Destabilization of ΔNp63α by Nedd4-mediated ubiquitination and Ubc9-mediated sumoylation, and its implications on dorsoventral patterning of the zebrafish embryo

We have recently identified the p53-related Delta Np63 gene as a transcriptional target of Bmp signaling that encodes a transcriptional repressor blocking neural development in the zebrafish ectoderm. However, in contrast to Bmps, the neural-repressing effect of forced Delta Np63 alpha expression is restricted to the presumptive forebrain, while posterior regions of the brain are not affected. Here, we show that this is due to instability of Delta Np63 alpha protein on the dorsal side of the embryo. In a yeast-two-hybrid screen, we isolated two Delta Np63 alpha-modifying enzymes, the SUMO-conjugating enzyme Ubc9 and the ubiquitin ligase Nedd4. The proteins bind to distinct sites in the C-terminal region of Delta Np63 alpha, which are absent in the shorter and more stable Delta Np63 gamma isoform. Similarly, mutant versions of Delta Np63 alpha unable to bind Nedd4 or Ubc9 are stabilized. Delta Np63 alpha is sumoylated and ubiquitinated both in HEK293 cells and in zebrafish embryos, and Nedd4 promotes ubiquitination and instability of Delta Np63 alpha protein, with lysine residue 637 serving as a potential alternative sumoylation and ubiquitination site that is crucial for Delta Np63 alpha destabilization. In zebrafish, ubc9.1 and nedd4 show restricted expression on the dorsal side of the embryo, where Delta Np63 alpha instability can be overcome upon blockage of endogenous Nedd4 activity, or upon injection of mutant versions of Delta Np63 alpha that are unable to bind Nedd4 or Ubc9. This results in a more widespread neural repression, affecting the entire Bmp-sensitive neuroectoderm. In sum, our data indicate that Delta Np63 alpha is ubiquitinated in a Nedd4- and sumoylated in a Ubc9-dependent fashion, and that these modifications can regulate Delta Np63 alpha stability in the zebrafish ectoderm.