Abstract
Cone-rod homeobox (CRX) protein is a “paired-like” homeodomain transcription factor that is essential for regulating rod and cone photoreceptor transcription. Mutations in human CRX are associated with the dominant retinopathies Retinitis Pigmentosa (RP), Cone-Rod Dystrophy (CoRD) and Leber Congenital Amaurosis (LCA), with variable severity. Heterozygous Crx Knock-Out (KO) mice (“+/−”) have normal vision as adults and fail to model the dominant human disease. To investigate how different mutant CRX proteins produce distinct disease pathologies, we generated two Crx Knock-IN (K-IN) mouse models: CrxE168d2 (“E168d2”) and CrxR90W (“R90W”). E168d2 mice carry a frameshift mutation in the CRX activation domain, Glu168del2, which is associated with severe dominant CoRD or LCA in humans. R90W mice carry a substitution mutation in the CRX homeodomain, Arg90Trp, which is associated with dominant mild late-onset CoRD and recessive LCA. As seen in human patients, heterozygous E168d2 (“E168d2/+”) but not R90W (“R90W/+”) mice show severely impaired retinal function, while mice homozygous for either mutation are blind and undergo rapid photoreceptor degeneration. E168d2/+ mice also display abnormal rod/cone morphology, greater impairment of CRX target gene expression than R90W/+ or +/− mice, and undergo progressive photoreceptor degeneration. Surprisingly, E168d2/+ mice express more mutant CRX protein than wild-type CRX. E168d2neo/+, a subline of E168d2 with reduced mutant allele expression, displays a much milder retinal phenotype, demonstrating the impact of Crx expression level on disease severity. Both CRX[E168d2] and CRX[R90W] proteins fail to activate transcription in vitro, but CRX[E168d2] interferes more strongly with the function of wild type (WT) CRX, supporting an antimorphic mechanism. E168d2 and R90W are mechanistically distinct mouse models for CRX-associated disease that will allow the elucidation of molecular mechanisms and testing of novel therapeutic approaches for different forms of CRX-associated disease.
Highlights
Cone-rod homeobox (CRX) (Accession: AAH53672.1) is an Otd/OTX-like ‘paired’ homeodomain transcription factor that is preferentially expressed in vertebrate rod and cone photoreceptor cells in the retina and pinealocytes in the brain [1,2]
CrxE168d2 (‘‘E168d2’’) mice carry a 2-bp deletion mutation, Glu168del2, which resulted in a codon frameshift and early truncation of the transactivation domains of CRX protein (Figure 1A–C)
Successful K-IN was confirmed by PCR amplification of neo (Primer set: Neo F/R) and the respective Crx allele (Table S1, Figure S1) and Sanger sequencing of homozygous mice (Figure 1C)
Summary
CRX (Accession: AAH53672.1) is an Otd/OTX-like ‘paired’ homeodomain transcription factor that is preferentially expressed in vertebrate rod and cone photoreceptor cells in the retina and pinealocytes in the brain [1,2]. The C terminal region of CRX (from the basic to the OTX-tail domains) is required for transactivation activity [4]. CRX interacts with transcription co-regulators including the rodspecific transcription factors NRL (Accession: NP_006168.1) [11][12], NR2E3 (Accession: AAH41421.1) [13][14], and general co-activator proteins GCN5, CBP and p300 (Accessions: AAC50641.1, AAC17736.1, NP_001420.2, respectively) [7] to coordinately control photoreceptor gene expression. In the homozygous Crx Knock-Out mouse (‘‘2/2’’), photoreceptors fail to form outer segments (OS), a highly specialized photoreceptor organelle which contains visual pigment opsins and other proteins required for phototransduction [15][16]. Gene expression profile studies showed that 2/2 mice have severely reduced
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