening approaches that detect specific
protein binding. Experiments
to further localize the onset of
the developmental defects and to
identify the L501P-sensitive
molecular targets of the GR DBD are
ongoing.
| |
ACKNOWLEDGMENTS |
We thank the many people who provided the plasmids used in this
work including, in particular, Q. Long, T. Zerucha, and M. Ekker but
also K. Yamamoto, W. Herr, C. Schild-Poulter, D. Grunwald, M. Halpern, V. Korzh, U. Strähle, M. Tada, D. Wilson, and E. Weinberg. We are grateful to the members of the Haché
laboratory and to Y. Lefebvre and M. Ekker for critical
comments on the manuscript.
This work was supported by an operating grant from the Medical Research
Council of Canada. J.M.W. has been funded by an L. Siminovitch
postdoctoral fellowship from The Loeb Health Research Institute at the
Ottawa Hospital and a fellowship from the Natural Sciences and
Engineering Research Council of Canada. G.G.P. is the recipient of an
MRC studentship. M.E.L. holds a junior postdoctoral fellowship from the
National Cancer Institute of Canada. R.J.G.H. is a Medical Research
Council of Canada scientist.
| |
FOOTNOTES |
*
Corresponding author. Mailing address for
Marie-Andrée Akimenko: The Loeb Health Research Institute, 725 Parkdale Ave., Ottawa, Ontario, Canada K1Y 4E9. Phone: (613) 761-5142. Fax: (613) 761-5036. E-mail: makimenko{at}lri.ca. Mailing address
for Robert J. G. Haché: The Loeb Health Research
Institute, 725 Parkdale Ave., Ottawa, Ontario, Canada K1Y 4E9.
Phone: (613) 761-5142. Fax: (613) 761-5036. E-mail:
rhache{at}lri.ca.
| |
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Molecular and Cellular Biology, October 1999, p. 7106-7122, Vol. 19, No. 10
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Abstract
Introduction
