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We have previously isolated Xretpos, a novel family of long terminal repeat (LTR)-retrotransposons in Xenopus laevis, whose transcript is restricted to ventro-posterior-specific regions and induced by bone morphogenetic protein-4 (BMP-4) signaling. To explore the molecular mechanism of the transcriptional regulation, we identified and characterized Xretpos promoter regions consisting of LTRs and a 5'-untranslated region. We demonstrated that this promoter region contains all the necessary regulatory elements for the spatial and temporal expression of XRETPOS: Sequence analysis of the Xretpos promoter revealed multiple Smad-binding elements and Olf-1/EBF-associated zinc finger (OAZ) binding sites similar to BMP-4 response element, which were identified and proved to be required for BMP-4 induction in the Xvent2 promoter. We further demonstrated that Smads and OAZ proteins bind to their response elements in the promoter and these bindings are essential for the BMP-4-induced activation of the Xretpos promoter. Furthermore, we showed that the endogenous expression of Xretpos protein indeed occurred and was temporally regulated and BMP-4-inducible during the early Xenopus development. Finally, overexpression and partial loss-of-function study revealed that Xretpos has a posterio-ventralizing activity. Together, our results place Xretpos downstream of BMP-4 and provide evidence for the conserved mechanism of transcriptional regulation of the BMP-4 target genes.
Figure 1. Stage†and tissueâ€specific expression of reporter constructs containing the LTR and 5′â€UTR of Xretpos promoter. The LTR is divided into three regions, U3, R and U5. (A) Activities of luciferase reporter constructs were measured from the extracts of embryos injected with pGL2â€Xretpos (LTRâ€UTR) or pGL2â€enhancer into all four blastomeres of the fourâ€cell stage embryos. One representative experiment is shown for this figure. (B) Xretpos reporter gene expression (left) was compared with endogenous Xretpos RNA (center). pgscâ€GFP expression is shown as control (right). pXretposâ€GFP was injected equatorially into all four blastomeres of fourâ€cell stage embryos, and the expression of GFP transcripts was visualized by wholeâ€mount in situ hybridization. The dorsal blastopore lip is indicated by arrowheads.
Figure 9. Phenotypic effects of overexpression (A) and partial loss (B and C) of Xretpos function. (A) Overexpression of Xretpos RNA resulted in reduced anteriorhead structures. Fourâ€cell stage embryos were injected into two dorsal animal or marginal regions at the fourâ€cell stage embryos with 4 ng of Xretpos RNA and allowed to develop until stage 43. Control embryos injected with 4 ng of PPL RNA produced normal tadpoles. (B) Rescue of axial structures in UVâ€irradiated embryos by antisense Xretpos RNA. Partial twinned axis structures were seen in UVâ€irradiated embryos injected into two nonâ€adjacent blastomeres at the fourâ€cell stage with antisense Xretpos RNA (1 ng/blastomere), but not in uninjected UVâ€irradiated controls. (C) Phenotypic effects of antisense Xretpos RNA injection in wildâ€type embryos. Anteroâ€dorsalized structures were observed in embryos which were injected diagonally at the fourâ€cell stage with antisense Xretpos RNA (1 ng/blastomere). Uninjected control embryos developed normally. (D) Reduction of endogenous Xretpos protein by antisense Xretpos RNA injection. The position of Xretpos protein is indicated by an arrow and Ponceau staining was performed as a loading control.
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