Validation of predicted CNBP target genes by in vitro and in vivo approaches.

ARMAS, P.; MARGARIT, E.; CALCATERRA, N. B.

Buenos Aires

Congreso; XLIX Annual Meeting of the Argentine Society for Biochemestry and Molecular Biology Research.; 2013

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 largely 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. Three genes were confirmed as CNBP targets in vitro and in vivo. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays followed by PCR (ChIP-PCR) showed that CNBP specifically binds to the consensus and to several consensus-like sequences present in the promoters of identified target genes. Finally, RT-qPCR and whole-mount in situ hybridizations performed on knocked-down zebrafish embryos confirmed tbx2b, wnt5b and smarca5 as a CNBP targets. In conclusion, we report the identification of three novel CNBP target genes functionally related in a regulatory network relevant for vertebrate embryonic development.