(from abstract): The long-term goal of this application is to identify those interactions between Pax-6 and other transcription factors that are required for normal lens development and transparency. Pax-6, a paired domain and homeodomain containing transcription factor, is essential for lens formation and function. Mutations in Pax-6 cause several human ocular diseases: aniridia, Peter's anomaly, autosomal dominant keratitis, foveal hypoplasia, and some forms of cataract. These defects lead to loss of vision. It is recognized that lens refraction and transparency depend on the accumulation of high concentrations and short-range interaction of multifunctional proteins, the crystallins. Recent in vivo studies demonstrated that missexpression of crystallins can lead to lens opacification resulting in cataracts. In order to carry out this long term goal of this project the following specific aims are proposed,-. (1) To identify and functionally characterize general trancription factors that interact with and mediate the transcriptional activity of Pax-6; (2) To identify and functionally characterize other DNA binding and non-DNA binding transcription factors that synergistically interact with Pax-6.
These specific aims will be achieved using an integrative approach involving functional in vitro (transcription using recombinant factors and nuclear extracts) and in vivo (transfections of cultured lens and non-lens cells) studies combined with protein-DNA binding assays using recombinant proteins and lens nuclear extracts, and protein-protein interactions studied in solution and on a solid matrix. The feasibility of the proposed study is demonstrated by preliminary data demonstrating those lens crystallin genes which are targets for Pax-6, identifying those other transcription factors that act in conjunction with Pax-6 (e.g. retinoic acid activated nuclear receptors), and by demonstrating the function of crystallin promoters fused to reporter genes in both transfected lens cells and in transgenic mice. Collectively, these studies form the basis for a functional model of Pax-6 mediated gene regulation that applies to both crystallin gene regulation and noncrystallin genes expressed in the lens.
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