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Alan Zhang, Nicholas Tran, Xiaodong Zhang, Shiming Chen; Distinct molecular mechanisms responsible for different retinopathies caused by CRX mutations DNH and LFH. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1580.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in the transcription factor CRX are associated with dominant retinopathies of varying severity in humans. We created two Knock-IN mouse models for CRX disease: Dominant-Negative High Expression (DNH) carries a 2-bp deletion at codon 168 resulting in a truncated CRX protein lacking the transactivation domains; Loss-of-function High Expression (LFH) carries an amino acid substitution, R90W in the DNA binding domain, compromising CRX’s ability to bind DNA. The Crx-DNH mouse models severe dominant Cone-Rod Dystrophy, while Crx-LFH models a recessive Leber Congenital Amaurosis. This study focuses on the distinct biochemical mechanisms by which the DNH and LFH proteins affect CRX target gene expression to cause distinct forms of CRX-associated disease.
Quantitative real time PCR (QRT-PCR) detected expression changes for selected CRX target genes in mutant vs WT retinas. Changes in DNA binding and transactivation activities of mutant vs WT CRX proteins were measured by retinal chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assays (EMSA), and Rhodopsin and Crx promoter dual luciferase reporter assays in transiently transfected HEK293 cells.
Crx-DNH/+ mice show severely decreased photoreceptor gene expression along with impaired rod and cone function and rapid retinal degeneration, while Crx-LFH/+ mice show normal retinal gene expression and function. Dual-luciferase assays revealed that neither LFH nor DNH protein can activate target gene expression in vitro and that DNH actively impairs WT CRX-mediated transactivation. EMSA showed DNH but not LFH binds DNA in vitro. Surprisingly, ChIP showed that DNH and LFH proteins both associate with the chromatin of CRX target genes in vivo. This supports our QRT-PCR findings that DNH/DNH cannot activate CRX target gene expression but LFH/LFH allows a low level of transcription.
DNH causes dominant disease by binding to DNA and interfering with WT CRX’s transactivation ability. LFH loses DNA binding and transactivation activity in vitro but can still associate with DNA in vivo to exert hypomorphic regulatory function. Crx DNH and LFH mice represent two mechanistically distinct models of CRX disease with vastly different pathologies, providing excellent models for testing novel therapeutic approaches.
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