Cooperative Interaction of Xvent-2 and GATA-2 in the Activation of the Ventral Homeobox Gene Xvent-1B
2002; Elsevier BV; Volume: 277; Issue: 26 Linguagem: Inglês
10.1074/jbc.m201831200
ISSN1083-351X
AutoresHenner Friedle, Walter Knöchel,
Tópico(s)Pluripotent Stem Cells Research
ResumoThe Xvent family of homeobox transcription factors is essential for the establishment of the dorsal-ventral body axis during Xenopus embryogenesis. In contrast toXvent-2B and other members of the Xvent-2 subfamily,Xvent-1B is not a direct response gene of bone morphogenetic protein-4 signaling. Xvent-1B is activated by Xvent-2, but CHX experiments revealed the requirement of additional factors. In this study, we report on the cooperative effect of Xvent-2 and the zinc finger transcription factor GATA-2 on the promoter of the Xvent-1B gene. We show thatGATA-2 is a direct target gene of bone morphogenetic protein-4 and that GATA-2 interacts with Xvent-2 to activate transcription of Xvent-1B. Both transcription factors bind to distinct elements within the Xvent-1B promoter, and GATA-2 physically interacts with the C-terminal domain of Xvent-2. Promoter/reporter studies in Xenopus embryos revealed that full activation of Xvent-1B requires both Xvent-2 and GATA-2. Moreover, the two factors are sufficient to direct transcription of Xvent-1B in the presence of CHX at the ventral side of the embryo. The failure of both factors to activateXvent-1B on the dorsal side suggests the existence of a dorsal inhibitor. This inhibitor is likely a component of the dorsal Wnt signaling pathway because nuclear translocation of β-catenin before midblastula transition results in a suppression ofXvent-1B transcription. The Xvent family of homeobox transcription factors is essential for the establishment of the dorsal-ventral body axis during Xenopus embryogenesis. In contrast toXvent-2B and other members of the Xvent-2 subfamily,Xvent-1B is not a direct response gene of bone morphogenetic protein-4 signaling. Xvent-1B is activated by Xvent-2, but CHX experiments revealed the requirement of additional factors. In this study, we report on the cooperative effect of Xvent-2 and the zinc finger transcription factor GATA-2 on the promoter of the Xvent-1B gene. We show thatGATA-2 is a direct target gene of bone morphogenetic protein-4 and that GATA-2 interacts with Xvent-2 to activate transcription of Xvent-1B. Both transcription factors bind to distinct elements within the Xvent-1B promoter, and GATA-2 physically interacts with the C-terminal domain of Xvent-2. Promoter/reporter studies in Xenopus embryos revealed that full activation of Xvent-1B requires both Xvent-2 and GATA-2. Moreover, the two factors are sufficient to direct transcription of Xvent-1B in the presence of CHX at the ventral side of the embryo. The failure of both factors to activateXvent-1B on the dorsal side suggests the existence of a dorsal inhibitor. This inhibitor is likely a component of the dorsal Wnt signaling pathway because nuclear translocation of β-catenin before midblastula transition results in a suppression ofXvent-1B transcription. lymphoid enhancer factor/T-cell factor bone morphogenetic protein cycloheximide cytomegalovirus glutathioneS-transferase midblastula transition reverse transcription truncated BMP type I receptor promoter mutated at a TCF target site The establishment of the dorsal-ventral body axis in vertebrate embryogenesis is the result of antagonisms among different growth factors and/or their mediators. One of the first steps in the early development in Xenopus laevis is the accumulation of β-catenin in the future dorsal signaling center, the Nieuwkoop center (1Larabell C.A. Torres M. Rowning B.A. Yost C. Miller J., Wu, R.M. Kimelman D. Moon R.T. J. Cell Biol. 1997; 136: 1123-1136Crossref PubMed Scopus (351) Google Scholar). The interaction of β-catenin with high mobility group box transcription factors of the LEF/TCF1 family leads to the activation of other dorsal factors such as the homeobox transcription factor siamois (2Brannon M. Kimelman D. Dev. Biol. 1996; 180: 344-347Crossref PubMed Scopus (122) Google Scholar). All of these molecules are components of the dorsal Wnt signaling pathway and result, when ectopically expressed on the ventral side, in a dorsalization of the embryo and formation of a second axis (3Behrens J. von Kries J.P. Kühl M. Bruhn D. Wedlich D. Grosschedl R. Birchmeier W. Nature. 1996; 382: 638-642Crossref PubMed Scopus (2595) Google Scholar). Another important signaling pathway in early embryogenesis is the BMP pathway which, in contrast to the dorsal Wnt signal, is responsible for the activation of ventral molecules (4Hogan B.L.M. Genes Dev. 1996; 10: 1580-1594Crossref PubMed Scopus (1722) Google Scholar, 5Graff J.M. Cell. 1997; 89: 171-174Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). In Xenopus, it has been shown that the autoregulatory loop of BMP-4 expression is mediated by the ventral homeobox protein Xvent-2 (6Schuler-Metz A. Knöchel S. Kaufmann E. Knöchel W. J. Biol. Chem. 2000; 275: 34365-34374Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Because Xvents can mimic all early BMP-2/4 effects, the Xvent transcription factors are regarded as downstream effectors of BMP-2/4 signaling in early amphibian development (7Lemaire P. Bioessays. 1996; 18: 701-704Crossref PubMed Scopus (14) Google Scholar). Although most of our knowledge about Vents is derived from experiments with Xenopus and zebrafish (8Gawantka V. Delius H. Hirschfeld K. Blumenstock C. Niehrs C. EMBO J. 1995; 14: 6268-6279Crossref PubMed Scopus (303) Google Scholar, 9Ault K.T. Dirksen M.-L. Jamrich M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6415-6420Crossref PubMed Scopus (85) Google Scholar, 10Rastegar S. Friedle H. Frommer G. Knöchel W. Mech. Dev. 1999; 81: 139-149Crossref PubMed Scopus (71) Google Scholar, 11Onichtchouk D. Gawantka V. Dosch R. Delius H. Hirschfeld K. Blumenstock C. Niehrs C. Development. 1996; 122: 3045-3053Crossref PubMed Google Scholar, 12Papalopulu N. Kintner C. Dev. Biol. 1996; 174: 104-114Crossref PubMed Scopus (89) Google Scholar, 13Ladher R. Mohun T.J. Smith J.C. Snape A.M. Development. 1996; 122: 2385-2394Crossref PubMed Google Scholar, 14Schmidt J.E. von Dassow G. Kimelman D. Development. 1996; 122: 1711-1721Crossref PubMed Google Scholar, 15Imai Y. Gates M.A. Melby A.E. Kimelman D. Schier A.F. Talbot W.S. Development. 2001; 128: 2407-2420PubMed Google Scholar), molecular cloning of a human Vent-like gene has recently been reported (16Moretti P.A.B. Davidson A.J. Baker E. Lilley B. Zon L.I. D'Andrea R.J. Genomics. 2001; 76: 21-29Crossref PubMed Scopus (19) Google Scholar). Based upon amino acid sequence comparisons, members of the Xvent family are divided into two subfamilies, the Xvent-1 (Xvent-1 (8Gawantka V. Delius H. Hirschfeld K. Blumenstock C. Niehrs C. EMBO J. 1995; 14: 6268-6279Crossref PubMed Scopus (303) Google Scholar), PV.1 (9Ault K.T. Dirksen M.-L. Jamrich M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6415-6420Crossref PubMed Scopus (85) Google Scholar), and Xvent-1B (10Rastegar S. Friedle H. Frommer G. Knöchel W. Mech. Dev. 1999; 81: 139-149Crossref PubMed Scopus (71) Google Scholar)) and Xvent-2 (Xvent-2 (11Onichtchouk D. Gawantka V. Dosch R. Delius H. Hirschfeld K. Blumenstock C. Niehrs C. Development. 1996; 122: 3045-3053Crossref PubMed Google Scholar), Xbr-1b (12Papalopulu N. Kintner C. Dev. Biol. 1996; 174: 104-114Crossref PubMed Scopus (89) Google Scholar), Xom (13Ladher R. Mohun T.J. Smith J.C. Snape A.M. Development. 1996; 122: 2385-2394Crossref PubMed Google Scholar), Vox 15 (14) and Xvent-2B (10Rastegar S. Friedle H. Frommer G. Knöchel W. Mech. Dev. 1999; 81: 139-149Crossref PubMed Scopus (71) Google Scholar)) subfamilies. Besides their sequence divergence, the two groups differ clearly in their expression patterns, which already suggests different regulatory mechanisms. It has been shown that Xvent-2B is a direct target gene of BMP signaling (10Rastegar S. Friedle H. Frommer G. Knöchel W. Mech. Dev. 1999; 81: 139-149Crossref PubMed Scopus (71) Google Scholar). The BMP mediator Smad1, which is phosphorylated by the BMP type I receptor, interacts with Smad4 to build a transcriptionally active complex on the Xvent-2B promoter (17Henningfeld K.A. Rastegar S. Adler G. Knöchel W. J. Biol. Chem. 2000; 275: 21827-21835Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 18Hata A. Seoane J. Lagna G. Montalvo E. Hemmati-Brivanlou A. Massagué J. Cell. 2000; 100: 229-240Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). This is part of a subsequent, indirect autoregulatory loop in which BMP-4 induces its own expression by using Xvent-2 as a mediator (6Schuler-Metz A. Knöchel S. Kaufmann E. Knöchel W. J. Biol. Chem. 2000; 275: 34365-34374Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) and a direct autoregulatory loop in which Xvent-2 activates its own expression (19Henningfeld K.A. Friedle H. Rastegar S. Knöchel W. J. Biol. Chem. 2002; 277: 2097-2103Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). In contrast, Xvent-1B is not up-regulated by BMP signaling in the absence of de novo protein synthesis and, therefore, is not regarded as a direct BMP-4 target (10Rastegar S. Friedle H. Frommer G. Knöchel W. Mech. Dev. 1999; 81: 139-149Crossref PubMed Scopus (71) Google Scholar). Instead,Xvent-1B can be activated by Xvent-2; and because it can rescue the phenotype caused by the dominant-negative Xvent-2 P(40) mutant, it has been suggested that Xvent-1 acts downstream of Xvent-2 (10Rastegar S. Friedle H. Frommer G. Knöchel W. Mech. Dev. 1999; 81: 139-149Crossref PubMed Scopus (71) Google Scholar). However, Xvent-2, like BMP-4, is not capable of activating members of the Xvent-1 family in the presence of cycloheximide (CHX). This suggests that either another or an additional factor is necessary for the activation of Xvent-1B. This factor could either be produced by a target gene of Xvent-2, or it might synergize with Xvent-2 in a cooperative manner. One possible candidate factor seemed to be the zinc finger protein GATA-2 (20Zon L.I. Mather C. Burgess S. Bolce M.E. Harland R.M. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10642-10646Crossref PubMed Scopus (151) Google Scholar), which activatesXvent-1, and when overexpressed as a dominant-interfering construct, had been shown to suppress specifically expression of theXvent-1 but not of the Xvent-2 gene (21Sykes T.G. Rodaway A.R.F. Walmsley M.E. Patient R.K. Development. 1998; 125: 4595-4605PubMed Google Scholar). TheGATA-2 gene is induced by BMP-4 (22Maeno M. Mead P.E. Kelley C., Xu, R.H. Kung H.F. Suzuki A. Ueno N. Zon L.I. Blood. 1996; 88: 1965-1972Crossref PubMed Google Scholar), and GATA-2 expression in ventral mesoderm starts at the early gastrula stage (23Kelley C. Yee K. Harland R. Zon L.I. Dev. Biol. 1994; 165: 193-205Crossref PubMed Scopus (114) Google Scholar),i.e. at the right time and at the right place to suggest this factor as an additional player in the in vivoregulation of Xvent-1 transcription. In the present report we have investigated the transcriptional regulation of the Xvent-1B gene by Xvent-2 and GATA-2. We have found that GATA-2 is able to activate the Xvent-1Bpromoter and that it rescues the effects induced by a dominant-negative Xvent-2 mutant. In contrast, blocking BMP signaling at the level of the BMP receptor, which leads to an inhibition of endogenous Xvent-2 gene activity, cannot be compensated for by GATA-2. Therefore, it seems unlikely that GATA-2 operates downstream of Xvent-2. Instead, this observation favors a model of a cooperative action between Xvent-2 and GATA-2 in the activation of Xvent-1B. To test this model we have performed protein-DNA binding assays. We could define target elements for both Xvent-2 and GATA-2 factors on the Xvent-1promoter. It could also be shown that GATA-2 interacts directly with the C-terminal domain of the Xvent-2 protein. Analysis of the Xvent-1B promoter by microinjection of different deletion mutants further supports the cooperative function of these two transcription factors. Whereas activation by GATA-2 is strictly dependent on Xvent-2 binding elements, Xvent-2 also seems to activate Xvent-1B in a GATA-2 independent manner, albeit at a much lower extent. Furthermore, although Xvent-2 is unable to activate Xvent-1B in the presence of CHX, a combination of Xvent-2 and GATA-2 is sufficient to up-regulate Xvent-1Bexpression if injected on the ventral side. However, overexpression of GATA-2 and Xvent-2 on the dorsal side does not lead to an activation ofXvent-1B transcription in CHX-treated embryos, even if coinjected with BMP-4. This suggests that activation ofXvent-1B on the ventral side of embryos in vivois the result not only of the presence of Xvent-2 and GATA-2 proteins, but also the absence of dorsal inhibitors. Xvent-1B promoter fragments were created by PCR using the following primers: upstream, –249 (5′-CGGGATCCATGGGATTCTGTGCCG-3′), −164 (5′-CGGGATCCACTGGAGCCAGGACCAGG-3′); downstream, +52 (5′-CCCAAGCTTCTGAAGGGAAGGCTGCT-3′), –164 (5′-CGGGATCCGAGTCTGTCAGGTTAGTG-3′). Nucleotide positions refer to the published sequence (10Rastegar S. Friedle H. Frommer G. Knöchel W. Mech. Dev. 1999; 81: 139-149Crossref PubMed Scopus (71) Google Scholar). The resulting PCR products were digested with BamHI/HindIII and cloned into BglII/HindIII of the pGL3-basic vector (Promega). The Xvent-1B/pGL3 construct −55/+52 was obtained by digesting the –164/+52 PCR fragment with Sau3A and the –249/Δ/+52 construct by fusing the –249/−164 and –55/+52 fragments. The GATA-mutated (Gm) Xvent-1B promoter fragment (−249/Gm/+52) was constructed by fusion of two PCR-generated fragments via an artificial EcoRI site using the following primers: upstream fragment, −249 (as described) and –192 (5′-GGAATTCTTGGAGGTTTCAGTTGGAG-3′); downstream fragment, –197 (5′-GGAATTCAAGGTGAAATCACTAACCTG-3′) and +52 (as described). The PCR products were digested withBamHI/EcoRI (−249/−192) or withEcoRI/HindIII (−197/+52) and ligated at theirEcoRI restriction sites. A mutation of the putative LEF/TCF binding site was introduced by fusing the –55/+52 promoter construct with a PCR fragment generated by using the –249 upstream primer (see above) and a mutated reverse primer starting at –52 (5′-CGGGATCCATATAAGCGGGAGAACCAGAA-3′; mutated positions are underlined). Microinjections were performed with in vitro fertilized Xenopusembryos, dejellied in 2% cysteine hydrochloride in 0.1 × MBSH (10 mm HEPES pH 7.4, 88 mm NaCl, 1 mm KCl, 2.4 mm NaHCO3, 0.82 mm MgSO4, 0.41 mmCaCl2, 0.66 mm KNO3) and staged according to Nieuwkoop and Faber (24Nieuwkoop P.D. Faber J. Normal Table of Xenopus laevis (Daudin). North Holland Publishing Co., Amsterdam1967Google Scholar). Before injection, embryos were placed into 1 × MBSH containing 4% Ficoll. Deletion mutants were injected dorsally or ventrally at the four-cell stage (20 pg/blastomere). In vitro transcribed capped mRNAs (mMessage-mMachine™ SP6 Kit, Ambion) were purified over RNeasy columns (Qiagen) for microinjection. Linearization and transcription of DNA were performed as indicated: pSP64T3-BMP-4 (Xenopus BMP-4,BamHI, SP6); pSP64T3-Xvent-2 (Xvent-2, EcoRI, SP6); pSP64-GATA-2 (NotI, SP6). RNA was injected at the following concentrations: 500 pg/blastomere BMP-4, 200–400 pg/blastomere Xvent-2, 50 pg/blastomere GATA-2. As an internal control, the pRL-CMV renilla reporter plasmid (Promega) was coinjected to allow for normalization of firefly luciferase values. Injected embryos were cultured until stage 11 and snap-frozen in liquid nitrogen. Luciferase assays were performed according to the manufacturer's protocol, except that 10 μl of passive lysis buffer was used per embryo (Dual Luciferase Assay System, Promega). Luciferase activities of firefly and renilla were determined separately using 20 μl of supernatant (centrifuged for 10 min at 4 °C). Fusion proteins were expressed inEscherichia coli BL21(DE3)Plus (Stratagene) and purified as described recently by Henningfeld et al. (19Henningfeld K.A. Friedle H. Rastegar S. Knöchel W. J. Biol. Chem. 2002; 277: 2097-2103Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar).35S-Labeled proteins were prepared using the TNT-coupled transcription-translation system (Promega). Protein extracts containing Myc-tagged Xvent-2 (MT-Xvent-2) were essentially prepared as reported previously (19Henningfeld K.A. Friedle H. Rastegar S. Knöchel W. J. Biol. Chem. 2002; 277: 2097-2103Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). To 20 μl of glutathione-Sepharose (Amersham Biosciences) in 500 μl of binding buffer (50 mmTris-HCl, pH 8.0, 50 mm KCl, 5 mmMgCl2, 1 mm dithiothreitol, 0.2% Nonidet P-40, and 10% glycerol) an equal amount (5 μg) of purified GST or GST fusion proteins and 5 μl of 35S-labeled proteins were added. After incubation at 4 °C for 2 h (rotating slowly), the reactions were washed four times with wash buffer (50 mmTris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, and 0.2% Nonidet P-40), and 30 μl of 2 × SDS loading buffer was added. The samples were boiled 5 min and analyzed on a 10% SDS-polyacrylamide gel. The gel was Coomassie stained to visualize GST proteins, dried, and subjected to PhosphorImager analysis. For pull-down assays from cell extracts, experiments were performed with 25 μg of total protein from Xenopus. Western analysis was performed with the anti-Myc antibody 9E10. Xenopus embryos were injected into both dorsal and ventral blastomeres at the four-cell stage with 500 pg of BMP-4, 400 pg of Xvent-2, or 50 pg of GATA-2 RNA. Lithium treatment was carried out at stage 5 for 15 min with 300 mm lithium chloride. At stage 7.0 embryos were treated with 25 μg/ml CHX (Sigma) until control embryos reached stage 10.5. Total RNA was isolated using RNeasy minicolumns (Qiagen; RNeasy protocol for isolation of total RNA from animal tissues). DNase digestion was performed by adding 1.5 μl of RNase-free DNase I (Roche Molecular Biochemicals) and 5 μl of 25 mm MgCl2 to 50 μl of total RNA. The reaction was incubated for 20 min at 37 °C followed by inactivation of DNase by heating for 10 min at 75 °C. cDNA synthesis was performed under the following conditions: 1 × RT reaction buffer (AmershamBiosciences), 10 ng of (dT)12–18, 10 ng of random primer, 0.2 mm dNTPs, 26.8 units of RNAguard™ RNase inhibitor (Amersham Biosciences), 10 units of Moloney murine leukemia virus reverse transcriptase (Amersham Biosciences), and 600 ng of total RNA. For PCRs the following primers and annealing conditions were used. For histone H4, the upstream primer was 5′-CGGGATAACATTCAGGGTATCACT-3′, and the downstream primer was 5′-ATCCATGGCGGTAACTGTCTTCCT-3′ with 56 °C annealing temperature. For Xvent-1B, the upstream primer was 5′-TTCCCTTCAGCATGGTTCAA-3′, and the downstream primer was 5′-GCATCTCCTTGGCATATTTGG-3′ with 56 °C annealing temperature. For GATA-2, the upstream primer was 5′-AGGAACTTTCCAGGTGCATGCAGGAG-3′, and the downstream primer was 5′-CGAGGTGCAAATTATTATGTTAC-3′ with 56 °C annealing temperature. LightCycler (Roche Molecular Biochemicals) reactions were set up according to the manufacturer (SYBR-green fast start protocol) and using the following primers: ODC (upstream primer, 5′-CAAAGCTTGTTCTACGCATAGCA-3′; downstream primer, 5′-GGTGGCACCAAATTTCACACT-3′) and Xvent-1B (upstream primer, 5′-GCTGCAGTATTTCAGTCCT-3′; downstream primer, 5′-ATCTGATTTGGTACTTTTCCCA-3′). The desired promoter fragments were excised from the pBSII KS+ (−249/−13,XbaI/DdeI; −249/−164,XbaI/EcoRI) or pGL3 vector (–164/−13,NheI/DdeI) and 3′-labeled on one strand by a fill-in reaction with [α-32P]dCTP and Klenow DNA polymerase. Binding reactions were carried out on ice for 30 min in 30 μl of binding buffer (30 mm Tris-HCl, pH 7.5, 30 mm KCl, 1 mm MgCl2, 1 mm dithiothreitol, 13.2% glycerol) containing 1 μg poly(dI-dC) and 1 ng of the gel-purified probe. After 5 min of preincubation, the protein was added and incubated for 30 min. The probes were separated on 7% native acrylamide gels in 0.5 × Tris-borate. Binding reactions for DNase I footprinting were prepared and incubated as described under "Electrophoretic Mobility Shift Assay." The concentration of MgCl2 was subsequently raised to 5 mm for DNase I footprinting, and 0.065 unit (free DNA) or 0.195 unit (DNA + protein) of DNase I was added at room temperature for 45 s. The DNase I digestion was stopped by the addition of an equal volume of sample buffer (66% deionized formamide, 20 mm EDTA, 660 mm sucrose). Sequencing reactions were performed according to the method of Maxam and Gilbert (25Maxam A.M. Gilbert W. Methods Enzymol. 1980; 65: 499-560Crossref PubMed Scopus (9013) Google Scholar). After preelectrophoresis for 2 h at 70 W, samples were analyzed by 7% denaturing PAGE at 60 W in 1 × Tris-borate-EDTA. Localization of Xvent-1B transcripts in gastrula stage embryos was demonstrated by whole mount in situ hybridization using a digoxygenin-labeled antisense RNA (10Rastegar S. Friedle H. Frommer G. Knöchel W. Mech. Dev. 1999; 81: 139-149Crossref PubMed Scopus (71) Google Scholar). Because Xvent-2 has been shown not to be sufficient to activate Xvent-1B in the presence of CHX (10Rastegar S. Friedle H. Frommer G. Knöchel W. Mech. Dev. 1999; 81: 139-149Crossref PubMed Scopus (71) Google Scholar), we have been searching for additional factors. One candidate was the zinc finger transcription factor GATA-2, which is induced by BMP signaling (22Maeno M. Mead P.E. Kelley C., Xu, R.H. Kung H.F. Suzuki A. Ueno N. Zon L.I. Blood. 1996; 88: 1965-1972Crossref PubMed Google Scholar). It has been shown that GATA-2 activates Xvent-1 and that a dominant-interfering GATA-2 (G2en) specifically suppressesXvent-1 but has no effect upon Xvent-2 expression (21Sykes T.G. Rodaway A.R.F. Walmsley M.E. Patient R.K. Development. 1998; 125: 4595-4605PubMed Google Scholar). This prompted us to analyze whether GATA-2 is a direct BMP target gene and to investigate the effects of GATA-2 on theXvent-1 gene and the Xvent-1 promoter. First, RT-PCR analysis was performed to study the BMP-4 induced activation of GATA-2. BMP-4 RNA was injected into embryos at the four-cell stage, CHX was added (10Rastegar S. Friedle H. Frommer G. Knöchel W. Mech. Dev. 1999; 81: 139-149Crossref PubMed Scopus (71) Google Scholar) at stage 7.0 prior to MBT, and the embryos were cultured until control embryos had reached midgastrula (stage 10.5). Fig. 1 A shows that BMP-4 leads to an increase of GATA-2 transcripts in both the absence and presence of CHX. This indicates that BMP-4 being translated at early cleavage stages is sufficient to activate zygotic transcription of GATA-2 after MBT even in the absence of protein biosynthesis. We next have analyzed whether GATA-2 is a regulator of Xvent-1B expression. In agreement with previous reports (21Sykes T.G. Rodaway A.R.F. Walmsley M.E. Patient R.K. Development. 1998; 125: 4595-4605PubMed Google Scholar) we find that GATA 2 leads to a distinct up-regulation ofXvent-1B; however, no transcripts are detected when injected embryos were treated with CHX (Fig. 1 B). Dorsal activation of Xvent-1B in GATA-2 or Xvent-2 RNA-injected embryos was also demonstrated by whole mount in situ hybridization (Fig.1, C–E). Although both factors led to an ectopic expression of Xvent-1B RNA within the dorsal marginal zone, only Xvent-2 renders expression within the most dorsal located region, the Spemann organizer. Taken together, our results suggest that GATA-2is a direct target of BMP-4 signaling and that GATA-2 activatesXvent-1B, but that additional factors are required because activation occurs only in the presence of protein biosynthesis. To investigate the involvement of GATA-2 in the activation of theXvent-1B promoter and to find out whether it acts downstream of Xvent-2 or in a parallel, cooperative mode, we have used the previously described −249/+52 Xvent-1B promoter fragment fused to a luciferase reporter (10Rastegar S. Friedle H. Frommer G. Knöchel W. Mech. Dev. 1999; 81: 139-149Crossref PubMed Scopus (71) Google Scholar) and performed coinjections of GATA-2 with the dominant-negative Xvent-2 P(40) RNA (26Onichtchouk D. Glinka A. Niehrs C. Development. 1998; 125: 1447-1456Crossref PubMed Google Scholar) or with truncated BMP type I receptor (tBR) RNA (27Graff J.M. Thies R.S. Song J.J. Celeste A.J. Melton D.A. Cell. 1994; 79: 169-179Abstract Full Text PDF PubMed Scopus (430) Google Scholar), respectively. As shown in Fig.2, GATA-2 can rescue the suppression caused by Xvent-2 P(40). This would still be in line with the downstream as well as with the parallel mechanism for the activation ofXvent-1B. However, coinjections of GATA-2 RNA with tBR RNA do not lead to an increase of luciferase activity compared with the lowered activity caused by injections of tBR. Because tBR injection suppresses Xvent-2 transcription, these results suggest that GATA-2 action needs endogenous Xvent-2 protein. They further argue for the cooperative model because blocking the BMP signaling cascade at the level of the BMP receptor leads to a loss of Xvent-2 protein in the affected cells and therefore prevents GATA-2 from up-regulating theXvent-1B promoter. To find out whether Xvent-2 physically interacts with GATA-2, we prepared a GST-Xvent-2 fusion construct and performed pull-down assays with radiolabeled GATA-2 protein. Fig. 3 A shows that GATA-2 can interact with the GST-Xvent-2 fusion protein, whereas GST alone is not able to bind to GATA-2 under these conditions. To confirm this result and to investigate whether in vivotranslated Xvent-2 binds to GATA-2, a GST-GATA-2 fusion protein was constructed and incubated with Xenopus extracts containing Myc-tagged Xvent-2 protein. As shown by immunoblotting in Fig.3 B, binding of Xvent-2 and GATA-2 was also detected under these circumstances. Additional experiments revealed that GST-GATA-2 was also able to bind to radiolabeled Xvent-2 P(40) (see Fig.3 C). This is an important finding because GATA-2 RNA injection resulted in a rescue of luciferase activity suppressed by injection of this dominant-negative Xvent-2 RNA. To gain more information about the interaction of GATA-2 and Xvent-2, we performed pull-down experiments with different Xvent-2 mutants lacking either the N-terminal (Xvent-2ΔN) or the C-terminal domains (Xvent-2ΔC) (Fig. 3 C). Whereas Xvent-2ΔN, which is missing almost half of the full-length protein, binds to GATA-2, Xvent-2ΔC does not, suggesting that interaction of GATA-2 with Xvent-2 requires the C-terminal part of Xvent-2 protein. Thus, it is concluded that GATA-2 binds to the C-terminal domain of Xvent-2 and that this interaction is not disrupted by the P(40) mutation within the homeodomain. The cooperative action of GATA-2 and Xvent-2 suggests that both transcription factors should bind to distinct elements in theXvent-1B promoter. We performed gel shift assays with different Xvent-1B promoter fragments together with bacterially expressed full-length GATA-2 and Xvent-2 proteins. As shown schematically in Fig. 4 A, Xvent-2 and GATA-2 proteins bind to −249/−13 and –249/−164Xvent-1B promoter fragments, respectively. When the 3′-part from positions –164 to −13 was used, only Xvent-2 protein leads to a shift. Using the Xvent-2 P(40) protein, no binding could be detected to any of these promoter fragments. These results suggest that the dominant-negative effect of this mutant is caused by a loss of DNA binding activity. To gain more detailed information about the binding sequences of GATA-2 and Xvent-2, we performed DNase I protection assays using theXvent-1B promoter fragment from position –249 to −13 (Fig.4, B and C). Fig. 4 B shows that GATA-2 protected only one region between positions –202 and –175 containing a canonical GATA-2 binding site 5′-TGATA-3′ (28Ko L.J. Engel J.D. Mol. Cell. Biol. 1993; 13: 4011-4022Crossref PubMed Scopus (508) Google Scholar). In contrast, Xvent-2 protects two regions (Fig. 4 C), a proximal region within positions –111 and −74 and a more distal region between –229 and –175. These findings confirm the results obtained from the gel shift experiments. The distal region, which overlaps with the GATA-2-protected region, was resolved further into three distinct elements by using a truncated −249/−164 promoter fragment (Fig.4 D). Inspection of all the Xvent-2 binding sites revealed the accumulation of nine motifs (see boxes in Fig. 4) that can be aligned to the consensus sequence 5′-CC/TAAT-3′. This sequence is in good agreement with results obtained from random oligonucleotide selection (29Trindade M. Tada M. Smith J.C. Dev. Biol. 1999; 216: 442-456Crossref PubMed Scopus (62) Google Scholar) as well as the recently reported 5′-CTAAT-3′ motif as an Xvent-2 target site on the BMP-4 and Xvent-2genes (6Schuler-Metz A. Knöchel S. Kaufmann E. Knöchel W. J. Biol. Chem. 2000; 275: 34365-34374Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 19Henningfeld K.A. Friedle H. Rastegar S. Knöchel W. J. Biol. Chem. 2002; 277: 2097-2103Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). DNA-protein binding assays have revealed that theXvent-1B promoter contains binding elements for Xvent-2 and GATA-2. To analyze the biological relevance of these elements, we have coinjected different promoter deletions with RNAs for these factors (Fig. 5). The longest mutant used for these experiments (–249/+52 Xvent-1B) contains the GATA-2 as well as the proximal and the distal Xvent-2-binding elements. Coinjections of this promoter fragment with GATA-2 or Xvent-2 RNA result in an increase of luciferase activity. A 5′-deletion starting at position –164 and missing the GATA-2 and distal Xvent-2 binding sites cannot be activated by GATA-2 but is still activated by Xvent-2 RNA injection. This activation is most likely the result of the proximal binding site because a −55/+52 promoter fragment was not activated. Vice versa, a promoter fragment with an internal deletion (−249/Δ/+52) missing the proximal Xvent-2 binding site is also activated by Xvent-2 RNA. Surprisingly, GATA-2 fails to activate this
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