GUANINE QUADRUPLEXES AND EMBRYO DEVELOPMENT

MARGARIT, E; DAVID, A; DOMIZI, P; BANCHIO, C; ARMAS, P; CALCATERRA NB

Congreso; 50th Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2014

Guanine quadruplexes (G4) are stable secondary structures of single-stranded DNA or RNA associated with gene expression regulation. The goal of this work was to assess the role of G4 as transcriptional regulatory elements present in genes required for embryonic development. An in silico search to find out sequences with the potential to form G4 was performed on promoter regions of human, mouse and zebrafish genes. The analysis yielded 23 genes present in the three species containing potential G4 within their promoters and displaying development gene ontology associated terms. Among them, col2a1a, fzd5, and nog3 were selected for further studies. The G4 formation was determined in vitro both by ThT fluorescence and circular dichroism assays. The G4 regulatory role was assessed in cellulo by cloning the sequences upstream of a constitutive promoter that governs the expression of luciferase. G4 located in template (-) strand reduced the expression of luciferase while those located in coding (+) strand had no effect. Furthermore, microinjection of zebrafish embryos with DNA oligonucleotides complementary to the selected G4s resulted in transcriptional reduction, as measured by RT-qPCR and revealed by morphological changes accordingly. The results suggest the existence of conserved G4 structures displaying transcriptional regulatory roles during the embryonic development of vertebrates