The long-term goal of this application is to elucidate the trancriptional control mechanisms that regulate the expression of aA-crystallin in the context of its locus. aA-crystallin is a lens-specific protein essential for normal lens transparency. Mutations in human aA-crystallin cause cataracts. Targeted deletion of the aA-crystallin gene in transgenic mouse likewise causes lens opacification. Expression of aA-crystallin occurs both in the lens epithelium (lens progenitor cells) and in lens fiber cells (terminally differentiated cells) and since it is up-regulated in the differentiating primary fibers it is an excellent marker for lens fiber cell differentiation. This study seeks to identify and characterize those regulatory regions controlling aA-crystallin expression in the lens epithelium and the lens fiber cells in vivo. In order to carry out this long-term goal the following specific aims are proposed: (1) To functionally delineate the mouse aA-crystallin in a transgenic mouse model using standardized and random integration sites, (2) To elucidate the temporal and spatial functions of evolutionary conserved distant control regions (DCRs) of the aA-crystallin locus in vivo, and (3) To map cis-regulatory sites and transcription factors interacting with the DCRs.
These aims will be achieved using an integrative approach involving transgenic mice, analyses of temporal and spatial gene expression patterns using a lac Z marker, and biochemical characterization of DNA-binding proteins interacting with the identified DCRs. Transgenic mice will be produced using the Recombinase Mediated Cassette Exchange, a method allowing insertion of a single copy of the transgene into a specific chromosomal site. The feasibility of the proposed study is supported by data demonstrating the presence of evolutionary conserved non-coding putative DCRs in the mouse and human aA-crystallin loci. These DCRs harbor """"""""enhancer-like"""""""" activities in transiently transfected lens cells. The long-range impact of this work is not only the elucidation of transcriptional regulation of aA-crystallin, but also collection of data that can be used for a rational design of tools used for transgenic studies in the lens to probe normal lens processes such as lens differentiation and maintenance, and to induce abnormal processes such as specific cataract models. ? ?
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