The long-term goal of this application is to elucidate the role of transcription factor Pax6 in lens biology and disease. Pax6 is essential for lens formation and function. Mutations or deletions in PAX6 cause ocular diseases including aniridia, early onset cataract, Peters'anomaly, and autosomal dominant keratitis. Pax6 regulates the expression of numerous genes that range from lens structural proteins including crystallins to other transcription factors including MafA, c-Maf, and Brg1. Evidence exists that Pax6 proteins functionally interact with Maf and Brgl proteins. Expression of Pax6 target genes is controlled through either gene activation or gene repression. Crystallin gene expression is directed by interactions of Pax6 with Mafs together with co-activators including ASC-2. To understand the function of Pax6 in lens, this proposal will: (1) Determine the molecular mechanism by which Pax6 activates the alpha B- and represses gamma F-crystallin genes;(2) Elucidate the function of chromatin remodeling factor Brg1, and transcriptional co-activator ASC-2 in vivo in lens;and (3) Identify and characterize genes regulated by Pax6 that control lens induction, invagination of the lens placode, and formation of the lens vesicle.
These aims will be achieved using functional analysis of gene expression including chromatin immunoprecipitations, promoter mutagenesis, transfections, and protein-DNA assays. The genetic experiments will be performed in vivo using transgenic mice. The function of Brg1 in lens will be determined using lens-specific inactivation of Brgl combined with an overexpression of the dominant negative (DN) Brgl mutant. The DN mutant of ASC-2 will also be expressed in lens, as it is required for the expression of specific crystallins. Differential gene expression between normal and Pax6 homozygous mouse embryos will be assessed using cDNA microarrays, followed by temporal and spatial analysis of the expression of selected genes. Collectively, these studies will refine our understanding of the genetic network regulated by Pax6 and mechanisms whereby Pax6 controls this genetic network in lens.
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