Pax6 and c-Maf regulate multiple stages of mammalian lens development. Here, we identified novel distal control regions (DCRs) of the aA-crystallin gene, a marker of lens fiber cell differentiation induced by FGF-signaling. DCR1 stimulated reporter gene expression in primary lens explants treated with FGF2 linking FGF-signaling with aA-crystallin synthesis. A DCR1/aA-crystallin promoter (including DCR2) coupled with EGFP virtually recapitulated the expression pattern of aA-crystallin in lens epithelium and fibers. In contrast, the DCR3/aA/EGFP reporter was expressed only in 'late' lens fibers. Chromatin immunoprecipitations showed binding of Pax6 to DCR1 and the aAcrystallin promoter in lens chromatin and demonstrated that high levels of aA-crystallin expression correlate with increased binding of c-Maf and CREB to the promoter and of CREB to DCR3, a broad domain of histone H3K9-hyperacetylation extending from DCR1 to DCR3, and increased abundance of chromatin remodeling enzymes Brg1 and Snf2h at the aA-crystallin locus. Our data demonstrate a novel mechanism of Pax6, c-Maf and CREB function, through regulation of chromatin-remodeling enzymes, and suggest a multistage model for the activation of aAcrystallin during lens differentiation.

Two lines of transgenic mice with one to two copies of a DNA fragment containing nucleotides -364 to +45 of the murine aA-crystallin gene linked to the bacterial chloramphenicol acetyltransferase (CAT) gene expressed the CAT gene only in their eye lenses. Both CAT activity and aA-crystallin were first detected in eyes at approximately 12.5 days of embryonic development, suggesting that the aA-CAT fusion gene and the endogenous aA-crystallin gene are coregulated during lens development in the transgenic mice. These experiments show that the murine aA-crystallin gene contains a short, cis-acting, tissue-specific regulatory sequence at its 5' end that can target the expression of the bacterial CAT gene, and probably foreign eukaryotic genes, specifically to the ocular lens.

Neoplastic tumors of the ocular lens of vertebrates do not naturally occur. Transgenic mice carrying a hybrid gene comprising the murine alpha A-crystallin promoter (-366 to +46) fused to the coding sequence of the SV40 T antigens developed lens tumors, which obliterated the eye cavity and even invaded neighboring tissue, thus establishing that the lens is not refractive to oncogenesis. Large-T antigen was detected early in lens development; it elicited morphological changes and specifically interfered with differentiation of lens fiber cells. Both alpha- and beta-crystallins persisted in many of the lens tumor cells, while gamma-crystallin was selectively reduced. Accessibility, characteristic morphology, and defined protein markers make this transparent epithelial eye tissue a potentially useful system for testing tumorigenicity of oncogenes and for studying malignant transformation from its inception until death of the animal.

During lens fiber cell differentiation, the regulation of crystallin gene expression is coupled with dramatic morphological changes. Here we report that Mafs, Prox1, and Pax6, which are essential transcription factors for normal lens development, bind to three functionally important cis elements, PL1, PL2, and OL2, in the chicken ␤B1-crystallin promoter and may cooperatively direct the transcription of this lens fiber cell preferred gene. Gel shift assays demonstrated that Mafs bind to the MARE-like sequences in the PL1 and PL2 elements, whereas Prox1, a sequence-specific DNA-binding protein like its Drosophila homolog Prospero, interacts with the OL2 element. Furthermore, Pax6, a known repressor of the chicken ␤B1-crystallin promoter, binds to all three of these cis elements. In transfection assays, Mafs and Prox1 activated the chicken ␤B1-crystallin promoter; however, their transactivation ability was repressed when co-transfected with Pax6. Taken together with the known spatiotemporal expression patterns of Mafs, Prox1, and Pax6 in the developing lens, we propose that Pax6 occupies and represses the chicken ␤B1-crystallin promoter in lens epithelial cells, and is displaced by Prox1 and Mafs, which activate the promoter, in differentiating cortical fiber cells.