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During vertebrate somitogenesis, various transcriptional factors function coordinately to determine the position of the somite boundary. Previously, we reported on the signaling crosstalk that occurs between two major transcription factors involved in somitogenesis, Tbx6 and mespb/mesp2. These factors synergistically activated the expression of a downstream gene, bowline/Ripply2, which is essential for precise formation of the somite boundary. However, the molecular mechanism underlying this synergistic effect remains unclear. In this report, we found that the Tbx6 and mespb proteins interacted physically with each other. Pulldown assays with various deletion mutants of these proteins identified the essential domains for this physical interaction. Finally, we found that interference with the physical interaction by a dominant-negative form of mespb, mespbDeltaDBD, abrogated the expression of the bowline gene during Xenopus somitogenesis. These results indicate that the appropriate expression of bowline/Ripply2 is regulated by a direct interaction between the Tbx6 and mespb proteins during Xenopus somitogenesis.
Fig. 1.
The Tbx6 and mespb–E47 proteins bind to the bowline cis-regulatory region in vivo. (A) Schematic diagram of Xenopus bowline genomic structure. Arrows indicate the primers used for the ChIP assays; the UP primers are for the bowlineproximal cis-regulatory region. The ORF1 and ORF2 primers are for the bowline fourth exon. The bowlineproximal cis-regulatory region and the bowline exons are indicated as white and gray boxes, respectively. (B) ChIP assays of the Tbx6 and mespb proteins. The bowlineproximal cis-regulatory region is specifically immunoprecipitated in the presence of myc-Tbx6 or myc-mespb and E47. (C) Schematic diagram of the structures of mespb and the mutant proteins. bHLH indicates the bHLH domain of the mespb protein. (D) Luciferase assays of the bowline promoter with the mespb mutant. COS7 cells were transfected with the bowlineproximal reporter −226Luc (100 ng) and a combination of the indicated expression vectors, Tbx6, mespb, and mespbδAD (200 ng each). Error bars represent the SEM of three independent experiments.
Fig. 2.
The Tbx6 and mespb proteins interact physically with each other both in vitro and in vivo. (A) Interaction of the Tbx6 and mespb proteins in COS7 cells. COS7 cells were transfected with 4 μg of myc-Tbx6 and HA-mespb, and the cell lysates were subjected to immunoprecipitation (IP) with the anti-myc antibody. Each lysate (1%) was loaded as the input. (B) Interaction between the Tbx6 and mespb proteins in Xenopus embryos. Protein extracts of Xenopus embryos co-injected with myc-Tbx6 mRNA and HA-mespb mRNA (500 pg each) were subjected to immunoprecipitation with the anti-myc antibody. (C–F) Subcellular localization of Tbx6-RFP and mespb-GFP in COS7 cells. COS7 cells were co-transfected with the indicated plasmids and analyzed for GFP or RFP expression by fluorescence microscopy. Nuclei are counterstained with DAPI.
Fig. 3.
The physical interaction between the Tbx6 and mespb proteins is mediated by the T-box and the bHLH domains, respectively. (A) GST-pulldown of in vitro-translated myc-Tbx6 and its deletion mutant proteins by immobilized GST-mespb protein. The asterisks indicate the specific bands. (B) GST-pulldown of in vitro-translated myc-mespb and its deletion mutant proteins by immobilized GST-T-box+α. The asterisks indicate the specific bands. (C) Structures of the myc-Tbx6 protein and deletion mutants thereof. T-box indicates the T-box domain of the Tbx6 protein. Numbers indicate the corresponding amino acids of Tbx6. (D) Structures of myc-mespb protein and deletion mutants thereof. bHLH indicates the bHLH domain of the mespb protein. Numbers indicate the corresponding amino acids of mespb.
Fig. 4.
A dominant-negative form of mespb, mespbδDBD, represses bowline expression both in vitro and in vivo. (A,B) Luciferase assays of the bowline promoter with the mespb mutant. (A) COS7 cells were transfected with the −226Luc construct (100 ng) and the combination of indicated expression vectors, Tbx6, mespb, and mespbδDBD (200 ng each). (B) COS7 cells were transfected with the −226Luc construct (150 ng) and the combination of indicated expression vectors. The numbers indicate the amounts (ng) of DNA used for transfection. (C) Whole-mount in situ hybridization for the bowline gene. Xenopus embryos were unilaterally injected with EGFP mRNA or mespbδDBD mRNA (500 pg each). The arrowhead indicates the reduced expression of the bowline gene. The embryos were injected on the left side, and dorsal views are shown with anterior towards the top. The numbers of embryos showing reduced bowline expression are shown at the bottom-right corner of each image.
