CHARGE syndrome-which stands for coloboma of the eye, heart defects, atresia of choanae, retardation of growth/development, genital abnormalities, and ear anomalies-is a severe developmental disorder with wide phenotypic variability, caused mainly by mutations in (chromodomain helicase DNA-binding protein 7), known to encode a chromatin remodeler. The genetic lesions responsible for mutation-negative cases are unknown, at least in part because the pathogenic mechanisms underlying CHARGE syndrome remain poorly defined. Here, we report the characterization of a mouse model for mutation-negative cases of CHARGE syndrome generated by insertional mutagenesis of (family with sequence similarity 172, member A). We show that Fam172a plays a key role in the regulation of cotranscriptional alternative splicing, notably by interacting with Ago2 (Argonaute-2) and Chd7. Validation studies in a human cohort allow us to propose that dysregulation of cotranscriptional alternative splicing is a unifying pathogenic mechanism for both mutation-positive and mutation-negative cases. We also present evidence that such splicing defects can be corrected in vitro by acute rapamycin treatment.

To gain further insight into chromatin‐mediated regulation of mammalian sex determination, we analyzed the role of the CHARGE syndrome‐associated proteins FAM172A and CHD7. This study is based on our prior discoveries that a subset of corresponding mutant mice display complete male‐to‐female sex reversal, and that both of these proteins regulate co‐transcriptional alternative splicing in neural crest cells. Here, we report that FAM172A and CHD7 are present in the developing gonads when sex determination normally occurs in mice. The interactome of FAM172A in pre‐Sertoli cells again suggests a role at the chromatin‐spliceosome interface, like in neural crest cells. Accordingly, analysis of Fam172a‐mutant pre‐Sertoli cells revealed transcriptional and splicing dysregulation of hundreds of genes. Many of these genes are similarly affected in Chd7‐mutant pre‐Sertoli cells, including several known key regulators of sex determination and subsequent formation of testis cords. Among them, we notably identified Sry as a direct transcriptional target and WNT pathway‐associated Lef1 and Tcf7l2 as direct splicing targets. The identified molecular defects are also associated with the abnormal morphology of seminiferous tubules in mutant postnatal testes. Altogether, our results thus identify FAM172A and CHD7 as new players in the regulation of male sex determination and differentiation in mice, and further highlight the importance of chromatin‐mediated regulatory mechanisms in these processes.

The poorly characterized protein FAM172A is mutated in some individuals affected by a disorder of neural crest development called CHARGE syndrome. We also know that FAM172A can interact with the main CHARGE syndrome-associated protein CHD7 and the small RNA-binding protein AGO2 at the chromatin-spliceosome interface. Focusing on this intriguing FAM172A-AGO2 interaction, we now report that FAM172A is one of the long sought-after regulator of AGO2 nuclear import. This FAM172A function relies on its nuclear localization signal, being enhanced by CK2-mediated phosphorylation and abrogated by a CHARGE syndrome-associated missense mutation. Accordingly, Fam172a and Ago2 genetically interact in mice, and neural crest-specific depletion of Ago2 is sufficient to phenocopy CHARGE syndrome without impacting post-transcriptional gene silencing. Rapamycin-mediated rescue suggests that observed morphological anomalies are instead due to alternative splicing defects. This work thus demonstrates that non-canonical nuclear functions of AGO2 and associated regulatory mechanisms may be clinically relevant.

CHARGE syndrome is a neural crest-related disorder mainly caused by mutation of the chromatin remodeler-coding geneCHD7. Alternative causes include mutation of other chromatin and/or splicing factors. One of these additional players is the poorly characterized FAM172A, which we previously found in a complex with CHD7 and the small RNA-binding protein AGO2 at the chromatin–spliceosome interface. Focusing on the FAM172A–AGO2 interplay, we now report that FAM172A is a direct binding partner of AGO2 and, as such, one of the long sought-after regulators of AGO2 nuclear import. We show that this FAM172A function mainly relies on its classical bipartite nuclear localization signal and associated canonical importin-α/β pathway, being enhanced by CK2-induced phosphorylation and abrogated by a CHARGE syndrome-associated missense mutation. Overall, this study thus strengthens the notion that noncanonical nuclear functions of AGO2 and associated regulatory mechanisms might be clinically relevant.