Pause-conférence | Les membres du CERMO-FC

Pause-conférence | Les membres du CERMO-FC

Finely regulated FAM172A-AGO2 interaction is key for co-transcriptional alternative splicing in the context of CHARGE syndrome.

Séphora Sallis, candidate au doctorat dans le laboratoire du Pr Nicolas Pilon à l’UQAM

Résumé: CHARGE Syndrome is a rare developmental disorder which affects 1/ 10 000 newborns and has a very complex clinical presentation. Heterozygous mutation of CHD7 (Chromodomain helicase DNA-binding domain) is currently the main genetic cause of CHARGE syndrome. This condition belongs to the neurocristopathies family (i.e., diseases of neural crest cell development), but underlying molecular mechanisms are not well understood. The Pr. Nicolas PILON’s Laboratory identified FAM172A as a new candidate gene for CHARGE syndrome. Preliminary studies revealed that FAM172A could regulate the co-transcriptional alternative splicing machinery, through interaction with Argonaute 2 (AGO2), CHD7, DNA and RNA.
BiFC (Bimolecular Fluorescence Complementation) experiments confirmed a direct interaction between FAM172A and AGO2, that starts in the cytoplasm and may translocate into the nucleus, where AGO2 is implicated in alternative splicing events. In addition, mass spectrometry studies identified a potential phosphorylation site on FAM172A by the Casein Kinase II. Preliminary studies suggest that the translocation of FAM172A-AGO2 into nuclei is facilitated by the phosphorylation of FAM172A. Moreover, the functional significance of this interaction was validated by genetic interaction in mice. Indeed, by breeding Fam172aTp/Tp mice (hypomorphic allele of Fam172a) with a model where Ago2 is specifically Knocked Out (KO) in neural crest cells, we observed more severe CHARGE syndrome-like phenotypes.
Our current working model is that perturbations of FAM172A-mediated translocation of AGO2 into the nucleus lead to global dysregulation of transcription and alternative splicing in neural crest cells, thereby causing CHARGE syndrome.

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Characterizing the role of Chd7 in GABA network development- CHARGE syndrome and beyond

Priyanka Jamadagni, candidate au doctorat dans le laboratoire du Pr Kessen Patten à l’INRS

Résumé: Mutations in the ATP-dependent chromatin remodeller chromodomain, helicase, DNA binding (CHD) 7 are the primary cause of CHARGE syndrome and have been associated with autism spectrum disorders (ASDs). However, the mechanisms by which mutations in CHD7 affect brain development and function that underlines the neurological deficits observed in CHARGE patients are poorly understood. To study this, employing the suitability of Zebrafish for the study of early neurodevelopment, we have developed a zebrafish chd7 CRISPR/Cas9 knockout model. chd7 knockout zebrafish larvae exhibit characteristic CHARGE features like a small head phenotype, defects in craniofacial cartilage development and displayed hyperactivity particularly during the dark phase of the light-dark cycle. We found this hyperactivity, is because of a defect in the number of GABAergic neurons and possibly their function, in the chd7 mutants. Further, using an unbiased whole transcriptomic approach, we identified genes involved in various biological processes and molecular pathways are dysregulated in the chd7 mutants. Particularly, a CHD7 target gene paqr3b is highly dysregulated and the MAPK/ERK signaling is upregulated in the chd7 mutants. An RT-qPCR analysis showed that PAQR3 gene expression was also highly decreased in CHD7 mutation-positive patient derived lymphoblastoid cell lines (LCLs) s compared to control LCLs. We further identified a small molecule via high throughput drug screen, that is able to restore MAPK/ERK signalling and improve both GABAergic defects and behavioural anomalies. Our findings thus indicate loss of chd7 results in a deficit of inhibitory neurons via a potentially new pathway, suggesting an essential role of chd7 in the brain neuronal network development.

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