CNBP, a nucleic acid chaperone protein, modulates the transcription of a gene involved in craneofacial development through G-quadruplex unfolding.

ARMAS, P.; WEINER, A.; CALCATERRA, N.B.; PASCUTTI, F.; GÓMEZ ZAMORANO, D.; DAVID, A.P.

Buenos Aires

Congreso; REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS; 2017

SOCIEDADES DE BIOCIENCIAS

In a previous work we found a set of developmental genes containing conserved G-quadruplexes (G4) in their promoters. The folding and transcriptional enhancer role of some of the identified G4 were addressed by in vitro and in cellulo studies, and further confirmed in vivo by G4-specific disruption through antisense oligonucleotides (ASO) microinjection in zebrafish embryos. ASO disruption of noggin3 (nog3, one of the studied genes) caused craniofacial malformation phenotypes consistent with nog3 function in pharyngeal development. In this work, an in silico analysis predicted that the G4 controlling noggin transcription (nog3-G4 for the zebrafish gene and NOG-G4 for the human gene) overlapped with the binding motif of cellular nucleic acid binding protein (CNBP). CNBP is a nucleic acid chaperone protein with preference for G-rich single stranded nucleic acids, reported as promoting cell proliferation and involved in craniofacial embryonic development. Electrophoretic mobility shift assays showed that CNBP bound to folded nog3-G4 and NOG-G4 with lower affinity than to the unfolded nucleic acids. Circular dichroism and polymerase stop assays revealed that CNBP promotes the unfolding of nog3-G4 and NOG-G4, in agreement with similar results obtained for DNA and RNA-G4 reported to control the expression of a set of proto-oncogenes. ChIP performed in HEK293 cells expressing CNBP-EGFP demonstrated the interaction of CNBP with the region containing NOG-G4. Furthermore, CNBP siRNA knockdown in HeLa cells led to an increased NOG transcription when compared with controls. In agreement, CNBP overexpression in zebrafish embryos caused a down-regulation of nog3 transcription in vivo. Results suggest that CNBP role in craniofacial embryonic development and cell proliferation is performed, at least in part, by resolving G4-DNA secondary structures. Besides, results demonstrate the existence of G4-DNA in vivo and their role in the transcriptional regulation of vertebrate genes.