Validation of predicted CNBP targets by in vitro and in vivo approaches

PABLO ARMAS; EZEQUIEL MARGARIT; NORA B CALCATERRA

Capital Federal

Congreso; 49th Annual Meeting Argentine Society for Biochemistry and Molecular Biology.; 2013

SAIB - Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Cellular Nucleic acid Binding Proteins (CNBP) is a highly conserved single-stranded nucleic acid binding chaperone. Although CNBP is essential for craniofacial development, its molecular targets are unknown. Monohybrid assays identified mouse and zebrafish genomic sequences as putative CNBP targets. Bioinformatic analyses revealed a G-enriched 14-nucleotide consensus binding site (NGGGGG(A/T)GGGGGGN) and predicted a reduced list of CNBP putative targets based on gene ontology terms and synexpression with CNBP. Here, we validated CNBP regulatory function on some putative target genes by studying their expression changes in CNBP knocked-down zebrafish embryos treated with specific morpholino. Among the tested genes, three of them (tbx2b, wnt5b and smarca5) were confirmed as CNBP targets in vitro and in vivo. Indeed, electrophoretic mobility shift assays and ChIP showed that CNBP specifically binds to the consensus and consensus-like sequences present in the promoters of identified genes and, RT-qPCR and whole-mount in situ hybridizations on knocked-down zebrafish embryos revealed changes in transcript abundance of the three candidate genes. In conclusion, we report the identification of three novel CNBP target genes functionally related in a regulatory network relevant for vertebrate embryonic development.