Novel transcriptional regulatory mechanism for a gene involved in craniofacial development: G-quadruplex modulation by a nucleic acid chaperone.

ALDANA DAVID; FEDERICO PASCUTTI; NORA CALCATERRA; ANDREA WEINER; PABLO ARMAS

Praga

Congreso; 6th International Meeting on Quadruplex Nucleic Acids; 2017

Accurate spatiotemporal gene transcriptional regulation is essential for the success of embryonic development and results from the combined action of cis- and trans-regulatory elements. In a previous work (1) 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 (2). The phenotype caused by nog3-ASO microinjection was fully rescued by co-injection with nog3-mRNA. Therefore we selected this gene for further studies. In silico analysis predicted that the G4 controlling nog3 transcription (nog3-G4) overlapped with the binding motif of cellular nucleic acid binding protein (CNBP), a nucleic acid chaperone protein with preference for G-rich single stranded nucleic acids (3,4). Electrophoretic mobility shift assays showed that CNBP bound folded nog3-G4 with lower affinity than the unfolded nucleic acid. Additionally, circular dicroism and polymerase stop assays established that CNBP was able to promote nog3-G4 unfolding, in agreement with similar results obtained for this protein with other G4. CNBP role on G4-mediated nog3 transcriptional control was further addressed in vivo by overexpression of CNBP in zebrafish embryos through microinjection of cnbp-egfp-mRNA. Embryos overexpressing CNBP showed down-regulated nog3 transcription consistent with CNBP G4-resolving role. In the same line of evidence, co-injection of cnbp-egfp-mRNA with nog3-ASO acted additively on nog3-G4 unfolding worsening the craneofacial malformation phenotypes. 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.