Abstract
BackgroundPax3 is a developmental transcription factor that is required for neural tube and neural crest development. We previously showed that inactivating the p53 tumor suppressor protein prevents neural tube and cardiac neural crest defects in Pax3-mutant mouse embryos. This demonstrates that Pax3 regulates these processes by blocking p53 function. Here we investigated the mechanism by which Pax3 blocks p53 function.Methodology/Principal FindingsWe employed murine embryonic stem cell (ESC)-derived neuronal precursors as a cell culture model of embryonic neuroepithelium or neural crest. Pax3 reduced p53 protein stability, but had no effect on p53 mRNA levels or the rate of p53 synthesis. Full length Pax3 as well as fragments that contained either the DNA-binding paired box or the homeodomain, expressed as GST or FLAG fusion proteins, physically associated with p53 and Mdm2 both in vitro and in vivo. In contrast, Splotch Pax3, which causes neural tube and neural crest defects in homozygous embryos, bound weakly, or not at all, to p53 or Mdm2. The paired domain and homeodomain each stimulated Mdm2-mediated ubiquitination of p53 and p53 degradation in the absence of the Pax3 transcription regulatory domains, whereas Splotch Pax3 did not stimulate p53 ubiquitination or degradation.Conclusions/SignificancePax3 inactivates p53 function by stimulating its ubiquitination and degradation. This process utilizes the Pax3 paired domain and homeodomain but is independent of DNA-binding and transcription regulation. Because inactivating p53 is the only required Pax3 function during neural tube closure and cardiac neural crest development, and inactivating p53 does not require Pax3-dependent transcription regulation, this indicates that Pax3 is not required to function as a transcription factor during neural tube closure and cardiac neural crest development. These findings further suggest novel explanations for PAX3 functions in human diseases, such as in neural crest-derived cancers and Waardenburg syndrome types 1 and 3.
Highlights
Understanding how regulators of embryonic development function on a molecular level is a major objective of developmental biology
It has been believed that the activity of Pax3 as a DNA-binding transcription factor is responsible for these functions, and that the phenotype of Pax3Sp/Sp embryos is due to loss of transcription factor activity of Splotch Pax3 [11,35]
Our previous studies demonstrated that neural tube closure, cardiac neural crest migration, and cardiac outflow tract septation proceeds normally in Pax3Sp/Sp and Pax3-null embryos as long as p53 is inactivated [25,27], demonstrating that Pax3 is required for these processes only to inactivate p53
Summary
Understanding how regulators of embryonic development function on a molecular level is a major objective of developmental biology. We showed that inactivation of p53 through germ-line mutation or chemical inhibition prevented the NTD, exencephaly and spina bifida, and COTD that are characteristic of Pax3Sp/Sp embryos, as well as associated apoptosis, in embryos expressing nonfunctional Pax alleles [25,27]. This indicates that Pax is not required in neuroepithelium and neural crest to regulate genes that direct morphogenesis or migration, but that it is required to block p53-dependent processes that lead to apoptosis. If expression of Pax causes a reduction in steadystate levels of p53 protein, differentiating ESC would be a valid cell culture model to study the mechanism by which Pax blocks p53 function in embryonic neuroepithelium and neural crest
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