Missense mutations in CRX homeodomain cause dominant retinopathies through two distinct mechanisms [ChIP-seq]

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 use Cone-Rod Homeobox (CRX) as a model to decipher the disease-causing mechanisms of two HD mutations, p.E80A and p.K88N, that produce severe dominant retinopathies. Through integrated analysis of molecular and functional evidence in vitro and in knock-in mouse models, we uncover two novel gain-of-function mechanisms: p.E80A increases transactivation of canonical CRX target genes in developing photoreceptors; p.K88N alters 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 hinders 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. CRX ChIP-seq experiments were performed on developing mouse retinas (P14) to understand how homeodomain missense mutations, p.E80A, p.K88N, p.R90W, each affects CRX chromatin binding in homozygous mutant mouse retinas.