Two independent and interactive DNA-binding subdomains of the Pax6 paired domain are regulated by alternative splicing.

Abstract

Vertebrate Pax proteins share a conserved 128-amino-acid DNA-binding motif, the paired domain. The PAX6 gene, which is mutated in the routine Small eye and human aniridia developmental defects, also encodes a second protein with a 14-amino-acid insertion in the paired domain. This protein, which arises by alternative mRNA splicing, exhibits unique DNA-binding properties. Unlike other paired domains, which bind DNA predominantly by their amino termini, the extended Pax6 paired domain interacts with DNA exclusively through its carboxyl terminus. This property can be simulated by deletion of 30 amino-terminal residues from the Pax6 or Pax2 paired domains. Thus, the insertion acts as a molecular toggle to unmask the DNA-binding potential of the carboxyl terminus. The functional nonequivalence of the two Pax6 proteins is underscored by a T ~ C mutation at position -3 of the alternative splice acceptor site that changes the ratio of the two isoforms and causes a distinct human ocular syndrome.

Autoregulation of Pax6 transcriptional activation by two distinct DNA‐binding subdomains of the paired domain

“…2c). As reported previously (Epstein et al 1994b), the 5a insertion abolished P6CON binding (Fig. 2b, lanes 8-13), but did not affect 5aCON binding (Fig.…”

“…Extending previous observations (Epstein et al 1994b), we clearly demonstrate that the two subdomains can bind independently to their cognate sites. However, the two functional subdomains are shown to negatively regulate their transactivation potentials to each other.…”

“…The CTS has recently been suggested to form a DNA-binding domain distinct from that of the NTS (Epstein et al 1994b), but this functional importance is not understood. To examine the CTS function, we prepared wild-type and R128C mutant paired domain (PrD) proteins fused to GST, and analysed their binding to P6CON and 5aCON by gel shift assay.…”

“…Recent X-ray structural analysis of paired has revealed that these subdomains contain helix-turn-helix motifs resembling the Hin recombinase, and are separated by a b-turn, and that the NTS and the b-turn make contacts with DNA (Xu et al 1995). In contrast to the other paired domain-containing proteins, however, both NTS and CTS of the Pax6 paired domain are suggested to have distinct DNA-binding activities (Epstein et al 1994b). Human and mouse PAX6 genes are alternatively spliced, to produce a Pax6-5a variant which has a 14 amino acid insertion into NTS encoded by exon 5a.…”

“…The high affinity binding sites for Pax6 and Pax6-5a were determined by a PCR-based method, and are called P6CON and 5aCON, respectively (Epstein et al 1994a,b). According to this model, NTS is involved in the P6CON binding, and the 5a insertion serves as a molecular switch to unmask the cryptic DNA-binding activity of CTS to 5aCON (Epstein et al 1994b).…”