The cone-rod homeobox (CRX) protein is a critical K50 homeodomain transcription factor responsible for the differentiation and maintenance of photoreceptor neurons in the vertebrate retina. Mutant alleles in the human gene encoding CRX result in a variety of distinct blinding retinopathies, including retinitis pigmentosa, cone-rod dystrophy, and Leber congenital amaurosis. Despite the success of using in vitro biochemistry, animal models, and genomics approaches to study this clinically relevant transcription factor over the past 25 years since its initial characterization, there are no high-resolution structures in the published literature for the CRX protein. In this study, we use bioinformatic approaches and small-angle X-ray scattering (SAXS) structural analysis to further understand the biochemical complexity of the human CRX homeodomain (CRX-HD). We find that the CRX-HD is a compact, globular monomer in solution that can specifically bind functional cis-regulatory elements encoded upstream of retina-specific genes. This study presents the first structural analysis of CRX, paving the way for a new approach to studying the biochemistry of this protein and its disease-causing mutant protein variants.

Roses are red, some eyes are blue, you wouldn’t see this without CRX too. The Cone‐Rod Homeobox protein or CRX is an essential transcription factor for the development of cells in the eye called photoreceptors. Photoreceptor cells (PRs) convert light into signals that the brain can understand. CRX plays a role in the differentiation of these photoreceptors into rods and cones, which detect achromatic and colored light, respectively. While there are details known about the molecular function of CRX, there is little known about the structural information of the protein. It is known that CRX contains a DNA binding domain (DBD) and two activation domains (ADs). Changes to the amino acid sequence in any of these regions are linked to a number of diseases associated with congenital blindness. We would like to better understand the structure and function of the AD and DBD to describe the regulation of photoreceptor development by CRX. We used Small‐Angle X‐Ray Scattering (SAXS) and DNA binding assays on the DBD showing that the DBD is globular and can bind to DNA in the absence of the activation domain. We crosslinked the DBD peptide to DNA with glutaraldehyde in an attempt to get a bound structure of the peptide‐DNA complex. The peptide‐DNA complex will show more accurately how the DBD binds to DNA and the stoichiometry of the complex. We are working to confirm preliminary findings suggesting a 2:1 DBD:DNA complex. These experiments will help us to understand the role that CRX plays in regulating transcriptional networks in PR neurons.

The cone-rod homeobox (CRX) protein is a critical K50 homeodomain transcription factor responsible for the differentiation and maintenance of photoreceptor neurons in the vertebrate retina. Mutant alleles in the human gene encoding CRX result in a variety of distinct blinding retinopathies, including retinitis pigmentosa, cone-rod dystrophy, and Leber congenital amaurosis. Despite the success of using in vitro biochemistry, animal models, and genomics approaches to study this clinically relevant transcription factor over the past 24 years since its initial characterization, there are no high-resolution structures in the published literature for the CRX protein. In this study, we use bioinformatic approaches and small-angle x-ray scattering (SAXS) structural analysis to further understand the biochemical complexity of the human CRX homeodomain (CRX-HD). We find that the CRX-HD is a compact, globular monomer in solution that can specifically bind functional cis-regulatory elements encoded upstream of retina specific genes. This study presents the first structural analysis of CRX, paving the way for a new approach to studying the biochemistry of this protein and its disease-causing mutant protein variants.