Reversal of Xenopus Oct25 Function by Disruption of the POU Domain Structure

Ying Cao,Franz Oswald,Stephan A Wacker,Karin Bundschu,Walter Knöchel

doi:10.1074/jbc.m109.064386

Ying Cao, Franz Oswald + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m109.064386

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Abstract

Xenopus Oct25 is a POU family subclass V (POU-V) transcription factor with a distinct domain structure. To investigate the contribution of different domains to the function of Oct25, we have performed gain of function analyses. Deletions of the N- or C-terminal regions and of the Hox domain (except its nuclear localization signal) result in mutants being indistinguishable from the wild type protein in the suppression of genes promoting germ layer formation. Deletion of the complete POU domain generates a mutant that has no effect on embryogenesis. However, disruption of the alpha-helical structures in the POU domain, even by a single amino acid mutation, causes reversal of protein function. Overexpression of such mutants leads to dorsalization of embryos and formation of secondary axial structures. The underlying mechanism is an enhanced transcription of genes coding for antagonists of the ligands for ventralizing bone morphogenetic protein and Wnt pathways. Corresponding deletion mutants of Xenopus Oct60, Oct91, or mouse Oct4 also exhibit such a dominant-negative effect. Therefore, our results reveal that the integrity of the POU domain is crucial for the function of POU-V transcription factors in the regulation of genes that promote germ layer formation.

Highlights

Nuclear Localization Signal Is Essential for the Activity of Oct25—Oct25 and other Oct4 homologous proteins reveal a similar domain structure, including the diverse N- and C-terminal regions and the well conserved POU-specific domain (POU)-specific and Hox-specific domains
We investigated the contribution of each region to the function of Oct25 by generating deletion constructs that were overexpressed in Xenopus embryos. quantitative RT-PCR (qRT-PCR) showed that deletion mutants either lacking the C-terminal region (Oct25⌬C), N-terminal region (Oct25⌬N), or lacking both the C- and N-terminal regions (Oct25PH) inhibited mesodermal and endodermal marker gene expression (Fig. 1A), which is reminiscent of the wild type protein effect (14 –16, 26)
Quite a few studies have been carried out on the functions of the mammalian Oct4 protein domains, we discovered a previously undefined phenomenon, i.e. when the POU-specific domain structure is disturbed, either by deletion of a few amino acids or by mutation of a single amino acid, the resulting mutated proteins exhibit dorsalizing activity in Xenopus embryos

Summary

Introduction

Experiments with whole embryos, animal cap explants, and luciferase reporters support the notion that the Oct25⌬POU[273–301] mutant stimulates transcription of genes responsible for mesendoderm formation and dorsalization of embryos. When Oct25⌬POU[273–301] RNA was co-injected with VegTMO, we did not observe transcriptional stimulation of Gsc, Chd, Xnr1, Xnr2, and Sia. The results suggest that stimulation of these genes by Oct25⌬POU[273–301] is dependent on VegT activity.

Results

Conclusion