CHARGE syndrome‐associated proteins FAM172A and CHD7 influence male sex determination and differentiation through transcriptional and alternative splicing mechanisms

Abstract

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 Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway

“…Second, by deubiquitinating and consequent regulation of the levels of the protein Ppil4, our findings suggest that USP7 may regulate RNA splicing in neurons, which will be the subject of future studies. Our findings in interaction and quantitative TMT-proteomics analyses revealing that USP7 interacts with and regulates the abundance of small nuclear ribonucleoproteins, the RNA helicase protein Dhx, and the RNA regulatory protein Fam172a 37,38 , further support a role for USP7 in the control of RNA processing. Recent studies suggest that Ppil4 is recruited to active spliceosomes to facilitate catalytic activation of the spliceosome by positioning the key RNA helicase Prp2 downstream of the branching site adenosine of introns 39,40 .…”

FAM172A controls the nuclear import and alternative splicing function of AGO2

“…Yet, the exact molecular mechanism by which AGO2 regulates alternative splicing, and the role of FAM172A in this process, are not fully understood. Both AGO2 (Ameyar-Zazoua et al, 2012) and FAM172A (Belanger et al, 2018, Belanger et al, 2022) are known to interact with many chromatin- and spliceosome-associated proteins, suggesting a role in bridging both machineries (Batsche & Ameyar-Zazoua, 2015, Berube-Simard & Pilon, 2018). Our rapamycin data are consistent with this possibility (Fig.7), further suggesting that it is possible to circumvent the loss of either AGO2 or FAM172A by increasing the pool of available spliceosome-associated proteins.…”

“…Indeed, CK2 activity is known to be enhanced by multiple cellular stressors (Ampofo, Sokolowsky et al, 2013, Sayed, Kim et al, 2000, Watabe & Nakaki, 2011). Moreover, it is noteworthy that about half of the proteins (43%; 29/67 proteins) composing the stress-induced response complex (SIRC) associated with AGO2 in HEK293 cells (Castanotto et al, 2018) are also included in the published FAM172A interactome from N2a (Belanger et al, 2018) and/or PGR9E11 cells (Belanger et al, 2022). One protein identified as being of special significance in the SIRC is Nucleolin (Castanotto et al, 2018), which we further validated as interactor for both FAM172A and AGO2 in N2a cells using co-IP (Fig.S6D).…”

“…Our mechanistic studies further allowed us to propose a model whereby FAM172A – which contains a large ARB2 domain (Argonaute Binding protein 2) split in two halves by a bipartite nuclear localization signal (NLS) – helps to stabilize the chromatin-spliceosome interface, notably by interacting with CHD7 and AGO2 (Belanger et al, 2018). These studies also suggested that perturbation of chromatin-mediated alternative splicing is a general pathological mechanism for CHARGE syndrome, regardless of involved gene defect (Belanger et al, 2018, Belanger, Cardinal et al, 2022, Berube-Simard & Pilon, 2018).…”

“…Yet, the exact molecular mechanism by which AGO2 regulates alternative splicing, and the role of FAM172A in this process, are not fully understood. Both AGO2 (Ameyar-Zazoua et al, 2012) and FAM172A (Belanger et al, 2018, Belanger et al, 2022 are known to interact with many chromatin-and spliceosome-associated proteins, suggesting a role in bridging both machineries (Batsche & Ameyar-Zazoua, 2015.…”