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Molecular and Cellular Biology, September 1998, p. 5579-5586, Vol. 18, No. 9
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Dual Roles for Pax-6: a Transcriptional Repressor of Lens Fiber Cell-Specific beta -Crystallin Genes

Melinda K. Duncan,1 John I. Haynes II,2,dagger Ales Cvekl,2 and Joram Piatigorsky2,*

Department of Biological Sciences, The University of Delaware, Newark, Delaware 19716,1 and Laboratory of Molecular and Developmental Biology, National Eye Institute, Bethesda, Maryland 20892-27302

Received 20 April 1998/Accepted 22 May 1998

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 (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 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.

* Corresponding author. Mailing address: Laboratory of Molecular and Developmental Biology, National Eye Institute, Building 6, Room 205, 6 Center Dr., MSC 2730, Bethesda, MD 20892-2730. Phone: (301) 496-9467. Fax: (301) 402-0781. E-mail: Joram{at}helix.nih.gov.

dagger Present address: Vaccine Analytical Research, Merck and Co., West Point, PA 19486.

Molecular and Cellular Biology, September 1998, p. 5579-5586, Vol. 18, No. 9
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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Copyright © 1998 by the American Society for Microbiology. All rights reserved.