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Mutation of PAX3

The molecular basis of the mouse mutation splotch (Sp), which is associated with spina bifida and exencephaly, was analyzed at three of its alleles, Sp, Sp2H, and Spr. The paired box gene Pax-3 maps within the Inha to Akp3 interval, near or at the Sp locus on chromosome 1, and Pax-3 appears to be deleted in heterozygous Spr/+ mice. Analysis of genomic DNA and cDNA clones constructed from Sp2H/Sp2H embryos identifies a deletion of 32 nucleotides in the Pax-3 mRNA transcript and gene. This deletion maps within the paired homeodomain of PAX-3 and is predicted to create a truncated protein as a result of a newly created termination codon at the deletion breakpoint. This study provides evidence for a causal link between deletion of the paired homeodomain of Pax-3 and the Sp2H mutation, and infers that Pax-3 plays a key role in normal neural development (Epstein, 1991).

The splotch (Sp) mouse mutant displays defects in neural tube closure in the form of exencephaly and spina bifida. A complex mutation in the Pax-3 gene including an A-->T transversion at the invariant 3' AG splice acceptor of intron 3 has been identified in the Sp/Sp mutant. This genomic mutation abrogates the normal splicing of intron 3, resulting in the generation of four aberrantly spliced mRNA transcripts. Two of these Pax-3 transcripts make use of cryptic 3' splice sites within the downstream exon, generating small deletions that disrupt the reading frame of the transcripts. A third aberrant splicing event results in the deletion of exon 4, while a fourth retains intron 3. These aberrantly spliced mRNA transcripts are not expected to result in functional Pax-3 proteins and are thus responsible for the phenotype observed in the Sp mouse mutant. (Epstein, 1993)

Waardenburg syndrome (WS) types I, II, and III are related autosomal dominant disorders characterized by sensorineural hearing loss, dystopia canthorum, pigmentary disturbances, and other developmental defects. Disease causing PAX3 mutations have been identified in a few families from each of the three disease subtypes, WS-I, WS-II, and WS-III. In others, although the mutations have not been pinpointed, linkage with the PAX3 locus on chromosome 2q35 has been demonstrated. Two mutations in the human PAX3 gene cause WS type I. One mutation is a deletion/frameshift in the paired-domain of PAX3 and results in a protein without functional DNA binding domains. The second mutation is a single-base substitution and results in a premature termination codon in the homeodomain of PAX3. This is the first demonstration of a mutation in the homeodomain DNA binding motif in this protein resulting in WS and one of the few examples of a mutation in a homeodomain of any protein that results in human disease (Baldwin, 1994).

Mutations in PAX3 have been described in human patients with Type 1 Waardenburg syndrome (hearing loss and pigmentary abnormalities). Splotch mice have mutations in the homologous mouse Pax-3 gene. A series of patients is described who have previously unidentified PAX3 mutations. These include a chromosomal deletion, a splice-site mutation and an amino acid substitution that closely correspond (respectively) to the molecular changes seen in the Splotch-retarded, Splotch and Splotch-delayed mouse mutants. These mutations confirm that Waardenburg syndrome is produced by gene dosage effects and show that the phenotypic differences between Splotch mice and humans with Waardenburg syndrome are caused by differences in genetic background rather than different primary effects of the mutations (Tassabehji, 1994).

Mutations in the human PAX3 gene have previously been associated with two distinct diseases: Waardenburg syndrome and alveolar rhabdomyosarcoma. The normal human PAX3 gene is encoded by 8 exons. Intron-exon boundary sequences were obtained for PAX3 exons 5, 6, 7, and 8 and together with previous work provide the complete genomic sequence organization for PAX3. Difficulties in obtaining overlapping genomic clone coverage of PAX3 were circumvented in part by RARE cleavage mapping, which shows that the entire PAX3 gene spans 100 kb of chromosome 2. Sequence analysis of the last intron of PAX3, which contains the previously mapped t(2;13)(q35;q14) translocation breakpoints of alveolar rhabdomyosarcoma, reveals the presence of a pair of inverted Alu repeats and a pair of inverted (GT)n-rich microsatellite repeats within a 5-kb region. This work establishes the complete structure of PAX3 and will permit high-resolution analyses of this locus for mutations associated with Waardenburg syndrome, alveolar rhabdomyosarcoma, and other phenotypes for which PAX3 may be a candidate locus (Macina, 1995).

CDC46/MCM5 (Drosophila homolog; Disc proliferation abnormal) encodes a protein that is highly conserved among yeast, plants, and animals. It is found in a complex that exhibits DNA replication licensing activity, which is proposed to regulate the synthesis of DNA once and only once per cell cycle. In yeast, loss of function mutations of CDC46/MCM5 decrease DNA synthesis. Very little is known about the regulation of CDC46/MCM5 in any species. In the mouse embryo, expression of cdc46 is increased in unfused portions of the neural tube when the gene encoding the transcription factor, Pax-3, is either nonfunctional or underexpressed. These results are observed both in embryos of diabetic mice, which express significantly reduced levels of Pax-3 mRNA, and in Splotch embryos, which carry loss of function Pax-3 alleles. This indicates that expression of cdc46 is negatively regulated as part of a Pax-3-dependent pathway. Since cdc46 appears to regulate DNA synthesis and cell cycle progression, it is possible that its overexpression is involved in defective embryonic development that is associated with loss of Pax-3 function (Hill, 1998).

Pax3 encodes a transcription factor expressed during mid-gestation in the region of the dorsal neural tube that gives rise to migrating neural crest populations. In the absence of Pax3, both humans and mice develop with neural crest defects. Homozygous Splotch embryos that lack Pax3 die by embryonic day 13.5 with cardiac defects that resemble those induced by neural crest ablation in chick models. This has led to the hypothesis that Pax3 is required for cardiac neural crest migration. A specific population of neural crest cells, defined by ablation studies in the chick, migrates to the rostral pole of the heart tube and mediates septation of the single great vessel emerging from the primitive heart. This results in division of the truncus arteriosus into the aorta and the pulmonary artery and is associated with rotation of the outflow tract, resulting in juxtaposition of the aorta with the left ventricle. Studies in chick embryos indicate that disruption of neural crest migration can result in cardiac outflow tract defects. Thus, ablation of premigratory cardiac neural crest cells emerging between the mid-otic placode and the caudal boundary of somite 3 results in the failure of cardiac outflow tract septation Such studies indicate that errors in this process may underlie relatively common forms of congenital heart disease in humans which are often associated with other neural crest related abnormalities and syndromes. Cardiac derivatives of Pax3-expressing precursor cells have not been previously defined, and Pax3-expressing cells within the heart have not been well demonstrated. Hence, the precise role of Pax3 during cardiac development remains unclear. A Cre-lox method has been used to fate map Pax3-expressing neural crest precursors to the cardiac outflow tract. Although Pax3 itself is extinguished prior to neural crest populating the heart, derivatives of these precursors contribute to the aorticopulmonary septum. Neural crest cells are found in the outflow tract of Splotch embryos, albeit in reduced numbers. This indicates that contrary to prior reports, Pax3 is not required for cardiac neural crest migration. Using a neural tube explant culture assay, it has been demonstrated that neural crest cells from Splotch embryos show normal rates of proliferation but altered migratory characteristics. These studies suggest that Pax3 is required for fine tuning the migratory behavior of the cardiac neural crest cells while it is not essential for neural crest migration (Epstein, 2000).

Pax-3 is a transcription factor that is expressed in the neural tube, neural crest, and dermomyotome. Apoptosis is associated with neural tube defects (NTDs) in Pax-3-deficient Splotch (Sp/Sp) embryos. p53 deficiency, caused by germ-line mutation or by pifithrin-alpha, an inhibitor of p53-dependent apoptosis, rescues not only apoptosis, but also NTDs, in Sp/Sp embryos. Pifithrin-alpha inhibits p53-dependent transcription and apoptosis. The precise mechanisms are not known, but given that nuclear accumulation of p53 is reduced, this suggests that pifithrin-alpha stimulates nuclear export, inhibits nuclear import, or decreases p53 stability. Pax-3 deficiency has no effect on p53 mRNA, but increases p53 protein levels. These results suggest that Pax-3 regulates neural tube closure by inhibiting p53-dependent apoptosis, rather than by inducing neural tube-specific gene expression (Pani, 2002).

Alternative splicing of Pax-3

Alternatively spliced isoforms of murine Pax-3 and Pax-7 have been identified which differ by the presence or absence of a single glutamine residue in a linker region that separates two distinct DNA-binding subdomains within the paired domain. By reverse transcription-PCR, these isoforms of Pax-3 and Pax-7 (Q+ and Q-) are detected at similar levels through multiple developmental stages in the early mouse embryo. DNA-binding studies using the Q+ and Q- isoforms of Pax-3 reveal that this alternative splicing event has no major effect on the ability of these isoforms to bind to an oligonucleotide specific for the Pax-3 homeodomain (P2) or to a paired domain recognition sequence (e5) that interacts primarily with the N-terminal subdomain of the paired domain. However, DNA-binding studies with sequences (P6CON and CD19-2/A) containing consensus elements for both the N-terminal and C-terminal subdomains reveals that the Q- isoform binds to these sequences with a two- to fivefold-higher affinity. Further mutation of the GTCAC core N-terminal subdomain recognition motif of CD19-2/A generates binding sites with a high degree of specificity for the Q- isoform. These differences in DNA binding in vitro are also reflected in the enhanced ability of the Q- isoform to stimulate transcription of a reporter containing multiple copies of CD19-2/A upstream of the thymidine kinase basal promoter. In support of the observations made with these naturally occurring Pax-3 isoforms, introducing a glutamine residue at the analogous position in PAX6 causes a fivefold reduction in binding to P6CON and a complete loss of binding to CD19-2/A and to the C-terminal subdomain-specific probe 5aCON. These studies therefore provide direct evidence for a role for the paired-domain linker region in DNA target site selection, and they identify novel isoforms of Pax-3 and Pax-7 that have the potential to mediate distinct functions in the developing embryo (Vogan, 1997).

The recognition of DNA targets by Pax-3 is achieved through the coordinate use of two distinct helix-turn-helix-based DNA-binding modules: a paired domain, composed of two structurally independent subdomains joined by a short linker, and a paired-type homeodomain. In mouse, the activity of the Pax-3 paired domain is modulated by an alternative splicing event in the paired domain linker region that generates isoforms (Q+ and Q-) with distinct C-terminal subdomain-mediated DNA-binding properties. Derivatives of a classical high affinity paired domain binding site (CD19-2/A) were used to derive an improved consensus recognition sequence for the Pax-3 C-terminal subdomain. This new consensus differs at six out of eight positions from the C-terminal subdomain recognition motif present in the parent CD19-2/A sequence, and includes a 5'-TT-3' dinucleotide at base pairs 15 and 16 that promotes high affinity binding by both Pax-3 isoforms. However, with a less favorable guanine at position 15, only the Q- isoform retains high affinity binding to this sequence, suggesting that this alternative splicing event might serve to stabilize binding to suboptimal recognition sequences. Mutagenic analysis of the linker demonstrates that both the sequence and the spacing in this region contribute to the enhanced DNA-binding properties of the Pax-3/Q- isoform. These studies establish a clear role for the Pax-3 C-terminal subdomain in DNA recognition and, thus, provide insights into an important mechanism by which Pax proteins achieve distinct target specificities (Vogan, 1997).

Post-transcriptional regulation of Pax3

Helicase UPF1 functions in both Staufen 1 (STAU1)-mediated mRNA decay (SMD) and nonsense-mediated mRNA decay (NMD), which are competitive pathways. STAU1- and UPF2-binding sites within UPF1 overlap so that STAU1 and UPF2 binding to UPF1 appear to be mutually exclusive. Furthermore, down-regulating the cellular abundance of STAU1, which inhibits SMD, increases the efficiency of NMD, whereas down-regulating the cellular abundance of UPF2, which inhibits NMD, increases the efficiency of SMD. Competition under physiological conditions is exemplified during the differentiation of C2C12 myoblasts to myotubes: The efficiency of SMD increases and the efficiency of NMD decreases, consistent with the finding that more STAU1 but less UPF2 bind UPF1 in myotubes compared with myoblasts. Moreover, an increase in the cellular level of UPF3X during myogenesis results in an increase in the efficiency of an alternative NMD pathway that, unlike classical NMD, is largely insensitive to UPF2 down-regulation. The remarkable balance NCC SMD and the two types of NMD are discussed in view of data indicating that PAX3 mRNA is an SMD target whose decay promotes myogenesis whereas myogenin mRNA is a classical NMD target encoding a protein required for myogenesis (Gong, 2009).

PAX3 interaction with pRB

The specific loss of pRB (see Drosophila Retinoblastoma-family protein) or p107 together with p130 disrupts the normal development of only a very limited spectrum of tissues. These developmental defects have been attributed primarily to deregulation of E2F activity and consequent uncontrolled proliferation. It was hypothesized, however, that the tissue-specific nature of these defects may also reflect deregulation of pRB-family associated factors that are specifically involved in determining cell fate. The pRB-family members are here reported to interact with transcription factors that contain paired-like homeodomains such as MHox, Chx10 and Pax-3. The interaction between the pRB-family and the paired-like homeodomain proteins was initially identified in a yeast two-hybrid screen where the N-terminal portion of p130 was used to isolate interacting factors from an embryonic mouse library. This interaction has been confirmed by in vitro binding and co-immunoprecipitation assays. Co-expression of Pax-3 dependent pRB, p107 or p130 with Pax-3 causes repression of activated transcription from the c-met promoter. These data demonstrate that the pRB-family proteins can modulate the activity of factors which specifically control cell fate and/or differentiation as well as controlling cell cycle regulators (Wiggan, 1998).

Other Pax3 interactions

The first neural crest cells to emigrate from the neural tube are specified as neurons and glial cells and are subsequently followed by melanocytes of the skin. It is important to understand how this fate switch is controlled. The transcriptional repressor FOXD3 is expressed exclusively in the neural/glial precursors and MITF is expressed only in melanoblasts. Moreover, FOXD3 represses melanogenesis. This study shows that avian MITF expression begins very early during melanoblast migration and that loss of MITF in melanoblasts causes them to transdifferentiate to a glial phenotype. Ectopic expression of FOXD3 represses MITF in cultured neural crest cells and in B16-F10 melanoma cells. FOXD3 does not bind directly to the MITF promoter, but instead interacts with the transcriptional activator PAX3 to prevent the binding of PAX3 to the MITF promoter. Overexpression of PAX3 is sufficient to rescue MITF expression from FOXD3-mediated repression. It is concluded that FOXD3 controls the lineage choice between neural/glial and pigment cells by repressing MITF during the early phase of neural crest migration (Thomas, 2009).

Methylation of the PAX3 gene

The developmental genes Pax7 and Pax3 are differentially methylated; the gene region that encodes the paired domain is hypomethylated, whereas the region that encodes the homeodomain is hypermethylated. For this reason, the known DNA sequence between the paired and homeoboxes was analysed for the presence of a conserved DNA motif to which a modifying protein could bind in order to direct the methylation or demethylation of surrounding gene sequences. The octapeptide-encoding region was found to contain several nucleotides that are highly conserved throughout the Pax gene family from phylogenetically distant species. The most conserved nucleotides are thought to comprise a motif TN8TCCT where N8=any combination of eight nucleotides. A conserved octapeptide-like-encoding sequence containing the TN8TCCT motif is also found in non-Pax genes of higher eukaryotes and in plants on the non-coding strand of DNA. Moreover, differential methylation seems to be associated with the presence of the TN8TCCT motif in p53 and the human oestrogen receptor genes. The presence of the TN8TCCT motif within an octapeptide-like-encoding sequence in human T-cell leukemia virus type 1 might suggest that the putative recognition motif may have been introduced into various host genomes via some form of retroviral agent (Ziman, 1998).

Transcriptional regulation of Pax3

The patterning of the cardiovascular system into systemic and pulmonic circulations is a complex morphogenetic process, the failure of which results in clinically important congenital defects. This process involves extensive vascular remodeling and coordinated division of the cardiac outflow tract (OFT). The homeodomain transcription factor Pbx1 orchestrates separate transcriptional pathways to control great-artery patterning and cardiac OFT septation in mice. Pbx1-null embryos display anomalous great arteries owing to a failure to establish the initial complement of branchial arch arteries in the caudal pharyngeal region. Pbx1 deficiency also results in the failure of cardiac OFT septation. Pbx1-null embryos lose a transient burst of Pax3 expression in premigratory cardiac neural crest cells (NCCs) that ultimately specifies cardiac NCC function for OFT development, but does not regulate NCC migration to the heart. Pbx1 directly activates Pax3, leading to repression of its target gene Msx2 in NCCs. Compound Msx2/Pbx1-null embryos display significant rescue of cardiac septation, demonstrating that disruption of this Pbx1-Pax3-Msx2 regulatory pathway partially underlies the OFT defects in Pbx1-null mice. Conversely, the great-artery anomalies of compound Msx2/Pbx1-null embryos remain within the same spectrum as those of Pbx1-null embryos. Thus, Pbx1 makes a crucial contribution to distinct regulatory pathways in cardiovascular development (Chang, 2008).

In vitro studies were conducted to further assess the potential role of Pbx1 in the transcriptional regulation of Pax3, which contains Pbx1 binding sites in its promoter. Electrophoretic mobility shift assays (EMSA) confirmed that Site A, which contains a consensus Pbx1/Meis1 binding sequence (5'-TGACAGTT-3'), supported robust cooperative binding by Pbx1 and Meis1, but not binding by either protein alone. By contrast, Pbx1 did not form binding complexes with several representative Hox proteins (HoxB2, HoxB4 or HoxB7) on Site A. Site B, which is located 1.1 kb upstream of the Pax3 transcriptional start site, was bound robustly by HoxB4 or Meis1 in the presence of Pbx1. DNA binding by HoxB2 and HoxB7 on Site B was also dependent on Pbx1. Pbx1-Meis1-Hox trimeric complexes did not form on either isolated Site A or Site B. The requirement for Pbx1 in regulating Pax3 promoter activity was assessed using a reporter gene containing the 1.6 kb Pax3 promoter fragment in PC12 pheochromocytoma cells, which are derivatives of NCCs. Whereas HoxB4 alone produced a modest increase in Pax3 promoter activity, co-transfection of Pbx1 and HoxB4 strongly activated transcription. Consistent with the binding studies, adding Meis1 to the transfection mixture did not further enhance the Pax3 transcriptional response. Thus, Pbx1 partners with Meis and Hox proteins to directly activate expression of Pax3 through its proximal promoter elements. Taken together, these results show that Pbx1 is essential for Pax3 proximal promoter activity and for transient premigratory cardiac NCC expression of Pax3. It is proposed that activity of the Pax3 1.6 kb proximal promoter in vivo partially reflects Pbx1-dependent Pax3 expression in premigratory cardiac NCCs. The broader and sustained dorsal neural tube expression of Pax3 is likely to require additional regulatory elements outside the 1.6 kb region that are not under Pbx1 control (Chang, 2008).

Table of contents

paired continued: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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