G-quadruplexes and Cellular Nucleic acid Binding Protein (CNBP) modulate TCOF1 (Treacher Collins Franceschetti 1) transcription

GIL ROSAS, MAUCO LUCAS; CENTOLA, CIELO L; MOUGUELAR, VALERIA S.; DAVID, ALDANA; PIGA, ERNESTO; GOMEZ ZAMORANO, DENNIS; CALCATERA, NORA B.; ARMAS P; COUX, G.

Rosario

Congreso; SAIB - SAMIGE Joint meeting 2021 on line; 2021

SAIB-SAMIGE

Treacher Collins Franceschetti Gene 1 (TCOF1) is involved in ribosomal RNA metabolism and is responsible for about 90% of mandibular dysostosis (MD) cases. Recently we identified a correlation in TCOF1 and CNBP (cellular nucleic acid binding protein, a nucleic acids chaperone involved in rostral development) expression in human mesenchymal cells. As CNBP is a transcriptional regulator of several genes, we investigated the possible modulation of TCOF1 expression by CNBP. Bioinformatic analysis yielded two CNBP consensus binding sites in TCOF1 promoter (Hs-791 and Hs-2160). The sites coincide with G-quadruplex (G4, stable secondary structures formed by G-rich sequences that are built around tetrads of Hoogsteen-type hydrogen-bonded guanine bases) putative forming sequences (PQS). We confirmed in vitro that synthetic oligonucleotides containing these PQS folded into G4 by circular dichroism and intrinsic fluorescence. EMSA analysis with purified CNBP confirmed binding to the target G4s with Kd values in the nM range. Also, spectroscopic studies suggested that CNBP acted as a G4-unfolding protein over Hs-2160 G4. ChIP studies in HeLa cells extracts detected that CNBP was bound to Hs-791 and Hs-2160 sites in TCOF1 promoter. HEK293 cell line expression studies revealed that Hs-2160 (but not Hs-791) PQS increased the transcription of luciferase controlled by the SV40 nonrelated promoter. Moreover, HEK293 cells treated with pyridostatin (a selective G4 stabilizing agent) showed increased endogenous TCOF1 mRNA expression. In zebrafish TCOF1 ortholog promoter we detected a site (Dr-2393) with equivalent properties to Hs-2160. G4 disruption in zebrafish embryos by microinjection of DNA oligonucleotides complementary to the G4 (antisense oligonucleotides or ASOs) resulted in decreased transcription of the tcof1 gene and larvae with phenotypes compatible with tcof1 knockdown. Finally, Morpholino-mediated cnbp knockdown in zebrafish induced tcof1 expression. The results gathered here suggest that TCOF1 transcriptional expression is modulated by CNBP through a mechanism involving G4 folding/unfolding. Also, that this regulation is active in vertebrates as distant as bonny fish and humans. These findings have implications in MD comprehension and treatment