Modeling CHARGE Syndrome with Human Inner Ear Organoids

PROJECT SUMMARY CHARGE syndrome is a congenital disorder characterized by dysmorphic features of inner ear structures, and caused primary by sporadic mutations in CHD7, a gene encoding an ATP-dependent chromatin remodeling enzyme. Results from our recent study revealed distinctive phenotypic differences between CHD7 KO and S834F mutant inner ear organoids, suggesting that CHD7 regulates inner ear development through multifaceted and context-dependent mechanisms. To investigate the genotype-phenotype relationship, we will establish human embryonic stem cell lines harboring the D1812G mutation in a putative WDR5-binding domain or the G1982W mutation in the SANT domain. Gene and protein expression profiles of D1812G and G1982W cochlear organoids will be compared with those of S834F organoids with abolished remodeling activity. Our previous results also revealed down-regulation of multiple deafness genes in CHD7-null otic progenitors. To address if down-regulation of known deafness genes could account for sensorineural hearing loss in individuals with CHARGE syndrome, we will test if these deafness genes are downregulated in hair cells of cochlear organoids carrying defective CHD7. Ultrastructural and functional properties of mutant hair cells will be compared to those of wild-type hair cells. Additionally, cell lineage tracing will be performed to characterize drifted otic lineage specification in mutant organoids. To elucidate the mechanisms underlying CHD7- dependent transcriptional regulation during inner ear development, we will determine CHD7-binding loci, their transcriptional activity states and chromatic accessibility in human otic progenitors through an integrated analysis of CUT&RUN, scATAC-seq and scRNA-seq data. A potential role for CHD7 in posttranslational histone modifications will be assessed by immunoprecipitation and ChIP-PCR. To assess possible contributions of CHD7-dependent cell non-autonomous factors to otic progenitor differentiation, we will employ a proteomic analysis of organoid conditioned media and test if some of the identified proteins can rescue defective otic differentiation in CHD7 KO organoids. The results obtained from the proposed experiments are expected to increase our understanding of the etiology of inner ear pathology in individuals with CHARGE syndrome and will shed valuable insights into the development of personalized clinical interventions.