CNBP, A NUCLEIC ACID CHAPERONE PROTEIN, MODULATES THE TRANSCRIPTION OF A GENE INVOLVED IN CRANEOFACIAL DEVELOPMENT THROUGH G-QUADRUPLEX UNFOLDING.

DENNIS GÓMEZ ZAMORANO; ALDANA P. DAVID; PABLO ARMAS; ANDREA WEINER; NORA B CALCATERRA; FEDERICO PASCUTTI

Cuernavaca

Simposio; ICGEB-LAZEN "V Latin American Zebrafish Network Course and Symposium"; 2018

ICGEB

Background. In a previous work we found a set of developmentally regulated genes containing conserved G-quadruplexes (G4) within their proximal promoter regions. The folding and a positive transcriptional regulatory role of those G4 were addressed by in vitro and in cellullo studies. Moreover, their role in gene transcriptional regulation was confirmed in vivo by G4-specific disruption through antisense oligonucleotides (ASO) microinjection in zebrafish embryos. This strategy led to reduction of transcriptional levels of the analyzed genes and mimicked morphological changes reported for their loss-of-function. ASO disruption of the G4 involved in controlling one of the studied genes, noggin3 (nog3), caused craneofacial malformation phenotypes consistent with nog3 function in chondrogenic progenitor survival during zebrafish pharyngeal development. This phenotype was fully rescued by co-injection with nog3-mRNA. Therefore we selected this gene for further studies. Objectives. Analyze the interaction of the cellular nucleic acid binding protein (CNBP) with the G4 present in the proximal promoter region of the gene noggin and its consequences in the transcriptional control of this developmental gene. Methods and Results. 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 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 HeLa cells demonstrated the interaction of CNBP with the region containing NOG-G4. This technique was also performed in HEK293 cells expressing CNBP-EGFP and this result was confirmed. 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. Conclusions. These results provide strong evidence of the participation of a G4-resolving protein in the G4-mediated transcription control of a developmental gene, and suggest that G-quadruplex modulation by a nucleic acid chaperone may be a novel transcriptional regulatory mechanism adding complexity to strictly regulated biological processes such as embryonic development.