Molecular basis of splotch and Waardenburg PAX-3 mutations

Max-Planck-Institute for Biophysical Chemistry, Department of Molecular Cell Biology, Göttingen, Federal Republic of Germany.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 05/1994; 91(9):3685-9. DOI: 10.1073/pnas.91.9.3685
Source: PubMed

ABSTRACT Pax genes control certain aspects of development, as mutations result in (semi)dominant defects apparent during embryogenesis. Pax-3 has been associated with the mouse mutant splotch (Sp) and the human Waardenburg syndrome type 1 (WS1). We have examined the molecular basis of splotch and WS1 by studying the effect of mutations on DNA binding, using a defined target sequence. Pax-3 contains two different types of functional DNA-binding domains, a paired domain and a homeodomain. Mutational analysis of Pax-3 reveals different modes of DNA binding depending on the presence of these domains. A segment of Pax-3 located between the two DNA-binding domains, including a conserved octapeptide, participates in protein homodimerization. Pax-3 mutations found in splotch alleles and WS1 individuals change DNA binding and, in the case of a protein product of the Sp allele, dimerization. These findings were taken as a basis to define the molecular nature of the mutants.

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    • "As diagrammed in Figure 2 E, the structural domains of Pax3 that have been previously identified include an N-terminal transcription inhibitory domain (ID), the paired domain (PD), a conserved octapeptide (OCT), the paired-type homeodomain (HD), and a C-terminal trans-activation domain (TAD). The PD and HD are each independent DNA-binding domains [3], [9], [10] that bind to DNA with higher affinity together than when only one of the domains is bound together [8], the OCT is necessary for homodimerization [11], and the ID and TAD possess transcription inhibition and transcription activation activities, respectively [33]. We constructed plasmids to express glutathione-S-transferase- (GST) and FLAG-tagged proteins fused with full-length w.t. "
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    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
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    • "To distinguish between these hypotheses, mice were bred to introduce the Pax3 Sp or Pax3 Sp-d mutant alleles into the Wnt1-Cre; Foxd3 flox/− background (Supplementary Fig. 1). Pax3 Sp is a null allele encoding a truncated Pax3 protein (Chalepakis et al., 1994). "
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    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.
    genesis 01/2011; 49(1):10-23. DOI:10.1002/dvg.20686 · 2.02 Impact Factor
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    • "A few mutations have been tested for their functional consequences. The tests mostly included DNA-binding activity and transactivation capabilities [Chalepakis et al., 1994; Corry and Underhill, 2005; Fortin et al., 1997; Watanabe et al., 1998]. "
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    ABSTRACT: Waardenburg syndrome (WS) is characterized by the association of pigmentation abnormalities, including depigmented patches of the skin and hair, vivid blue eyes or heterochromia irides, and sensorineural hearing loss. However, other features such as dystopia canthorum, musculoskeletal abnormalities of the limbs, Hirschsprung disease, or neurological defects are found in subsets of patients and used for the clinical classification of WS. Six genes are involved in this syndrome: PAX3 (encoding the paired box 3 transcription factor), MITF (microphthalmia-associated transcription factor), EDN3 (endothelin 3), EDNRB (endothelin receptor type B), SOX10 (encoding the Sry bOX10 transcription factor), and SNAI2 (snail homolog 2), with different frequencies. In this review we provide an update on all WS genes and set up mutation databases, summarize molecular and functional data available for each of them, and discuss the applications in diagnostics and genetic counseling. Hum Mutat 31, 1–16, 2010. © 2010 Wiley-Liss, Inc.
    Human Mutation 04/2010; 31(4):391 - 406. DOI:10.1002/humu.21211 · 5.14 Impact Factor
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