Affiliation:
1. Department of Biological Sciences, The University of Delaware, Newark, Delaware 19716,1and
2. Laboratory of Molecular and Developmental Biology, National Eye Institute, Bethesda, Maryland 20892-27302
Abstract
ABSTRACT
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 αB-, αA-, δ1-, and ζ-crystallin genes in the lens. Here we have examined the possibility that the inverse relationship between the expression of Pax-6 and β-crystallin genes within the developing chicken lens reflects a negative regulatory role of Pax-6. Cotransfection of a plasmid containing the βB1-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 βB1-crystallin promoter activity as effectively as the full-length protein, but the PD alone or Pax-6 (5a), 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 βB1-crystallin promoter. Earlier experiments showed that the βB1-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 βB1-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 βB1-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.
Publisher
American Society for Microbiology
Subject
Cell Biology,Molecular Biology
Cited by
127 articles.
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