Functional properties of natural human PAX6 and PAX6(5a) mutants.

Pax6 is a highly conserved member of a Pax family of genes encoding transcription factors.1 Nine mammalian Pax genes (Pax1 to Pax9) share an N-terminal 128 amino acid DNA-binding domain called the paired domain (PD). The PD of Pax proteins can be structurally and functionally separated into two independent DNA-binding subdomains, PAI and RED.2 Pax6 also contains an internal, paired-type homeodomain (HD), allowing different modes of Pax6 binding to DNA. Mammalian Pax6 genes encode predominantly two forms of the Pax6 protein, Pax6 and Pax6(5a).3-5 Pax6 contains a canonical PD. In contrast, Pax6(5a) contains a 14 amino acid insertion within the PAI domain (see Fig. 1A). Pax6 plays critical roles in the organogenesis of the brain, visual, and olfactory systems, as well as peptide hormone gene expression in the pancreas.6-10 Numerous studies have shown that Pax6 is essential for morphogenesis of the eye from its earliest stages and subsequent formation of all major ocular tissues.7,11,12 Figure 1 Structure of PAX6 and PAX6(5a) and position of human mutations. (A) Schematic representation of PAX6 (amino acid residues 1-422); PAX6(5a) (amino acid residues 1-436); PAX6 PD, HD (yellow); and transcriptional activation domain (P/S/T, green); and subdivision ... In humans, heterozygous mutations in PAX6 cause a wide spectrum of ocular defects13-22 and subtle changes in the olfactory epithelium and brain.23 Mutations generating truncated PAX6 proteins and deletions of one allele typically result in aniridia. The prominent feature of aniridia is iris hypoplasia, often combined with cataracts, glaucoma, nystagmus, and foveal and optic nerve hypoplasia.24,25 Missense mutations generating single amino acid substitutions, representing approximately 10% of total mutations, cause less severe abnormalities (e.g., foveal hypoplasia, Peters’ anomaly, congenital cataracts, and autosomal dominant keratitis).10,26 A rare case of a human PAX6 compound homozygote resulted in anophthalmia and lethal brain defects.14 Hence, it has been proposed that a PAX6 gene dosage effect is responsible for phenotypes associated with mutations in one or both alleles of PAX6.8,10 Molecular studies of natural PAX6 mutants are useful in understanding the molecular mechanisms of PAX6 and other PAX proteins; however, earlier studies have focused only on a single or a few mutants tested in a single common cell line. Notably, two missense mutations, R26G and R128C, tested in P19 cells, reduced PAX6 but increased PAX6(5a)-activation potential, suggesting that a specific mutant can act as a hypermorph.27 Crystal structure of Pax6 PD in complex with DNA provides a model to understand the function of individual structural motifs comprising the PD and may ultimately predict impact of disease-causing single amino acid substitutions.28 In this report, we sought to determine transactivation and DNA-binding properties of 10 representative natural PAX6 and PAX6(5a) mutants with different reporters using a variety of DNA-binding mechanisms in different cell types. The results revealed an unexpected range of reduced or increased transactivation properties of each mutant tested. Our data indicate that cellular environment might influence whether an individual mutation acts as loss- or gain-of-function or neutrally. The results also suggest that some mutations not only impair the respective subdomain but also the overall conformation of Pax6.