Beyond the Binding Site: In Vivo Identification of tbx2, smarca5 and wnt5b as Molecular Targets of CNBP during Embryonic Development
Author
dc.contributor.author
Armas, Pablo
Author
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Margarit, Ezequiel
es_CL
Author
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Mouguelar, Valeria S.
es_CL
Author
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Allende Connelly, Miguel
es_CL
Author
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Calcaterra, Nora B.
es_CL
Admission date
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2014-02-05T13:54:07Z
Available date
dc.date.available
2014-02-05T13:54:07Z
Publication date
dc.date.issued
2013
Cita de ítem
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PLoS ONE 8(5): e63234
en_US
Identifier
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doi:10.1371/journal.pone.0063234
Identifier
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https://repositorio.uchile.cl/handle/2250/119757
General note
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Artículo de publicación ISI
en_US
Abstract
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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.