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

GIL ROSAS, MAUCO S; MOUGUELAR, VALERIA S.; DAVID, ALDANA ; GOMEZ ZAMORANO, DENNIS; ARMAS PABLO; CALCATERRA, NORA B; COUX, GABRIELA

Buenos Aires

Congreso; REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS; 2017

Sociedades Biociencias

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 expressionin 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.