The cleft palate candidate gene BAG6 supports FoxO1 acetylation to promote FasL-mediated apoptosis during palate fusion

Cleft palate is a common craniofacial defect, which occurs when the palate fails to fuse during development. During fusion, the palatal shelves migrate towards the embryonic midline to form a seam. Apoptotic elimination of medial edge epithelium (MEE) cells along this seam is required for the completion of palate fusion.Whole exome sequencing (WES) of six Chinese cleft palate families was applied to identify novel cleft palate-associated gene variants. Palatal fusion and immunofluorescence studies were performed in a murine palatal shelf organ culture model. Gene and protein expression were analyzed by qPCR and immunoblotting in murine MEE cells during seam formation in vivo. Mechanistic immunoprecipitation studies were performed in murine MEE cells in vitro.WES identified Bcl-2 associated anthanogene 6 (BAG6) as a novel cleft palate-associated gene. In murine MEE cells, we discovered upregulation of Bag6 and the transcription factor forkhead box protein O1 (FoxO1) during seam formation in vivo. Using a palatal shelf organ culture model, we demonstrate that nuclear-localized Bag6 enhances MEE cell apoptosis by promoting p300's acetylation of FoxO1, thereby promoting transcription of the pro-apoptotic Fas ligand (FasL). Subsequent gain- and loss-of-function studies in the organ culture model demonstrated that FasL is required for Bag6/acFoxO1-mediated activation of pro-apoptotic Bax/caspase-3 signaling, MEE apoptosis, and palate fusion. Palatal shelf contact was shown to enhance Bag6 nuclear localization and upregulate nuclear acFoxO1 in MEE cells.These findings demonstrate that nuclear-localized Bag6 and p300 co-operatively enhance FoxO1 acetylation to promote FasL-mediated MEE apoptosis during palate fusion.