title: Beyond the binding site: in vivo identification of tbx2, smarca5 and wnt5b as molecular targets of CNBP during embryonic development.
abstract: CNBP is a nucleic acid chaperone implicated in vertebrate craniofacial development, as well as in myotonic dystrophy type 2 (DM2) and sporadic inclusion body myositis (sIBM) human muscle diseases. CNBP is highly conserved among vertebrates and has been implicated in transcriptional regulation; however, its DNA binding sites and molecular targets remain elusive. The main goal of this work was to identify CNBP DNA binding sites that might reveal target genes involved in vertebrate embryonic development. To accomplish this, we used a recently described yeast one-hybrid assay to identify DNA sequences bound in vivo by CNBP. Bioinformatic analyses revealed that these sequences are G-enriched and show high frequency of putative G-quadruplex DNA secondary structure. Moreover, an in silico approach enabled us to establish the CNBP DNA-binding site and to predict CNBP putative targets based on gene ontology terms and synexpression with CNBP. The direct interaction between CNBP and candidate genes was proved by EMSA and ChIP assays. Besides, the role of CNBP upon the identified genes was validated in loss-of-function experiments in developing zebrafish. We successfully confirmed that CNBP up-regulates tbx2b and smarca5, and down-regulates wnt5b gene expression. The highly stringent strategy used in this work allowed us to identify new CNBP target genes functionally important in different contexts of vertebrate embryonic development. Furthermore, it represents a novel approach toward understanding the biological function and regulatory networks involving CNBP in the biology of vertebrates.
authors.affiliation: Instituto de Biología Molecular y Celular de Rosario (IBR), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)-Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario (UNR), Ocampo y Esmeralda, (S2000FHQ) Rosario, Argentina.
authors.forename: Pablo
authors.initials: P
authors.lastname: Armas
authors.forename: Ezequiel
authors.initials: E
authors.lastname: Margarit
authors.forename: Valeria S
authors.initials: VS
authors.lastname: Mouguelar
authors.forename: Miguel L
authors.initials: ML
authors.lastname: Allende
authors.forename: Nora B
authors.initials: NB
authors.lastname: Calcaterra
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019295:Computational Biology; D016384:Consensus Sequence / Q000235:genetics; D004247:DNA / Q000378:metabolism; D047108:Embryonic Development / Q000235:genetics*; D054856:G-Quadruplexes; D018507:Gene Expression Regulation, Developmental; D015723:Gene Library; D053263:Gene Regulatory Networks / Q000235:genetics; D016678:Genome / Q000235:genetics; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016601:RNA-Binding Proteins / Q000378:metabolism*; D015203:Reproducibility of Results; D020825:T-Box Domain Proteins / Q000378:metabolism*; D051153:Wnt Proteins / Q000378:metabolism*; D000071818:Wnt-5a Protein; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics; D029961:Zebrafish Proteins / Q000378:metabolism*
chemical_list: D000251:Adenosine Triphosphatases; D004247:DNA; D016601:RNA-Binding Proteins; D020825:T-Box Domain Proteins; D051153:Wnt Proteins; D000071818:Wnt-5a Protein; D029961:Zebrafish Proteins; C499012:Cnbp protein, mouse; C480282:Wnt5a protein, zebrafish; C499019:cnbpa protein, zebrafish; C491461:tbx2a protein, zebrafish; C584808:Smarca5 protein, zebrafish