Modulation of PAX6 Homeodomain Function by the Paired Domain

Abstract

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 ...

Molecular analysis of a human PAX6 homeobox mutant

“…HeLa cells were transfected with different concentrations of wild-type or R242T mutant expression plasmids together with a luciferase reporter plasmid, containing the HD-binding site P3. 29 Figure 3c and d show the transcriptional activity of the wild-type and R242T mutant, respectively. For both constructs, the transcriptional behaviour is described by a linear regression, and the ratio between the corresponding angular coefficients (R242T/wild-type) was 1.72.…”

“…It has been reported that the HD may influence the DNA-binding properties of the PD. 29 Thus, by using bacterially expressed proteins containing both the PD and the HD (PAX6 2 -271 construct) we tried to assess whether the R242T mutation may affect the PD DNA-binding activity. Figure 1c shows the results of the DNA binding of PAX6 2 -271 peptides: the R242T mutant peptide has the same binding activity as the wild-type PAX6 2 -271 , both to the P3 and Cr oligonucleotides.…”

“…We used two radiolabelled double-stranded oligonucleotide target probes: P3, containing the HD-binding site 29 and Cr, containing the PD-binding site 30 ( Figure 1a). The R242T mutant peptide reveals the same binding activity to the P3 as the wild-type HD, as shown in Figure 1b.…”

“…25 Electophoretic mobility shift assays DNA-binding properties of PAX6 wild-type and mutant peptides were investigated using a series of doublestranded oligonucleotides (top strand only is shown): P3(5 0 -TCGAGGGCATCAGGATGCTAATTGGATTAGCAT CCGATCGGG-3 0 ) and Cr (5 0 -AAGCATTTTCACGCATGAG TGCACAG-3 0 ), which contain consensus DNA-binding sequences for PAX6 HD and PD, respectively. 29,30 The oligonucleotides were radiolabelled at the 5 0 end terminal by incubation with [g-32P]-dATP. Protein and DNA (both at the final concentration of 0.1 mM) were incubated for 30 min at room temperature in a total volume of 20 ml, using a buffer containing 20 mM Tris-HCl (pH 7.6), 75 mM KCl, 0.25 mg/ml bovine serum albumin (BSA), 5 mM dithiotreitol (DTT), 10 mg/ml calf thymus DNA, and 10% glycerol.…”

“…The PAX6-responsive promoter (P3) contains a PAX6 HD-binding sequence and luciferase as reporter gene. 29 The activity of P3 was normalized by cotransfecting the RSV-CAT promoter, in which the reporter gene is the chloramphenycol-acetyl-transferase gene (CAT). The plasmids were used in the following amounts for dish: RSV-CAT, 2 mg; PAX6 wild-type and R242T mutant expression vector, 1, 2 or 4 mg; P3 reporter, 9 mg.…”