DUAL ROLES FOR PAX-6 - A TRANSCRIPTIONAL REPRESSOR OF LENS FIBER CELL-SPECIFIC BETA-CRYSTALLIN GENES

It has been demonstrated previously that Pax-6, a paired domain (PD)/h omeodomain (HD) transcription factor critical for eye development, con tributes to the activation of the alpha B-, alpha A-, delta 1-, and ze ta-crystallin genes in the lens. Here we have examined the possibility that the inverse relationship between the expression of Pax-6 and bet a-crystallin genes within the developing chicken lens reflects a negat ive regulatory role of Pax-6. Cotransfection of a plasmid containing t he beta B1-crystallin promoter fused to the chloramphenicol acetyltran sferase reporter gene and a plasmid containing the full length mouse P ax-6 coding sequences into primary embryonic chicken lens epithelial c ells or fibroblasts repressed the activity of this promoter by as much as 90%, Pax-6 constructs lacking the C-terminal activation domain rep ressed beta B1-crystallin promoter activity as effectively as the full -length protein, but the PD alone or Pax-6 (ja), a splice variant with an altered PD affecting its DNA binding specificity, did not. DNase f ootprinting analysis revealed that truncated Pax-6 (PD+HD) binds to th ree regions (-183 to -152, -120 to -48, and -30 to +1) of the beta B1- crystallin promoter. Earlier experiments showed that the beta B1-cryst allin promoter sequence from -120 to -48 contains a cis element (PL2 a t -90 to -76) that stimulates the activity of a heterologous promoter in lens cells but not in fibroblasts. In the present study, we show by electrophoretic mobility shift assay and cotransfection that Pax-6 bi nds to PL2 and represses its ability to activate promoter activity; mo reover, mutation of PL2 eliminated binding by Pax-6, Taken together, o ur data indicate that Pax-6 (via its PD and HD) represses the beta B1- crystallin promoter by direct interaction with the PL2 element. We thu s suggest that the relatively high concentration of Pax-6 contributes to the absence of beta B1-crystallin gene expression in lens epithelia l cells and that diminishing amounts of Pax-6 in lens fiber cells duri ng development allow activation of this gene.