This is a competitive revision application in response to the Notice NOT-OD-09-058 """"""""NIH Announces the Availability of Recovery Act Funds for Competitive Revision"""""""". Our funded R01 project (5R01 EY012543-09) investigates the role of the homeodomain transcription factor Crx in photoreceptor development and maintenance. Crx is a key member of the photoreceptor transcription factor network that regulates the transcription of many photoreceptor genes essential for rod/cone function. CRX mutations are linked in a dominant fashion to several forms of retinal degeneration affecting both cones and rods, but the mechanism that leads to photoreceptor disease is unclear. To begin to understand this mechanism, we previously proposed two specific aims to test our hypothesis that Crx acts by recruiting co-activators for chromatin remodeling.
In Aim 1, we proposed to determine the role of three Crx-interacting co- activators, using loxP/Cre-mediated conditional knockout in photoreceptors and ectopic expression studies.
In Aim 2, we proposed to determine the role of chromatin remodeling, including histone modifications and intra-chromosomal loop formation, in regulating opsin gene transcription. These two aims are expected to provide insight into Crx's mechanism of action, but they do not directly address how CRX mutations cause dominant disease. In this revision application, we propose to add Aim 3 to address this question directly as an extension of the first two aims. For this purpose, we have created two new mouse lines, each carrying a disease-causing point mutation, R90W or E168d2, knocked into the Crx locus. In contrast to heterozygous Crx knockout mice that do not develop dominant photoreceptor defects, mice carrying a single copy of either knocked-in allele show a delay in photoreceptor development. The homozygous mutants do not develop photoreceptor outer segments and are blind at P21. This is followed by rapid retinal degeneration, more severe than the phenotype of homozygous Crx knockout (Crx-/-) mice. We thus hypothesize that R90W or E168d2 mutation causes disease by producing a malfunctioning protein that has a dominant-negative effect on wild-type CRX and/or other target proteins. To test this hypothesis, we will first carefully characterize the phenotypes of heterozygous and homozygous mutant mice using morphological, biochemical and electrophysiological measures. We will then identify molecular targets of mutant CRX using genome-wide microarray and chromatin immunoprecipitation assays. Finally, based on the knowledge gained from our previous two aims and these two new subaims, we will analyze two best classes of candidate protein targets for their genetic interactions with mutant Crx. These approaches together will significantly advance our understanding of how the Crx regulatory network functions in photoreceptor gene expression, development, maintenance and diseases. It will also provide well-characterized new animal models that better model the pathogenesis of CRX- associated diseases and can be used for developing new therapeutic treatments.
Human CRX mutations cause three forms of photoreceptor disease in a dominant fashion, but the mechanisms are unknown. We have generated two new mouse lines, each carrying a disease-causing mutation knocked into the Crx locus. Both lines develop photoreceptor defects in the heterozygous and homozygous states. We hypothesize that these CRX mutations cause disease by a dominant-negative mechanism, and propose to test this hypothesis by thoroughly characterizing the phenotypes of the mutant mice using morphological, biochemical, and molecular genetics approaches. The outcome will establish these mouse lines as new animal models for investigating pathogenesis and treatments for CRX-linked diseases.
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