Fishing the molecular targets of CNBP during zebrafish development

EZEQUIEL MARGARIT; PABLO ARMAS; MIGUEL L ALLENDE; NORA B CALCATERRA

Rosario

Congreso; Latin American Zebrafish Network Meeting (LAZEN); 2012

LAZEN-Latin American Zebrafish Network

CNBP is a small and highly conserved single-stranded nucleic acid binding protein required for proper craniofacial development in zebrafish, mouse and chicken. CNBP is capable of chaperoning the formation of guanine quadruplexes (G4) which are related to gene expression regulation. Although biochemical and biological data is available for CNBP, its targets remain unknown. Using yeast inverse one-hybrid assays, we rescued and sequenced a total of 85 and 207 clones from zebrafish and mouse genomic DNA libraries, respectively. This resulted in a total of 11 unique clones from zebrafish and 85 from mouse which led us to identify a consensus motif of 14 nucleotides highly enriched in guanines. In addition, clones had a 2-fold higher G4 frequency in comparison to their corresponding genomes. In silico analyses showed that sequences bound by CNBP mapped inside or around 233 genes. A subset of 155 were further studied regarding available gene ontology (GO) and gene co-expression data. A final list of 5 genes was retrieved (tbx2, wnt5b, asph, smarca5, and col10a1). Zebrafish embryos were micro-injected with Morpholinos against CNBP and were analyzed both by real time qRT-PCR and whole-mount in situ hybridization. Three genes (smarca5, tbx2b, wnt5b) showed changes in their mRNA levels pointing them as novel CNBP gene targets. This work represents a novel approach toward understanding the biological function and regulatory networks involving CNBP in the biology of vertebrates.