INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

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-2_HD) 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-2_HD. 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.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

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 pGEX2TGR_C500Y (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. pTL2OCT2HD 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-2_HD (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.

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.

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 GR_C460Y (GR carrying a C460Y substitution) or GR_C460Y/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).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

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-GR_C500Y (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).

View larger version (33K): [in this window] [in a new window]   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.

View larger version (30K): [in this window] [in a new window]   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).

View larger version (49K): [in this window] [in a new window]   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.

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-GR_L501P fusion protein had no effect on reporter gene expression.

View larger version (28K): [in this window] [in a new window]   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).

View larger version (33K): [in this window] [in a new window]   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.

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.

View larger version (27K): [in this window] [in a new window]   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).

View larger version (118K): [in this window] [in a new window]   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.

Expression of the GR_C460Y 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.

View larger version (109K): [in this window] [in a new window]   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.

View larger version (94K): [in this window] [in a new window]   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.

View larger version (57K): [in this window] [in a new window]   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.

View larger version (39K): [in this window] [in a new window]   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.

Developmental defects induced by ectopic expression of the GR DBD are rescued by coexpression of Oct-2_HD. To begin to assess the specificity of the developmental defects induced by the GR DBD peptides, we tested whether the phenotypes induced by the GR_C460Y peptide could be influenced by the coexpression of a homeodomain peptide. For this experiment, we injected the mRNA encoding GR_C460Y alone (180 pg) or together with a twofold excess of the mRNA encoding Oct-2_HD (360 pg). Oct-2_HD 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-2_HD almost completely rescued the embryos from the effects of GR_C460Y. 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 GR_C460Y-encoding mRNA alone that failed to develop or displayed the severely malformed phenotypes illustrated in Fig. 7.

View larger version (24K): [in this window] [in a new window]   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).

View larger version (49K): [in this window] [in a new window]   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.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

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-2_HD 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).

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-2_HD counteracted the effects of the GR DBD to allow normal embryonic development.