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Cellular nucleic-acid-binding protein, a transcriptional enhancer of c-Myc, promotes the formation of parallel G-quadruplexes

G-rich sequences that contain stretches of tandem guanines can form four-stranded, intramolecular stable DNA structures called G-quadruplexes (termed G4s). Regulation of the equilibrium between single-stranded and G4 DNA in promoter regions is essential for control of gene expression in the cell. G4s are highly stable structures; however, their folding kinetics are slow under physiological conditions. CNBP (cellular nucleic-acid-binding protein) is a nucleic acid chaperone that binds the G4-forming G-rich sequence located within the NHE (nuclease hypersensitivity element) III of the c-Myc proto-oncogene promoter. Several reports have demonstrated that CNBP enhances the transcription of c-Myc in vitro and in vivo; however, none of these reports have assessed the molecular mechanisms responsible for this control. In the present study, by means of Taq polymerase stop assays, electrophoretic mobility-shift assays and CD spectroscopy, we show that CNBP promotes the formation of parallel G4s to the detriment of anti-parallel G4s, and its nucleic acid chaperone activity is required for this effect. These findings are the first to implicate CNBP as a G4-folding modulator and, furthermore, assign CNBP a novel mode-of-action during c-Myc transcriptional regulation.

CNBP: A multifunctional nucleic acid chaperone involved in cell death and proliferation control

Cellular nucleic acid binding protein (CNBP) has been implicated in vertebrate craniofacial development and in myotonic dystrophy type 2 (DM2) and sporadic inclusion body myositis (sIBM) human diseases. In these seemingly unrelated biological processes, CNBP appears to be involved in controlling cell death and proliferation rates. Low levels of CNBP may reduce rate of global protein synthesis, thereby reducing proliferation and increasing apoptosis. Conversely, CNBP might affect transcription of genes required for cell proliferation. Experimental evidences gathered so far make it difficult to ascertain or rule out any of these possibilities. Moreover, both possibilities may not be mutually exclusive. CNBP is a small and strikingly conserved single‐stranded nucleic acid binding protein that is able to bind DNA as well as RNA. CNBP has a broad spectrum of targets, ranging from regulatory sites in gene promoters to translational regulatory elements in mRNA untranslated regions. Biochemical experiments have recently shed light on the possible mechanism of action for CNBP, which may act as a nucleic acid chaperone catalyzing the rearrangement of G‐rich nucleic acid secondary structures likely relevant for transcriptional and/or translational gene regulation. This review focuses on the involvement of CNBP in vertebrate craniofacial development and human DM2 and sIBM diseases, as well as on the biochemical and structural features of CNBP and its cellular and molecular mechanism of action.

Cellular nucleic acid binding protein binds G‐rich single‐stranded nucleic acids and may function as a nucleic acid chaperone

Cellular nucleic acid binding protein (CNBP) is a small single‐stranded nucleic acid binding protein made of seven Zn knuckles and an Arg‐Gly rich box. CNBP is strikingly conserved among vertebrates and was reported to play broad‐spectrum functions in eukaryotic cells biology. Neither its biological function nor its mechanisms of action were elucidated yet. The main goal of this work was to gain further insights into the CNBP biochemical and molecular features. We studied Bufo arenarum CNBP (bCNBP) binding to single‐stranded nucleic acid probes representing the main reported CNBP putative targets. We report that, although bCNBP is able to bind RNA and single‐stranded DNA (ssDNA) probes in vitro, it binds RNA as a preformed dimer whereas both monomer and dimer are able to bind to ssDNA. A systematic analysis of variant probes shows that the preferred bCNBP targets contain unpaired guanosine‐rich stretches. These data expand the knowledge about CNBP binding stoichiometry and begins to dissect the main features of CNBP nucleic acid targets. Besides, we show that bCNBP presents a highly disordered predicted structure and promotes the annealing and melting of nucleic acids in vitro. These features are typical of proteins that function as nucleic acid chaperones. Based on these data, we propose that CNBP may function as a nucleic acid chaperone through binding, remodeling, and stabilizing nucleic acids secondary structures. This novel CNBP biochemical activity broadens the field of study about its biological function and may be the basis to understand the diverse ways in which CNBP controls gene expression. J. Cell. Biochem. 103: 1013–1036, 2008.

Dissecting CNBP, a Zinc-Finger Protein Required for Neural Crest Development, in Its Structural and Functional Domains

Cellular nucleic-acid-binding protein (CNBP) plays an essential role in forebrain and craniofacial development by controlling cell proliferation and survival to mediate neural crest expansion. CNBP binds to single-stranded nucleic acids and displays nucleic acid chaperone activity in vitro. The CNBP family shows a conserved modular organization of seven Zn knuckles and an arginine-glycine-glycine (RGG) box between the first and second Zn knuckles. The participation of these structural motifs in CNBP biochemical activities has still not been addressed. Here, we describe the generation of CNBP mutants that dissect the protein into regions with structurally and functionally distinct properties. Mutagenesis approaches were followed to generate: (i) an amino acid replacement that disrupted the fifth Zn knuckle; (ii) N-terminal deletions that removed the first Zn knuckle and the RGG box, or the RGG box alone; and (iii) a C-terminal deletion that eliminated the three last Zn knuckles. Mutant proteins were overexpressed in Escherichia coli, purified, and used to analyze their biochemical features in vitro, or overexpressed in Xenopus laevis embryos to study their function in vivo during neural crest cell development. We found that the Zn knuckles are required, but not individually essential, for CNBP biochemical activities, whereas the RGG box is essential for RNA-protein binding and nucleic acid chaperone activity. Removal of the RGG box allowed CNBP to preserve a weak single-stranded-DNA-binding capability. A mutant mimicking the natural N-terminal proteolytic CNBP form behaved as the RGG-deleted mutant. By gain-of-function and loss-of-function experiments in Xenopus embryos, we confirmed the participation of CNBP in neural crest development, and we demonstrated that the CNBP mutants lacking the N-terminal region or the RGG box alone may act as dominant negatives in vivo. Based on these data, we speculate about the existence of a specific proteolytic mechanism for the regulation of CNBP biochemical activities during neural crest development.

Primary structure and developmental expression of Bufo arenarum cellular nucleic acid‐binding protein: Changes in subcellular localization during early embryogenesis

A Bufo arenarum cellular nucleic acid‐binding protein (bCNBP) full‐length cDNA was cloned. bCNBP is a 19.4 kDa protein containing seven CCHC zinc finger motifs, an RGG box and a Ser‐rich region. Amino acid comparisons showed high values of homology in vertebrates and smaller values in insects or inferior eukaryotes. Northern blot analysis during oogenesis and early development revealed two transcripts with different expressions of pattern behavior. One of them is present in all stages analyzed, whereas the other is only detected from the beginning of zygotic transcription. Immunocytochemistry assays carried out on sections of ovary and early embryos showed that there was no specific staining of previtellogenic oocytes. In early vitellogenic oocytes, in oocytes at stages V/VI and in embryos at early blastula stage, reaction was observed inside the cytoplasm. At mid‐blastula stage, CNBP was mainly detected in the epiblast. At the late gastrula stage, two layers of cells were stained in the archenteron roof, in which the internal one presented as strong staining. Nuclei in this layer were stained even stronger than the cytoplasm. Changes in mRNA expression patterns, accompanied by changes in subcellular localization, suggest that CNBP might interact with both nuclear and cytoplasmic nucleic acids.

Set-up of an infrared fast behavioral assay using zebrafish (Danio rerio) larvae, and its application in compound biotoxicity screening.

Zebrafish (Danio rerio) is increasingly employed for evaluating toxicity and drug discovery assays. Commonly experimental approaches for biotoxicity assessment are based on visual inspection or video recording. However, these techniques are limited for large‐scale assays, as they demand either a time‐consuming detailed inspection of the animals or intensive computing resources in order to analyze a considerable amount of screenshots. Recently, we have developed a simple methodology for tracking the locomotor activity of small animals cultured in microtiter plates. In this work, we implemented this automatic methodology, based on infrared (IR) microbeam scattering, for measuring behavioral activity in zebrafish larvae. We determined the appropriate culture conditions, number of animals and stage of development to get robust results. Furthermore, we validated this methodology as a rapid test for evaluating toxicity. By measuring the effects of reference compounds on larvae activity, we were able to estimate the concentration that could cause a 50% decrease in activity events values (AEC50), showing a strong linear correlation (R2 = 0.91) with the LC50 values obtained with the standard DarT test. The toxicity order of the measured compounds was CuSO4 > 2,4‐dinitrophenol > 3,4‐dichloroaniline > SDS > sodium benzoate > EDTA > K2CrO4; regarding solvents, EtOH ≈ DMSO. In this study, we demonstrate that global swimming behavior could be a simple readout for toxicity, easy to scale‐up in automated experiments. This approach is potentially applicable for fast ecotoxicity assays and whole‐organism high‐throughput compound screening, reducing the time and money required to evaluate unknown samples and to identify leading pharmaceutical compounds. Copyright

G-quadruplexes as novel cis-elements controlling transcription during embryonic development

G-quadruplexes are dynamic structures folded in G-rich single-stranded DNA regions. These structures have been recognized as a potential nucleic acid based mechanism for regulating multiple cellular processes such as replication, transcription and genomic maintenance. So far, their transcriptional role in vivo during vertebrate embryonic development has not yet been addressed. Here, we performed an in silico search to find conserved putative G-quadruplex sequences (PQSs) within proximal promoter regions of human, mouse and zebrafish developmental genes. Among the PQSs able to fold in vitro as G-quadruplex, those present in nog3, col2a1 and fzd5 promoters were selected for further studies. In cellulo studies revealed that the selected G-quadruplexes affected the transcription of luciferase controlled by the SV40 nonrelated promoter. G-quadruplex disruption in vivo by microinjection in zebrafish embryos of either small ligands or DNA oligonucleotides complementary to the selected PQSs resulted in lower transcription of the targeted genes. Moreover, zebrafish embryos and larvae phenotypes caused by the presence of complementary oligonucleotides fully resembled those ones reported for nog3, col2a1 and fzd5 morphants. To our knowledge, this is the first work revealing in vivo the role of conserved G-quadruplexes in the embryonic development, one of the most regulated processes of the vertebrates biology.

In vitro embryonic developmental phosphorylation of the cellular nucleic acid binding protein by cAMP-dependent protein kinase, and its relevance for biochemical activities

The zinc‐finger cellular nucleic acid binding protein (CNBP) is a strikingly conserved single‐stranded nucleic acid binding protein essential for normal forebrain formation during mouse and chick embryogenesis. CNBP cDNAs from a number of vertebrates have been cloned and analysed. CNBP is mainly conformed by seven retroviral Cys‐Cys‐His‐Cys zinc‐knuckles and a glycine/arginine rich region box. CNBP amino acid sequences show a putative Pro‐Glu‐Ser‐Thr site of proteolysis and several putative phosphorylation sites. In this study, we analysed CNBP phosphorylation by embryonic kinases and its consequences on CNBP biochemical activities. We report that CNBP is differentially phosphorylated by Danio rerio embryonic extracts. In vitro CNBP phosphorylation is basal and constant at early embryonic developmental stages, it begins to increase after mid‐blastula transition stage reaching the highest level at 48 hours postfertilization stage, and decreases thereafter to basal levels at 5 days postfertilization. The cAMP‐dependent protein kinase (PKA) was identified as responsible for phosphorylation on the unique CNBP conserved putative phosphorylation site. Site‐directed mutagenesis replacing the PKA phospho‐acceptor amino acid residue impairs CNBP phosphorylation, suggesting that phosphorylation may not only exist in D. rerio but also in other vertebrates. CNBP phosphorylation does not change single‐stranded nucleic acid binding capability. Instead, it promotes in vitro the annealing of complementary oligonucleotides representing the CT element (CCCTCCCC) from the human cellular myelocytomatosis oncogene (c‐myc) promoter, an element responsible for c‐myc enhancer transcription. Our results suggest that phosphorylation might be a conserved post‐translational modification that allows CNBP to perform a fine tune expression regulation of a group of target genes, including c‐myc, during vertebrate embryogenesis.

CNBP modulates the transcription of Wnt signaling pathway components

BACKGROUND Cellular nucleic acid binding protein (CNBP) is a small and highly conserved protein with nucleic acid chaperone activity that binds single-stranded nucleic acids. Data collected so far suggests that CNBP is required for proper craniofacial development. Despite the advances achieved in the last decade, the identity of the molecular targets of CNBP responsible for its role in rostral head development remains elusive. METHODS In this work we used the CNBP single-stranded DNA-consensus binding sequence to find out putative CNBP target genes present in the human, mouse, chicken, Xenopus and zebrafish genomes. RESULTS Most of the identified genes are associated with embryonic developmental processes, being three of them (cdk14, ptk7 and tcf7l2) members of the Wnt signaling pathway. This finding, along with previous one showing that CNBP down-regulates the transcription of Wnt5, aimed our work to address the role of CNBP on the WNT signaling players and pathway regulation. Experiments carried out in zebrafish developing embryos revealed that craniofacial morphology was more adversely affected as CNBP abundance decreased. Furthermore, we observed that CNBP up-regulated in a dose-dependent fashion the transcription of cdk14, ptk7 and tcf7l2, which in turn was reflected in c-myc, ccnd1 and axin2 expression. CONCLUSIONS RESULTS reveal a role of CNBP in transcriptional control of components of the Wnt signaling pathway, which might explain its requirement for proper craniofacial development.

Overexpression of KLC2 due to a homozygous deletion in the non-coding region causes SPOAN syndrome

SPOAN syndrome is a neurodegenerative disorder mainly characterized by spastic paraplegia, optic atrophy and neuropathy (SPOAN). Affected patients are wheelchair bound after 15 years old, with progressive joint contractures and spine deformities. SPOAN patients also have sub normal vision secondary to apparently non-progressive congenital optic atrophy. A potential causative gene was mapped at 11q13 ten years ago. Here we performed next-generation sequencing in SPOAN-derived samples. While whole-exome sequencing failed to identify the causative mutation, whole-genome sequencing allowed to detect a homozygous 216-bp deletion (chr11.hg19:g.66,024,557_66,024,773del) located at the non-coding upstream region of the KLC2 gene. Expression assays performed with patients fibroblasts and motor neurons derived from SPOAN patients showed KLC2 overexpression. Luciferase assay in constructs with 216-bp deletion confirmed the overexpression of gene reporter, varying from 48 to 74%, as compared with wild-type. Knockdown and overexpression of klc2 in Danio rerio revealed mild to severe curly-tail phenotype, which is suggestive of a neuromuscular disorder. Overexpression of a gene caused by a small deletion in the non-coding region is a novel mechanism, which to the best of our knowledge, was never reported before in a recessive condition. Although the molecular mechanism of KLC2 up-regulation still remains to be uncovered, such example adds to the importance of non-coding regions in human pathology.