PAX6 is a transcription activator that regulates eye development in animals ranging from Drosophila to human. The C-terminal region of PAX6 is proline/serine/ threonine-rich (PST) and functions as a potent transactivation domain when attached to a heterologous DNAbinding domain of the yeast transcription factor, GAL4. The PST region comprises 152 amino acids encoded by four exons. The transactivation function of the PST region has not been defined and characterized in detail by in vitro mutagenesis. We dissected the PST domain in two independent systems, a heterologous system using a GAL4 DNA-binding site and the native system of PAX6. Our data consistently showed that in both systems all four constituent exons of the PST domain are responsible for the transactivation function. The four exon fragments act synergistically to stimulate transcription, although none of them can function individually as an independent transactivation domain. Combinations of two or more exon fragments can reconstitute substantial transactivation activity when fused to the DNAbinding domain of GAL4, but they surprisingly do not produce much activity in the context of native PAX6, although the mutant PAX6 proteins are stable and their DNA-binding function remains unaffected. Our data suggest that these mutants may antagonize the wildtype PAX6 activity by competing for target DNA-binding sites. We conclude that the PAX6 protein contains an unusually large transactivation domain that is evolutionarily conserved to a high degree and that its full transactivation activity relies on the synergistic action of the four exon fragments.
PAX6 is a transcription factor with two DNA-binding domains (paired box and homeobox) and a proline-serine-threonine (PST)-rich transactivation domain. PAX6 regulates eye development in animals ranging from jellyfish to Drosophila to humans. Heterozygous mutations in the human PAX6 gene result in various phenotypes, including aniridia, Peter's anomaly, autosomal dominant keratitis, and familial foveal dysplasia. It is believed that the mutated allele of PAX6 produces an inactive protein and aniridia is caused due to genetic haploinsufficiency. However, several truncation mutations have been found to occur in the C-terminal half of PAX6 in patients with Aniridia resulting in mutant proteins that retain the DNA-binding domains but have lost most of the transactivation domain. It is not clear whether such mutants really behave as loss-of-function mutants as predicted by haploinsufficiency. Contrary to this theory, our data showed that these mutants are dominant-negative in transient transfection assays when they are coexpressed with wild-type PAX6. We found that the dominant-negative effects result from the enhanced DNA binding ability of these mutants. Kinetic studies of binding and dissociation revealed that various truncation mutants have 3-5-fold higher affinity to various DNA-binding sites when compared with the wild-type PAX6. These results provide a new insight into the role of mutant PAX6 in causing aniridia.PAX6 is an evolutionarily conserved gene that regulates the development of the eye in animals ranging from jellyfish to Drosophila to humans (for review, see Ref. 1). The induction of ectopic compound eyes by overexpressing mouse and squid Pax6 (2, 3) clearly indicates that not only is the structure of PAX6 conserved but also its biochemical properties are conserved. Recent reports have shown that PAX6 is also involved in pancreas development (4, 5).Like other members of the PAX family, PAX6 functions as a transcriptional activator. Structural analysis of PAX6 has identified two DNA-binding domains (a paired domain at the N terminus and a paired like homeodomain in the middle), a glycine-rich hinge region that links the two DNA-binding domains, and a proline-serine-threonine-rich (PST) transactivation domain at the C terminus. Our recent studies designed to characterize the transactivation domain revealed that the four exons, which constitute the PST domain, synergistically stimulate transcriptional activation and that the transactivation potential is not localized but distributed throughout the PST domain (6). The transcription factors of the PAX family recognize their target genes via the DNA binding function of the paired domain (7,8). Several PAX6 paired-domain binding sequences have already been identified (reviewed in Ref. 1). However, studies of Czerny and Busslinger (9) identifying the P3 site as the optional binding site for cooperative binding and transactivation by the PAX6 homeodomain, conservation of P3 in eye-specific promoters (10), and the presence and requirement of the P3 site in the r...
PAX6 is required for proper development of the eye, central nervous system, and nose. PAX6 has two DNA binding domains, a glycine-rich region that links the two DNA binding domains, and a transactivation domain. There is evidence that the different DNA binding domains of PAX6 have different target genes. However, it is not clear if the two DNA binding domains function independently. We have studied the effect of structural changes in the paired domain on the function of PAX6 mediated through its homeodomain. The R26G and I87R mutations have been reported in different human patients with clinically different phenotypes and are in the N-and the C-terminal halves of the paired domain, respectively. Surprisingly, we found that the I87R mutant protein not only lost the transactivation function but also failed to bind DNA by either of its DNA binding domains. In contrast, the R26G mutant protein lost DNA binding through its paired domain but had greater DNA binding and transactivation than wild-type PAX6 on homeodomain binding sites. Like R26G, the 5a isoform showed higher DNA binding than wild-type PAX6. This study demonstrates that the two subdomains of the paired domain influence the function of the homeodomain differentially and also provides an explanation for the difference in phenotypes associated with these mutations.Pax6 is considered the master control gene for morphogenesis and evolution of the eye (1). It is an evolutionarily conserved gene in both vertebrates and invertebrates. The Pax6 genes cloned from representatives of at least eight animal phyla are structurally and functionally similar (2), and ectopic expression of mouse and squid Pax6 in Drosophila results in ectopic eye formation (3, 4). Pax6 is expressed in the developing eye, nose, pancreas, and central nervous system (5-10). Loss of PAX6 function leads to severe brain abnormalities, microencephaly, early postnatal death, and the absence of eyes and nose in rodents (11, 12) and humans (13). In addition, PAX6 is also essential for the differentiation of ␣-cells and the formation of the islets in the pancreas (14,15). Heterozygous mutations in the PAX6 gene are responsible for several naturally occurring phenotypes including aniridia.The PAX6 protein can be subdivided into several distinct domains. It has two DNA binding domains (a paired domain (PD) 1 at the NH 2 terminus and a paired-like homeodomain (HD) in the middle), a glycine-rich region that links the two DNA binding domains, and a transactivation domain at the COOH terminus (5, 16). There are two major alternatively spliced forms of PAX6 which differ by the presence or absence of 14 amino acids within the PD which are coded by an exon known as 5a (17). Studies suggest that the target genes of PAX6 can be regulated by three types of DNA binding sites: (i) those identified by the PD., e.g. mouse neural cell adhesion molecule (N-CAM) (18); (ii) those identified by the HD, e.g. rhodopsin (19); and (iii) those identified by cooperative interaction of the PD and HD e.g. N-CAM L1 (20).The PD ...
Mutations in the human PAX6 gene produce various phenotypes, including aniridia, Peters' anomaly, autosomal dominant keratitis and familial foveal dysplasia. The various phenotypes may arise from different mutations in the same gene. To test this theory, we performed a functional analysis of two missense mutations in the paired domain: the R26G mutation, previously reported in a case of Peters' anomaly, and an unreported I87R mutation, which we identified in a patient with aniridia. While both the R26 and the I87 positions are conserved in the paired boxes of all known PAX genes, X-ray crystallography has shown that only R26 makes contact with DNA. We showed that the R26G mutant failed to bind a subset of paired domain binding sites but, surprisingly, bound other sites and successfully transactivated promoters containing those sites. In contrast, the I87R mutant had lost the ability to bind DNA at all tested sites and failed to transactivate promoters. Our data support the haploid-insufficiency hypothesis of aniridia, and the hypothesis that R26G is a hypomorphic allele.
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