paired
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).
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).
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).
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).
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