Missense mutations in CRX homeodomain cause dominant retinopathies through two distinct mechanisms [RNA-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. Bulk RNA-seq experiments were performed on developing (P10) and adult (P21) mouse retinas to uncover the transcriptome alterations resulted from CRX homeodomain missense mutations, p.E80A, p.K88N, p.R90W, in both heterozygous and homozygous mutant retinas.同源域转录因子（Homeodomain Transcription Factors, HD TFs）在脊椎动物发育过程中发挥关键调控作用。HD TFs的突变与人类疾病密切相关，但其致病机制仍不甚明确。本研究以视锥视杆同源盒（Cone-Rod Homeobox, CRX）为模型，解析两种可引发严重显性遗传性视网膜病变的HD突变——p.E80A与p.K88N的致病机制。通过整合体外实验与敲入小鼠模型中的分子与功能证据，本研究揭示了两种全新的功能获得性致病机制：p.E80A可增强发育阶段感光细胞内CRX经典靶基因的转录激活活性；p.K88N则改变CRX的DNA结合特异性，使其发生异位结合并严重扰乱CRX靶基因的表达模式。两种机制均可引发新型视网膜形态学缺陷，并阻碍感光细胞成熟，其表型与功能丧失型模型截然不同。本研究阐明了CRX同源域中E80与K88残基在其调控功能中的独特作用，并强调了转录精准性在正常发育进程中的重要意义。为揭示CRX同源域错义突变p.E80A、p.K88N及p.R90W在杂合与纯合突变小鼠视网膜中引发的转录组改变，本研究针对发育阶段（出生后第10天，P10）与成年阶段（出生后第21天，P21）的小鼠视网膜开展了批量RNA测序（Bulk RNA-seq）实验。