TREACHER COLLINS FRANCESCHETTI GENE 1 (TCOF1), THE MAIN GENE ASSOCIATED WITH TREACHER-COLLINS SYNDROME, IS REGULATED BY CELLULAR NUCLEIC ACID BINDING PROTEIN (CNBP) THROUGH G-QUADRUPLEXES

GIL ROSAS, MAUCO LUCAS; CENTOLA, CIELO L; MOUGUELAR, VALERIA S.; DAVID, ALDANA; PIGA, ERNESTO; ARMAS PABLO; COUX, GABRIELA

Chascomus

Taller; V Taller de Biología Celular y del Desarrollo; 2022

INTECH

TCOF1 is responsible for about 80% of mandibulofacial dysostosis cases. Recently, we identified a correlation between TCOF1 and CNBP (a nucleic acids chaperone involved in G-quadruplex unfolding) expression in human cells. As CNBP has been indicated as a regulator of several genes during rostral development, our aim here was to examine the possible modulation of TCOF1 expression by CNBP. Computational analysis for the detection of CNBP binding sites yielded several sequences in TCOF1 putative promoter. Two of them (named Hs-791 and Hs-2160) overlapped with G-quadruplex putative forming sequences (PQS). We validated the folding of these PQS by biophysical approaches, and the addition of purified CNBP provoked unfolding of Hs-2160 (analyzed by circular dichroism signal). In vitro assays (EMSA) with purified CNBP and in vivo studies (chromosome immunoprecipitation) confirmed binding to both target PQS. Luciferase reporter plasmids were constructed by cloning either the PQS (ora mutated control version) upstream of the SV40 promoter. Plasmids were transfected in HEK-293 cells and luciferase activity was measured as a transcriptional reporter. Hs-2160 (but not Hs-791 or the mutated controls) increased the transcription of luciferase controlled by the SV40 nonrelated promoter. HEK-293 cells incubated with Pyridostatin, a G-quadruplex stabilizer, induced TCOF1 transcriptional expression. In the zebrafish TCOF1 orthologue promoter, we detected a site (Dr-2393). with comparable properties to Hs-2160. G-quadruplex disruption in zebrafish embryos by microinjection of DNA oligonucleotides complementary to Dr-2393 resulted in decreased transcription of the tcof1 gene and altered cranial morphology. These results suggest that TCOF1 transcriptional expression is modulated by CNBP through a mechanism involving G4 folding/unfolding