Dual roles for Pax-6:: a transcriptional repressor of lens fiber cell-specific β-crystallin genes

It has been demonstrated previously that Pax-6, a paired domain (PD)/homeodomain (HD) transcription factor critical for eye development, contributes to the activation of the alpha B-, alpha A-, delta 1-, and zeta-crystallin genes in the lens. Here we have examined the possibility that the inverse relationship between the expression of Pax-6 and beta-crystallin genes within the developing chicken lens reflects a negative regulatory role of Pax-6. Cotransfection of a plasmid containing the beta B1-crystallin promoter fused to the chloramphenicol acetyltransferase reporter gene and a plasmid containing the full length mouse Pax-6 coding sequences into primary embryonic chicken lens epithelial cells or fibroblasts repressed the activity of this promoter by as much as 90%, Pax-6 constructs lacking the C-terminal activation domain repressed 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 footprinting analysis revealed that truncated Pax-6 (PD+HD) binds to three regions (-183 to -152, -120 to -48, and -30 to +1) of the beta B1-crystallin promoter. Earlier experiments showed that the beta B1-crystallin promoter sequence from -120 to -48 contains a cis element (PL2 at -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 binds to PL2 and represses its ability to activate promoter activity; moreover, mutation of PL2 eliminated binding by Pax-6, Taken together, our data indicate that Pax-6 (via its PD and HD) represses the beta B1-crystallin promoter by direct interaction with the PL2 element. We thus suggest that the relatively high concentration of Pax-6 contributes to the absence of beta B1-crystallin gene expression in lens epithelial cells and that diminishing amounts of Pax-6 in lens fiber cells during development allow activation of this gene.