Beyond the Binding Site: In Vivo Identification of tbx2, smarca5 and wnt5b as Molecular Targets of CNBP during Embryonic Development.

• Autores: Armas, Pablo; Margarit, Ezequiel; Mouguelar, Valeria Soraya; Allende, Miguel L.; Calcaterra, Nora Beatriz
• Año de publicación: 2013
• Idioma: inglés
• Tipo de recurso: artículo
• Materia: CNBP; Molecular Targets; Embryonic Development
• Institución: Consejo Nacional de Investigaciones Científicas y Técnicas

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.
Fil: Armas, Pablo. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Rosario. Instituto de Biologia Molecular y Celular de Rosario;
Fil: Margarit, Ezequiel. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Rosario. Instituto de Biologia Molecular y Celular de Rosario;
Fil: Mouguelar, Valeria Soraya. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Rosario. Instituto de Biologia Molecular y Celular de Rosario;
Fil: Allende, Miguel L.. FONDAP. Center for Genome Regulation. Facultad de Ciencias. Universidad de Chile. Santiago, Chile;
Fil: Calcaterra, Nora Beatriz. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Rosario. Instituto de Biologia Molecular y Celular de Rosario;