The DNA-binding properties of these PAX6 and PAX6(5a) mutants were tested using the “optimal” DNA-binding sites P6CON,
34 5aCON,
4 and HDCON
30 for recombinant PD, PD(5a), and HD proteins, respectively. These “optimal” binding sites were obtained with GST-fusion proteins representing individual DNA-binding subdomains using enrichment of binding sites from pools of random sequences.
4 30 34 Optimal binding sites for full-length PAX6 and PAX6(5a) remain to be determined. P6CON is a bipartite binding site
31 34 in which the 5′-portion is strongly recognized by the PAI subdomain, and the 3′-half is recognized weakly by the RED subdomain
(Fig. 2) . In contrast, 5aCON is recognized by RED subdomain,
4 5 and HDCON is bound through the HD homodimer.
30 Natural Pax6-binding sites are usually recognized by a combination of PAI, RED, and HD for PAX6 or RED and HD for PAX6(5a).
31 The P6CON binding site is closest to the Pax6-binding site in the guinea pig ζ-crystallin promoter.
35 The 5aCON binding site, as a tetramer for PD5a, was shown in mouse γE- and γF-crystallin promoters.
5 36 Binding of Pax6 through both its HD and PD is important for the G1 element in the rat glucagon promoter.
37 A sequence of
ATTATGC
TAAT (the HD-binding nucleotides are italic), along with three PD-binding sites, is present in the Pax6-responsive region of the rat c-Maf promoter.
38 Thus, P6CON, 5aCON, and HDCON represent well the diversity of natural Pax6 DNA-binding sites.