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

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

Praga

Congreso; G4thering - 6th International Meeting on Quadruplex Nucleic Acids.; 2017

6th International Meeting on Quadruplex Nucleic Acids.

Accuratespatiotemporal gene transcriptional regulation is essential for the success ofembryonic development and results from the combined action of cis- andtrans-regulatory elements. In a previous work (1) we found a set ofdevelopmentally regulated genes containing conserved G-quadruplexes (G4) withintheir proximal promoter regions. The folding and a positive transcriptionalregulatory role of those G4 were addressed by in vitro and in cellullostudies.  Moreover, their role in genetranscriptional regulation was confirmed invivo by G4-specific disruption through antisense oligonucleotides (ASO)microinjection in zebrafish embryos. This strategy led to reduction oftranscriptional levels of the analyzed genes and mimicked morphological changesreported for their loss-of-function. ASO disruption of the G4 involved incontrolling one of the studied genes, noggin3(nog3), caused craneofacialmalformation phenotypes consistent with nog3function in chondrogenic progenitor survival during zebrafish pharyngealdevelopment (2). The phenotype caused by nog3-ASOmicroinjection was fully rescued by co-injection with nog3-mRNA. Therefore we selected this gene for further studies. In silico analysis predicted that the G4controlling nog3 transcription (nog3-G4) overlapped with the bindingmotif of cellular nucleic acid binding protein (CNBP), a nucleic acid chaperoneprotein with preference for G-rich single stranded nucleic acids (3,4).Electrophoretic mobility shift assays showed that CNBP bound folded nog3-G4with lower affinity than the unfolded nucleic acid. Additionally, circulardicroism  and polymerase stop assaysestablished that CNBP was able to promote nog3-G4unfolding, in agreement with similar results obtained for CNBP with other G4.CNBP role on G4-mediated nog3transcriptional control was further addressed in vivo by overexpression of CNBP in zebrafish embryos throughmicroinjection of cnbp-egfp-mRNA. Embryosoverexpressing CNBP showed down-regulated nog3transcription consistent with CNBP G4-resolving role. In the same line ofevidence, co-injection of cnbp-egfp-mRNAwith nog3-ASO acted additively on nog3-G4 unfolding worsening thecraneofacial malformation phenotypes. These results provide strong evidence ofthe participation of a G4-resolving protein in the G4-mediated transcriptioncontrol of a developmental gene, and suggest that G-quadruplex modulation by anucleic acid chaperone may be a novel transcriptional regulatory mechanismadding complexity to strictly regulated biological processes such as embryonicdevelopment.