CONICET | Buscador de Institutos y Recursos Humanos

INVESTIGADORES

WEINER Andrea Maria Julia

congresos y reuniones científicas

Título:

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

Autor/es:

DAVID AP; AMJ WEINER; PASCUTTI F; CALCATERRA NB; ARMAS, P

Lugar:

Praga

Reunión:

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

Resumen:

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 we found a set of developmentally regulated genescontaining conserved G-quadruplexes (G4) within their proximal promoter regions.The folding and a positive transcriptional regulatory role of those G4 wereaddressed by in vitro and in cellullostudies. Moreover, their role in gene transcriptional regulation was confirmed in vivoby G4-specific disruption through antisenseoligonucleotides (ASO) microinjection in zebrafish embryos. This strategy ledto reduction of transcriptional levelsof the analyzed genes and mimicked morphologicalchanges reported for their loss-of-function. ASOdisruption of the G4 involved in controlling one of the studied genes, noggin3 (nog3), caused craneofacial malformation phenotypes consistentwith nog3 function in chondrogenic progenitorsurvival during zebrafish pharyngeal development. The phenotype caused by nog3-ASO microinjection was fullyrescued 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 acidbinding protein (CNBP), a nucleic acid chaperone protein with preference forG-rich single stranded nucleic acids. Electrophoretic mobility shift assaysshowed 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. CNBProle on G4-mediated nog3 transcriptionalcontrol was further addressed in vivo byoverexpression of CNBP in zebrafish embryos by microinjection of cnbp-egfp-mRNA. CNBP overexpressingembryos showed down-regulated nog3transcription consistent with CNBP G4-resolving role. In thesame line of evidence, co-injection of cnbp-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 mechanism addingcomplexity to strictly regulated biological processess such as embryonicdevelopment.

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