Two key transcription factors, homeodomain protein CRX and basic leucine zipper protein NRL, are at the center of gene regulation during photoreceptor differentiation and homeostasis. CRX is essential for specifying commitment of postmitotic photoreceptor precursors to the development of photoreceptor cells, whereas NRL determines the rod cell fate. In orchestrating the transcriptional program of photoreceptor development, CRX and NRL cooperate functionally and physically via a direct protein-protein interaction. Defects in photoreceptor transcriptional regulation due to mutations in the genes encoding CRX and NRL cause severe retinal diseases including retinitis pigmentosa, cone-rod dystrophy, and Leber congenital amaurosis. Despite our advanced understanding of the biology and transcriptional networks of CRX and NRL, mechanistic insight into the functions and unique synergy of these transcription factors at the atomic level is lacking. In the proposed studies, we seek to determine the crystal and solution structures of the individual DNA-bound complexes of CRX and NRL, as well as the structure of the ternary CRX/NRL/DNA complex. Although mutations in these TFs have been identified, they have not been mechanistically linked to regulation of key genes. The mechanistic predictions from the structures on how disease-causing mutations in CRX and NRL may alter DNA-binding specificity at cis-regulatory elements will be validated in the follow-up assays, including high- throughput approaches such as Spec-seq. These studies will enhance our knowledge of the functions of CRX and NRL, define the molecular nature of their synergy, and allow us to delineate specific mechanisms whereby mutant CRX and NRL proteins cause retinal diseases. We hypothesize that ultimately the structures of CRX and NRL complexed with their cis-regulatory elements will enable targeted design of therapeutics to treat visual disorders via modulation of transcriptional activities at specific promoters.