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The Xvent-2B promoter is regulated by a BMP-2/4-induced transcription complex comprising Smad signal transducers and specific transcription factors. Using a yeast one-hybrid screen we have found that Oct-25, a Xenopus POU domain protein related to mammalian Oct-3/4, binds as an additional factor to the Xvent-2B promoter. This interaction was further confirmed by both in vitro and in vivo analyses. The Oct-25 gene is mainly transcribed during blastula and gastrula stages in the newly forming ectodermal and mesodermal germ layers. Luciferase reporter gene assay demonstrated that Oct-25 stimulates transcription of the Xvent-2B gene. This stimulation depends on the Oct-25 binding site and the bone morphogenetic protein-responsive element. Furthermore, Oct-25 interacts in vitro with components of the Xvent-2B transcription complex, like Smad1/4 and Xvent-2. Overexpression of Oct-25 results in anterior/posterior truncations and lack of differentiation for neuroectoderm- and mesoderm-derived tissues including blood cells. This effect is consistent with an evolutionarily conserved role of class V POU factors in the maintenance of an undifferentiated cell state. In Xenopus, the molecular mechanism underlying this process might be coupled to the expression of Xvent proteins.
FIG. 1.
Xvent-2B promoter. A, proteins that bind to the 5â²-flanking sequence and regulate Xvent-2B transcription (for details, see text). B, schematic drawing of yeast reporter constructs used for the one-hybrid screen. Three copies of the -267 to -214 region of the Xvent-2B gene were cloned in front of the His or LacZ gene, respectively.
FIG. 2.
Localization of Oct-25 transcripts in Xenopus laevis embryos. Whole mount in situ hybridizations were performed with embryos of various developmental stages (24). A, stage 9. B, stage 11. C, stage 12. D-F, stage 15. E, double whole mount with Krox-20/Oct-25(BM-purple/5-bromo-4-chloro-3-indoyl-phosphate (BCIP)); rhombomeres 3 and 5 are indicated. F, double whole mount with en-2/Oct-25(BM-purple/BCIP), the former staining the midbrain/hindbrain boundary. G, stage 19. H, stage 23. I, stage 27. K, stage 30. L, tip of the tail at stage 35. M, transverse section of a stage 14 embryo. no, notochord.
FIG. 3.
Overexpression of Oct-25. Oct-25 RNA was injected into all four blastomeres of 4-cell stage embryos at a total amount of 150 pg (A), 300 pg (B), or 1 ng (C). Dorsal (d) and ventral (v) views of injected embryos are shown. The inset in A shows an uninjected control embryo.
FIG. 4.
Oct-25 overexpression inhibits terminal differentiation. Embryos were injected (inj.) with 600 pg of Oct-25 RNA at the 4-cell stage and grown until uninjected (uninj.) control embryos had reached stage 19 (st. 19) or stage 30 (st. 30), respectively. Total RNA was prepared from uninjected and injected embryos and analyzed by RT-PCR for indicated transcripts. Ornithine decarboxylase (ODC) was used as internal standard, and control reaction without reverse transcriptase (RT-) was used as a control for the absence of endogenous DNA. N-CAM, neural cell adhesion molecule; XAG, Xenopus anterior gradient.
FIG. 5.
DNase I footprint of the Oct-25 binding site on the Xvent-2B promoter. The -305 to -123 upstream region of the Xvent-2B gene was cloned in sense and in antisense orientation. After 5â²-end labeling with 32P the DNA fragments were submitted to DNase I footprinting using bacterially expressed Oct-25 protein. The M lanes denote the G/A chemical sequencing reactions, and the next lane contains DNase I-digested free DNA. The next four lanes contain the reactions with increasing amounts of Oct-25 protein (triangle). The vertical bars indicate protected regions, which at the bottom are shown to correlate for the sense and antisense strands (horizontal bars). Note that the protected region contains two putative POU factor binding motifs, each of them containing a binding site for the POU-specific domain (POUS) and for the homeodomain (POUH).
FIG. 6.
Mutational analysis of the Oct-25 binding site. A, the oligonucleotides containing the wild type or mutated -234 to -204 region of the Xvent-2B gene were 32P end-labeled and annealed. wt is the wild type sequence, and mu1 and mu2 are mutated at the indicated positions (nucleotides in lowercase letters). B, samples were incubated with increasing concentrations of the POU/homeodomain of Oct-25 protein (filled triangle) and submitted to EMSA. Note the failure of mu1 to bind the protein. C, binding of the POU/homeodomain of Oct-25 with wt or mu2 was competed for by increasing concentrations of unlabeled wt, mu1, or mu2 targets (open triangles). Note that binding of labeled wt or mu2 is efficiently reduced by unlabeled wt and mu2 but not by mu1.
FIG. 7.
Oct-25 stimulates Xvent-2B transcription. The Xvent-2B upstream region (-275/+44), the 5â²-deletion (-174/+44), or the -275/+44 region carrying an internal deletion (δ-200/-32) was fused to the luciferase reporter gene. 20 pg of DNA were co-injected into both blastomeres at the 2-cell stage of Xenopus embryos with Oct-25 RNA (250 pg) and/or BMP-4 RNA (300 pg) as indicated. Luciferase activity was measured at stage 12. The reporter activities determined in the absence of RNA were set as 100%. All values are averaged from at least three independent experiments.
FIG. 8.
Oct-25 is bound to the Xvent-2B promoter in vivo. myc-tagged Oct-25 (MT-Oct-25) RNA or RNA encoding the myc tag fused to a nuclear localization signal (NLS-MT) was injected into all blastomeres of 4-cell stage embryos. Embryos were collected for ChIP assay when control sibling embryos had reached stage 19. The presence of the Xvent-2B promoter was detected by PCR from the DNA samples after anti-myc antibody precipitation (anti-myc) and without antibody precipitation (no Ab) and from the cross-linked chromatin supernatant before immunoprecipitation (input), respectively. Another ChIP control was performed in parallel under identical conditions with uninjected embryos.
FIG. 9.
Interaction of Oct-25 with components of the BMP signaling pathway. Oct-25, Smad1, Smad4, Xvent-2, N terminus-truncated (δN) and C terminus-truncated (δC) versions of Xvent-2 and Oct-25, and an Oct-25 mutant lacking the POU and the homeodomain were labeled with [35S]methionine by in vitro transcription/translation and incubated with GST or GST fusion proteins as indicated. The pull-down reactions were washed and analyzed by 10% SDS-PAGE.
