Reciprocal Effect of Waardenburg Syndrome Mutations on DNA Binding by the Pax-3 Paired Domain and Homeodomain

The Drosophila pair-rule gene paired is required for the correct expression of the segment polarity genes wingless, engrailed and gooseberry. It encodes a protein containing three conserved motifs: a homeodomain (HD), a paired domain (PD) and a PRD (His/Pro) repeat. We use a rescue assay in which paired (or a mutated version of paired in which the functions of the conserved motifs have been altered) is expressed under the control of its own promoter, in the absence of endogenous paired, to dissect the Paired protein in vivo. We show that both the HD and the N- terminal subdomain of the PD (PAI domain) are absolutely required within the same molecule for normal paired function. In contrast, the conserved C-terminal subdomain of the PD (RED domain) appears to be dispensable. Furthermore, although a mutation abolishing the ability of the homeodomain to dimerize results in an impaired Paired molecule, this molecule is nonetheless able to mediate a high degree of rescue. Finally, a paired transgene lacking the PRD repeat is functionally impaired, but still able to rescue to viability. We conclude that, while Prd can use its DNA-binding domains combinatorially in order to achieve different DNA-binding specificities, its principal binding mode requires a cooperative interaction between the PAI domain and the homeodomain.

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Characterization of quail Pax-6 (Pax-QNR) proteins expressed in the neuroretina.

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7257 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7257-7266 ◽

Cited By ~ 64

Author(s):

C Carriere ◽

S Plaza ◽

P Martin ◽

B Quatannens ◽

M Bailly ◽

...

Keyword(s):

Dna Binding ◽

Autosomal Dominant ◽

Paired Domain ◽

Terminal Part ◽

Dominant Mutation ◽

Dna Binding Domains ◽

Binding Domains ◽

Carboxyl Terminal

After differential screening of a cDNA library constructed from quail neuroretina cells (QNR) infected with the v-myc-containing avian retrovirus MC29, we have isolated a cDNA clone, Pax-QNR, homologous to the murine Pax-6, which is mutated in the autosomal dominant mutation small eye of mice and in the disorder aniridia in humans. Here we report the characterization of the Pax-QNR proteins expressed in the avian neuroretina. From bacterially expressed Pax-QNR peptides, we obtained rabbit antisera directed against different domains of the protein: paired domain (serum 11), domain between the paired domain and homeodomain (serum 12), homeodomain (serum 13), and carboxyl-terminal part (serum 14). Sera 12, 13, and 14 were able to specifically recognize five proteins (48, 46, 43, 33, and 32 kDa) in the neuroretina. In contrast to proteins of 48, 46, and 43 kDa, proteins of 33 and 32 kDa were not recognized by the paired antiserum (serum 11). Paired-less and paired-containing proteins exhibited the same half-life (6 h) and were phosphorylated mostly on serine residues. Immunoprecipitations performed with subcellular fractions of neuroretinas showed that the paired-containing proteins were located in the nucleus, whereas the 33- and 32-kDa proteins were found essentially in the cytoplasmic compartment. However, immunofluorescence experiments performed after transient transfections showed that p46 and p33/32 were also located in vivo into the nucleus. Thus, the Pax-QNR/Pax-6 gene can produce proteins with two DNA-binding domains as well as proteins containing only the DNA-binding homeodomain.

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Identification of a novel mutation in the paired domain of PAX3 in an Iranian family with Waardenburg syndrome Type I

Ophthalmic Genetics ◽

10.1076/1381-6810(200003)21:1;1-i;ft025 ◽

2000 ◽

Vol 21 (1) ◽

pp. 25-28

Author(s):

Vihra N. Sotirova ◽

Tayebeh Rezaie ◽

M.R. Khoshsorour ◽

Mansoor Sarfarazi

Keyword(s):

Novel Mutation ◽

Type I ◽

Syndrome Type ◽

Waardenburg Syndrome ◽

Paired Domain ◽

Iranian Family

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A frameshift mutation in the HuP2 paired domain of the probable human homolog of murine Pax-3 is responsible for Waardenburg syndrome type 1 in an Indonesian family

Human Molecular Genetics ◽

10.1093/hmg/1.4.243 ◽

1992 ◽

Vol 1 (4) ◽

pp. 243-247 ◽

Cited By ~ 55

Author(s):

Robert Morell ◽

Thomas B. Friedman ◽

Sukarti Moeljopawiro ◽

Hartono ◽

Soewito ◽

...

Keyword(s):

Frameshift Mutation ◽

Syndrome Type ◽

Waardenburg Syndrome ◽

Human Homolog ◽

Paired Domain

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The PAX3 and 7 homeodomains have evolved unique determinants that influence DNA-binding, structure and communication with the paired domain

10.1101/701656 ◽

2019 ◽

Author(s):

Gareth N. Corry ◽

Brian D. Sykes ◽

D. Alan Underhill

Keyword(s):

Dna Binding ◽

Protein Interactions ◽

Ternary Complex ◽

Phylogenetic Analyses ◽

Inhibitory Effect ◽

Binding Assays ◽

Paired Domain ◽

Functional Diversification ◽

H Nmr

ABSTRACTThe PAX (paired box) family is a collection of metazoan transcription factors defined by the paired domain, which confers sequence-specific DNA-binding. Ancestral PAX proteins also contained a homeodomain, which can communicate with the paired domain to modulate DNA-binding. In the present study, we sought to identify determinants of this functional interaction using the paralogous PAX3 and 7 proteins. First, we evaluated a group of heterologous paired domains and homeodomains for the ability to bind DNA cooperatively through formation of a ternary complex (paired domain:homeodomain:DNA). This revealed that capacity for ternary complex formation was unique to the PAX3 and 7 homeodomains and therefore not simply a consequence of DNA-binding. We also found PAX3 and 7 were distinguished by an extended region of conservation N-terminal to the homeodomain (NTE). Phylogenetic analyses established the NTE was restricted to PAX3/7 orthologs of segmented metazoans, indicating it arose in a bilaterian precursor prior to separation of deuterostomes and protostomes. In DNA-binding assays, presence of the NTE caused a decrease in monomeric binding by the PAX3 homeodomain that reflected a lack of secondary structure in 1D-1H-NMR. Nevertheless, this inhibitory effect could be overcome by homeodomain dimerization or cooperative binding with the paired domain, establishing that protein interactions could induce homeodomain folding in the presence of the NTE. Strikingly, the PAX7 counterpart did not impair homeodomain binding, revealing inherent differences that could account for its distinct target profile in vivo. Collectively, these findings identify critical determinants of PAX3 and 7 activity, which contribute to their functional diversification.

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Both the paired domain and homeodomain are required for in vivo function of Drosophila Paired

Development ◽

10.1242/dev.122.9.2709 ◽

1996 ◽

Vol 122 (9) ◽

pp. 2709-2718 ◽

Cited By ~ 4

Author(s):

P. Miskiewicz ◽

D. Morrissey ◽

Y. Lan ◽

L. Raj ◽

S. Kessler ◽

...

Keyword(s):

Dna Binding ◽

Target Genes ◽

Ectopic Expression ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Paired Domain ◽

Mutant Proteins ◽

Segment Polarity ◽

Negative Effect

Drosophila paired, a homolog of mammalian Pax-3, is key to the coordinated regulation of segment-polarity genes during embryogenesis. The paired gene and its homologs are unusual in encoding proteins with two DNA-binding domains, a paired domain and a homeodomain. We are using an in vivo assay to dissect the functions of the domains of this type of molecule. In particular, we are interested in determining whether one or both DNA-binding activities are required for individual in vivo functions of Paired. We constructed point mutants in each domain designed to disrupt DNA binding and tested the mutants with ectopic expression assays in Drosophila embryos. Mutations in either domain abolished the normal regulation of the target genes engrailed, hedgehog, gooseberry and even-skipped, suggesting that these in vivo functions of Paired require DNA binding through both domains rather than either domain alone. However, when the two mutant proteins were placed in the same embryo, Paired function was restored, indicating that the two DNA-binding activities need not be present in the same molecule. Quantitation of this effect shows that the paired domain mutant has a dominant-negative effect consistent with the observations that Paired protein can bind DNA as a dimer.

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Characterization of quail Pax-6 (Pax-QNR) proteins expressed in the neuroretina

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7257-7266.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7257-7266

Author(s):

C Carriere ◽

S Plaza ◽

P Martin ◽

B Quatannens ◽

M Bailly ◽

...

Keyword(s):

Dna Binding ◽

Autosomal Dominant ◽

Paired Domain ◽

Terminal Part ◽

Dominant Mutation ◽

Dna Binding Domains ◽

Binding Domains ◽

Carboxyl Terminal

After differential screening of a cDNA library constructed from quail neuroretina cells (QNR) infected with the v-myc-containing avian retrovirus MC29, we have isolated a cDNA clone, Pax-QNR, homologous to the murine Pax-6, which is mutated in the autosomal dominant mutation small eye of mice and in the disorder aniridia in humans. Here we report the characterization of the Pax-QNR proteins expressed in the avian neuroretina. From bacterially expressed Pax-QNR peptides, we obtained rabbit antisera directed against different domains of the protein: paired domain (serum 11), domain between the paired domain and homeodomain (serum 12), homeodomain (serum 13), and carboxyl-terminal part (serum 14). Sera 12, 13, and 14 were able to specifically recognize five proteins (48, 46, 43, 33, and 32 kDa) in the neuroretina. In contrast to proteins of 48, 46, and 43 kDa, proteins of 33 and 32 kDa were not recognized by the paired antiserum (serum 11). Paired-less and paired-containing proteins exhibited the same half-life (6 h) and were phosphorylated mostly on serine residues. Immunoprecipitations performed with subcellular fractions of neuroretinas showed that the paired-containing proteins were located in the nucleus, whereas the 33- and 32-kDa proteins were found essentially in the cytoplasmic compartment. However, immunofluorescence experiments performed after transient transfections showed that p46 and p33/32 were also located in vivo into the nucleus. Thus, the Pax-QNR/Pax-6 gene can produce proteins with two DNA-binding domains as well as proteins containing only the DNA-binding homeodomain.

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Cooperative interactions between paired domain and homeodomain

Development ◽

10.1242/dev.122.9.2639 ◽

1996 ◽

Vol 122 (9) ◽

pp. 2639-2650 ◽

Cited By ~ 13

Author(s):

S. Jun ◽

C. Desplan

Keyword(s):

Dna Binding ◽

Dna Sequences ◽

Target Genes ◽

Cooperative Interaction ◽

Paired Domain ◽

Cooperative Interactions ◽

Dna Binding Domains ◽

Binding Domains

The Pax proteins are a family of transcriptional regulators involved in many developmental processes in all higher eukaryotes. They are characterized by the presence of a paired domain (PD), a bipartite DNA binding domain composed of two helix-turn-helix (HTH) motifs, the PAI and RED domains. The PD is also often associated with a homeodomain (HD) which is itself able to form homo- and hetero-dimers on DNA. Many of these proteins therefore contain three HTH motifs each able to recognize DNA. However, all PDs recognize highly related DNA sequences, and most HDs also recognize almost identical sites. We show here that different Pax proteins use multiple combinations of their HTHs to recognize several types of target sites. For instance, the Drosophila Paired protein can bind, in vitro, exclusively through its PAI domain, or through a dimer of its HD, or through cooperative interaction between PAI domain and HD. However, prd function in vivo requires the synergistic action of both the PAI domain and the HD. Pax proteins with only a PD appear to require both PAI and RED domains, while a Pax-6 isoform and a new Pax protein, Lune, may rely on the RED domain and HD. We propose a model by which Pax proteins recognize different target genes in vivo through various combinations of their DNA binding domains, thus expanding their recognition repertoire.

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