Missense mutations in CRX homeodomain cause dominant retinopathies through two distinct mechanisms

Homeodomain transcription factors (HD TFs) are instrumental to vertebrate development. Mutations in HD TFs have been linked to human diseases, but their pathogenic mechanisms remain elusive. Here we useCone-Rod Homeobox (CRX)as a model to decipher the disease-causing mechanisms of two HD mutations,p.E80Aandp.K88N, that produce severe dominant retinopathies. Through integrated analysis of molecular and functional evidencein vitroand in knock-in mouse models, we uncover two novel gain-of-function mechanisms:p.E80Aincreases CRX-mediated transactivation of canonical CRX target genes in developing photoreceptors;p.K88Nalters CRX DNA-binding specificity resulting in binding at ectopic sites and severe perturbation of CRX target gene expression. Both mechanisms produce novel retinal morphological defects and hinder photoreceptor maturation distinct from loss-of-function models. This study reveals the distinct roles of E80 and K88 residues in CRX HD regulatory functions and emphasizes the importance of transcriptional precision in normal development.