TREACHER-COLLINS-FRANCESCHETTI SYNDROME GENE 1 (TCOF1): REGULATION BY G-QUADRUPLEXES AND CNBP (CELLULAR NUCLEIC ACID BINDING PROTEIN).

GÓMEZ ZAMORANO, DENNIS; MOUGUELAR, VALERIA; CALCATERRA, NORA; DAVID, ALDANA; CALCATERRA, NORA; DAVID, ALDANA; COUX, GABRIELA; COUX, GABRIELA; ARMAS, PABLO; ARMAS, PABLO; GIL ROSAS, MAUCO; GIL ROSAS, MAUCO; GÓMEZ ZAMORANO, DENNIS; MOUGUELAR, VALERIA

Buenos Aires

Congreso; I Reunion Conjunta de Sociedades de Biociencias 2017; 2017

Treacher Collins Syndrome (TCS) is a congenital disease characterized by craniofacial defects. Over 90% of the cases are due to mutations in the TCOF1 gene, which codifies a nucleolar protein. We have reported a TCS model in zebrafish that fully recapitulates the craniofacial abnormalities observed in patients. Cnbp overexpression rescued the TCS-like phenotype in zebrafish, in a dose-dependent manner. A positive correlation between CNBP and TCOF1 expression in mesenchymal cells from both control and TCS subjects was found. Based on these data, we speculated a possible regulation of TCOF1 expression by Cnbp. Cnbp is a nucleic acid chaperone that interacts with and unfolds G-quadruplexes (G4) non-canonical nucleic acid structures. Consensus binding sites for Cnbp were detected both in Danio rerio (z2393) and Homo sapiens (h791 & h2160) Tcof1 promoters. These sites overlap with putative G-quadruplexes sequences (PQS). Synthetic single-stranded oligodeoxyribonucleotide sequences representing the human and zebrafish PQSs were used to assess in vitro whether they fold as G4 by Circular Dichroism (CD) and DNA intrinsic fluorescence. CDs and fluorescence performed on PQSs folded in the presence of 100 mM K+ showed spectra indicative of parallel topologies of G4. Addition of purified Cnbp decreased CD signals of z2393 and h2160 suggesting an interaction and unfolding. Quenching of Cnbp tryptophan fluorescence by both oligos also suggested such interactions (Kd in Nm range). ChIP analysis in zebrafish embryos confirmed binding of Cnbp to the tcof1 promoter in vivo. RT-qPCR experiments in zebrafish embryo depleted or overexpressing Cnbp showed altered expression of Tcof1. Moreover, Hela cells treated with siRNA to knock-down CNBP showed decreased TCOF1 expression measured by RT-qPCR. In conclusion, results suggest that Cnbp contributes to the TCOF1 expression by binding to G4 located in the promoter region and likely by unfolding these DNA secondary structures.