Treacher-Collins-Franceschetti Syndrome gene 1 (TCOF1) is regulated by CNBP and G-quadruplexes during zebrafish development

GIL ROSAS, MAUCO S; GOMEZ ZAMORANO, DENNIS; COUX, GABRIELA; DAVID, ALDANA; ARMAS PABLO; CENTOLA, CIELO L; CALCATERRA, NORA B

Buenos Aires

Congreso; LASDB Meeting 2019; 2019

Latin American Society for Developmental Biology

CNBP is a DNA/RNA chaperone involved in craniofacialdevelopment that interacts with and unfolds non-canonical nucleic acidstructures known as G-quadruplexes (G4). Recently, we found evidencessuggesting CNBP as a regulator of TCOF1 expression. TCOF1 is the causative gene in 90% ofTreacher Collins Syndrome (TCS) cases, a genetic disease characterized bycraniofacial defects. Therefore, our aim was to elucidate CNBP and G4 roles in TCOF1expression. Bioinformatic analysis indicated consensus binding sites for CNBPin TCOF1 promoters in Danio rerio (Dr2393) and Homo sapiens(Hs791 & Hs2160) in coincidence with putative G4 forming sequences.Biophysical studies confirmed that these sequences are able to fold as G4 andare specifically bound in vitro by CNBP both folded as G4 and unfolded. Invitro incubation of pre-folded Dr2393 and Hs2160 with CNBP induced G4unfolding. To assess CNBP role in TCOF1 transcriptional regulation, we performed CNBP knock-down in HeLacells by siRNA, showing a TCOF1 transcriptional reduction. Moreover, CNBPbinding to Dr2393 and Hs791 and Hs2160 was demonstrated by Chromatin-IP studiesin zebrafish embryos and HeLa cells, respectively. Finally, Antisenseoligonucleotides (ASO) microinjection disrupting Dr2393 G4 in zebrafish embryoscaused craniofacial phenotypes consistent with those produced by tcof1translation blocking Morpholino. Results suggest that TCOF1 expressionis modulated by CNBP through a complex interplay with G4 folding/unfolding.