Genes and Development Group, Centres for Integrative Physiology and Neuroscience Research, School of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, EH8 9XD, UK.
The development of the neural crest is orchestrated by a complex interplay between intercellular signalling molecules and transcription factors. Here, we demonstrate a direct interaction between two such factors, the paired-type transcription factor Pax3 and the secretory glycoprotein Wnt1. We found that the Wnt1 promoter can be regulated by Pax3 in a dose-dependent manner. Sequence analysis predicted a conserved binding site for Pax3 within the Wnt1 promoter region. Deletion or mutation of this sequence abolished the promoter response to Pax3. Using chromatin immunoprecipitation (ChIP) assays, we demonstrated that Pax3 interacts with the Wnt1 promoter in vivo. These data indicate that Pax3 directly regulates the expression of Wnt1 in the developing embryo.
"Some of the Pax proteins, including Pax3, contain a paired-type homeodomain that also binds to DNA, and a conserved octapeptide . It has traditionally been accepted that Pax3 regulates developmental processes by operating as a transcriptional regulator because: (i) Pax3 contains sequence-specific DNA-binding domains that are capable of directing trans-activation , , ; (ii) the protein product of the mutant Splotch Pax3 allele is trans-activation defective ; and (iii) several genes have been identified that are directly or indirectly regulated by Pax3 , , , , , , , , , , , . However, exactly how Pax3 regulates formation of the neural tube and neural crest-dependent structures has not yet been determined. "
[Show abstract][Hide abstract] ABSTRACT: Pax3 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.
We 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.
Pax3 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.
PLoS ONE 12/2011; 6(12):e29379. DOI:10.1371/journal.pone.0029379 · 3.23 Impact Factor
"Use of the classic Pax3 Sp and Pax3 Sp-d alleles confirms that we have uncovered a true genetic interaction rather than differences between Wnt1-Cre and Pax3 Cre expression domains, despite the fact that there are known differences in Cre expression in these two mouse lines (Engleka et al., 2005; Jiang et al., 2000). Pax3 is expressed before Wnt1 in the NC (Fenby et al., 2008; Osorio et al., 2009), and Pax3 binds directly to the Wnt1 promoter to activate Wnt1 transcription (Fenby et al., 2008), suggesting it is upstream. Wnt1-Cre is more commonly used for NC-specific deletion, but will act on cells within the developing roof plate and midbrain and does not label NCCs from the posterior-most levels of the NT (Jiang et al., 2000). "
[Show abstract][Hide abstract] ABSTRACT: The transcription factors Foxd3 and Pax3 are important early regulators of neural crest (NC) progenitor cell properties. Homozygous mutations of Pax3 or a homozygous NC-specific deletion of Foxd3 cause marked defects in most NC derivatives, but neither loss of both Foxd3 alleles nor loss of one Pax3 allele alone greatly affects overall development of cardiac NC derivatives. In contrast, compound mutant embryos homozygous for a NC-specific Foxd3 mutation and heterozygous for Pax3 have fully penetrant persistent truncus arteriosus, severe thymus hypoplasia, and midgestation lethality. Foxd3; Pax3 compound mutant embryos have increased cell death in the neural folds and a drastic early reduction of NC cells, with an almost complete absence of NC caudal to the first pharyngeal arch. The genetic interaction between these genes implicates gene dosage-sensitive roles for Foxd3 and Pax3 in cardiac NC progenitors. Foxd3 and Pax3 act together to affect survival and maintenance of cardiac NC progenitors, and loss of these progenitors catastrophically affects key aspects of later cardiovascular development.
"In line with this function, it regulates the expression of the muscle-specific TF, myoD, and myf-5 [Bajard et al., 2006; Bendall et al., 1999]. PAX3 also controls key regulators of NC development, such as c-RET, TGF-b2, and WNT1 [Fenby et al., 2008; Lang and Epstein, 2003; Mayanil et al., 2006]. The dependence on PAX3 function for proper NC development is highlighted by the phenotype induced by PAX3 mutations in human and mice. "
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