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Molecular and Cellular Biology, September 1998, p. 5579-5586, Vol. 18, No. 9
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
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Copyright © 1998, American Society for Microbiology. All rights reserved.
Dual Roles for Pax-6: a Transcriptional Repressor
of Lens Fiber Cell-Specific
-Crystallin Genes

B-,
A-,
1-, and
-crystallin genes in the lens. Here we have examined the possibility that the inverse relationship between the expression of
Pax-6 and
-crystallin genes within the developing chicken lens
reflects a negative regulatory role of Pax-6. Cotransfection of a
plasmid containing the
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
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
B1-crystallin promoter. Earlier
experiments showed that the
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
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
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.
Present address: Vaccine Analytical Research, Merck and Co.,
West Point, PA 19486.
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