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

Pablo Armas,Miguel L Allende,Ezequiel Margarit,Nora B Calcaterra,Valeria S Mouguelar,Nicholas S Foulkes

doi:10.1371/journal.pone.0063234

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.

Highlights

CNBP, formerly ZNF9, is a highly conserved nucleic acid chaperone [1] involved in proper organization of the zebrafish, chick, and mouse forebrain [2,3,4,5]
Lower levels of CNBP in DM2 muscle cells lead to a reduction of proteins of the translational apparatus, which results in an overall reduction of global protein synthesis [9,10]. sporadic inclusion body myositis (sIBM) is an inflammatory muscle disease characterized by abnormal accumulation of intra-muscle fiber aggregates composed mainly of amyloid precursor protein (APP) and b-amyloid peptide (Ab) [11]
Whole-genome Screening for CNBP Target Genes In order to identify CNBP DNA targets in the zebrafish and mouse genomes, screens were performed using a recently described assay based on libraries containing random genomic fragments upstream of the yeast URA3 gene in yeast cells [13]

Summary

Introduction

CNBP, formerly ZNF9, is a highly conserved nucleic acid chaperone [1] involved in proper organization of the zebrafish, chick, and mouse forebrain [2,3,4,5]. CNBP is involved in muscle human diseases, such as myotonic dystrophy type 2 (DM2, proximal myotonic myopathy, OMIM # 602668) [6] and the age-related sporadic inclusion body myositis (sIBM) [7]. DM2 is an autosomal dominant multisystemic disease caused by an expansion of intronic CCTG repeats in intron 1 of the human CNBP gene, which leads to a disruption of RNA metabolism in patients’ tissues by accumulation of untranslated CCUGn RNAs [8]. Lower levels of CNBP in DM2 muscle cells lead to a reduction of proteins of the translational apparatus, which results in an overall reduction of global protein synthesis [9,10]. The molecular mechanisms responsible for this reciprocal regulation have not been completely elucidated yet

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