The otd/Otx gene family encodes paired-like homeodomain proteins that are involved in the regulation of anterior head structure and sensory organ development. Using the yeast one-hybrid screen with a bait containing the Ret 4 site from the bovine rhodopsin promoter, we have cloned a new member of the family, Crx (Cone rod homeobox). Crx encodes a 299 amino acid residue protein with a paired-like homeodomain near its N terminus. In the adult, it is expressed predominantly in photoreceptors and pinealocytes. In the developing mouse retina, it is expressed by embryonic day 12.5 (E12.5). Recombinant Crx binds in vitro not only to the Ret 4 site but also to the Ret 1 and BAT-1 sites. In transient transfection studies, Crx transactivates rhodopsin promoter-reporter constructs. Its activity is synergistic with that of Nrl. Crx also binds to and transactivates the genes for several other photoreceptor cell-specific proteins (interphotoreceptor retinoid-binding protein, beta-phosphodiesterase, and arrestin). Human Crx maps to chromosome 19q13.3, the site of a cone rod dystrophy (CORDII). These studies implicate Crx as a potentially important regulator of photoreceptor cell development and gene expression and also identify it as a candidate gene for CORDII and other retinal diseases.

Crx is a novel paired-like homeodomain protein that is expressed predominantly in retinal photoreceptors and pinealocytes. Its gene has been mapped to chromosome 19q13.3, the site of a disease locus for autosomal dominant cone-rod dystrophy (CORDII). Analysis of the proband from a family with autosomal dominant CORD revealed an Arg41Trp substitution in the third residue of the CRX homeodomain. The sequence change cosegregated with the disease phenotype and was not detected in 247 normal controls. Recombinant CRX homeodomain containing the Arg41Trp substitution showed decreased DNA binding activity. Analysis of another 169 CORD probands identified three additional CRX sequence variations (Arg41Gln, Val242Met, and a 4 bp deletion in codons 196/7) that were not found among the controls. This data suggests that mutations in the CRX gene are associated with photoreceptor degeneration and that the Crx protein is necessary for the maintenance of normal cone and rod function.

In vitro DNA binding assays and transient transfection analysis with monkey kidney cells have implicated Nrl, a member of the Maf-Nrl subfamily of bZIP transcription factors, and the Nrl response element (NRE) in the regulation of rhodopsin expression. We have now further explored the role of the NRE and surrounding promoter elements. Using the yeast one-hybrid screen with integrated NRE and flanking DNA as bait, the predominant clone obtained was bovine Nrl. Recovery of truncated clones in the screen demonstrated that the carboxyl-terminal half of Nrl, which contains the basic and leucine zipper domains, is sufficient for DNA binding. To functionally dissect the rhodopsin promoter, transient expression studies with primary chick retinal cell cultures were performed. Deletion and mutation analyses identified two positive regulatory sequences: one between ؊40 and ؊84 base pairs (bp) and another between ؊84 and ؊130 bp. Activity of the ؊40 to ؊84 region was shown to be largely due to the NRE. On co-transfection with an NRL expression vector, there were 3-5-fold increases in the activity of rhodopsin promoter constructs containing an intact NRE but little or no effect with rhodopsin promoters containing a mutated or deleted NRE. Nrl was more effective than the related bZIP proteins, c-Fos and c-Jun, in stimulating rhodopsin promoter activity. The ؊84-to ؊130-bp region acted synergistically with the NRE to enhance both the level of basal expression and the degree of Nrl-mediated trans-activation. These studies support Nrl as a regulator of rhodopsin expression in vivo, identify an additional regulatory region just upstream of the NRE, and demonstrate the utility of primary retinal cell cultures for characterizing both the cis-acting response elements and trans-acting factors that regulate photoreceptor gene expression.