CONVERGENT EVOLUTION OF CRYSTALLIN GENE-REGULATION IN SQUID AND CHICKEN - THE AP-1 ARE CONNECTION

Previous experiments have shown that the minimal promoters required fo r function of the squid SL20-1 and SL11 crystallin genes in transfecte d rabbit lens epithelial cells contain an overlapping AP-1/antioxidant responsive element (ARE) upstream of the TATA box. This region resemb les the PL-1 and PL-2 elements of the chicken beta B1-crystallin promo ter which are essential for promoter function in transfected primary c hicken lens epithelial cells. Here we demonstrate by site-directed mut agenesis that the AP-1/ARE sequence is essential for activity of the s quid SL20-1 and SL11 promoters in transfected embryonic chicken lens c ells and fibroblasts. Promoter activity was higher in transfected lens cells than in fibroblasts. Electrophoretic mobility shift and DNase p rotection experiments demonstrated the formation of numerous complexes between nuclear proteins of the embryonic chicken lens and the AP-1/A RE sequences of the squid SL20-1 and SL11 crystallin promoters. One of these complexes comigrated and cross-competed with that formed with t he PL-1 element of the chicken beta B1-crystallin promoter. This compl ex formed with nuclear extracts from the lens, heart, brain, and skele tal muscle of embryonic chickens and was eliminated by competition wit h a consensus AP-1 sequence. The nonfunctional mutant AP-1/ARE sequenc es did not compete for complex formation. These data raise the intrigu ing possibility that entirely different, nonhomologous crystallin gene s of the chicken and squid have convergently evolved a similar cisacti ng regulatory element (AP-1/ARE) for high expression in the lens.