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Biochim Biophys Acta
2014 Nov 01;183911:1151-60. doi: 10.1016/j.bbagrm.2014.08.009.
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CNBP modulates the transcription of Wnt signaling pathway components.
Margarit E
,
Armas P
,
García Siburu N
,
Calcaterra NB
.
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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.
Fig. 1. Search for putative CNBP target genes conserved in five vertebrate species. (A) Strategy used for searching promoter regions (− 1000 bp from the transcription start site) of five vertebrate species (Homo sapiens, Gallus gallus, Xenopus tropicalis, Mus musculus and Danio rerio) containing at least one CNBP–CBS. Promoter sequences were retrieved from Ensembl by using the BioMart tool and CNBP–CBS was searched among sequences using the MEME/MAST algorithm. Data were grouped in five lists of promoters, one for each analyzed species. (B) Chart showing the numbers of total promoters analyzed for each of the five species and the absolute and relative (percentage) numbers of promoters containing the CNBP–CBS. GC-percentage observed in the respective genomes is shown in the right column. (C) The five gene lists corresponding to the resulting promoters were intersected using BioVenn, retrieving 16 shared genes. Functions of found genes were then addressed using PubMed and Gene Ontology (Cytoscape/BiNGO tool). (D) Chart showing the names, main function and reference for the three conserved putative CNBP target genes that are members of the Wnt signaling pathway.
Fig. 2. CNBP–CBS mapping in promoters of putative target genes. The location of the CNBP–CBS into the promoter regions of cdk14, tcf7l2 and ptk7 in the five species analyzed is shown with green lines. Relative positions to the transcription start sites and p-values of the MEME/MAST prediction are indicated. Boxed CNBP–CBS indicate conservation in relative positions.
Fig. 3. Direct interaction between CNBP and the promoters of target genes. (A) Relative abundance of cnbp-mRNA determined by RT-qPCR using total RNA from 24 hpf zebrafish embryos expressing CNBP fused to eGFP. Abundance of transcript was normalized using ef1a and rpl13a and relativized to the control condition (injected with the saline solution KCl 0.2 M). Bars represent mean of relative abundance and standard deviation (SD), n = 3. **p < 0.05 (Student's t-test). (B) Scheme of the location of CNBP–CBS in the promoter regions of zebrafish CNBP target genes cdk14, ptk7a and tcf7l2 and a negative control (actb2). Green lines indicate the position of CNBP–CBS. Red bars indicate regions studied by PCR in ChIP experiments. Numbers indicate positions relative to the transcription start sites and the p-values of the MEME/MAST prediction. (C) CNBP–ChIP assays performed on CNBP-overexpressing 24-hpf zebrafish embryos. Antibody (Ab), no antibody (−) and input samples were analyzed by PCR for the indicated regions (red bars in B).
Fig. 4. The phenotypes of 24 hpf-staged zebrafish embryos change according to the CNBP abundance. (A) Control embryo microinjected with miss-paired MO (mis). (B) Morphant embryo microinjected with spl-MO and showing mild phenotype (spl-1). (C) Morphant embryo microinjected with spl-MO and showing strong phenotype (spl-2). Lateral views of microinjected embryos were registered under stereoscopic microscope (whole body at the upper left of each panel) or with two different magnifications under differential interference contrast microscope (anterior-most region at the upper and lower right of each panel). Dotted lines mark regions between eyes (ey) and otic vesicle (ov), which include midbrain–hindbrain border (mhb) and showed darkened tissue in morphants, suggesting the presence of apoptotic cells. Scale bars represent 200 μm. (D) Numbers (and percentages) of embryos injected with mis-MO and spl-MO and their phenotypes. (E) Relative abundance of cnbp-mRNA was determined in 24 hpf morphants by RT-qPCR. Gene expression levels were normalized using ef1a and rpl13a and relativized to the mis-MO condition. Bars represent mean of relative abundance and standard deviation (SD), n = 3. ****p < 0.001 (ANOVA test).
Fig. 5. CNBP depletion adversely affects craniofacial cartilage formation in a dose-dependent fashion. Ventral views with the heads facing up of mis-MO condition (A), spl-1 condition (B) and spl-2 condition (C) 4 dpf larvae stained with Alcian blue. Red lines, distance between ceratohyal cartilages and lateral fins (cd); green lines, distance between Meckel cartilage and lateral fins (md); blue lines, distance between snout and lateral fins (sd); dotted white lines, angle formed by ceratohyal cartilages (ca); gray lines, length of ceratohyal cartilages (cl). Scale bars represent 200 μm. (D) Values of the angle formed between ceratohyalcartilage in controls and CNBP-depleted larvae. (E) Relative variations of cartilage development in controls and CNBP-depleted larvae. (F) Number of ceratobranchial arches observed in controls and CNBP-depleted larvae. Abbreviations: cb1–5, ceratobranchial arches 1–5; ch, ceratohyalcartilage; m, Meckel's cartilage; pq, palatoquadratecartilage. Bars in (D) and (E) represent mean of angle values and relative length values, respectively, and standard deviation (SD), n = 39 for mis, n = 44 for spl-1, n = 34 for spl-2 (n were twice these values for cl because both ceratohyal cartilages were measured in each embryo). * p < 0.05 (ANOVA test).
Fig. 6. CNBP controls the transcription of cdk14, ptk7a and tcf7l2 in zebrafish developing embryos. Relative abundance of genes was determined by RT-qPCR using total RNA extractions from 24 hpf embryos and specific primers. Actb2 expression was assessed as CNBP non-target control. Gene expression levels were normalized using ef1a and rpl13a and relativized to the corresponding controls. (A) Expression analysis on CNBP-depleted embryos obtained from spl-1, spl-2 or mis conditions. (B) Expression analysis on CNBP-overexpressing embryos obtained from injection of CNBP-eGFP-mRNA. Bars represent mean of relative abundance and standard deviation (SD), n = 3. *p < 0.1, **p < 0.05, ***p < 0.01, ****p < 0.001 (ANOVA test).
Fig. 7. Expression analysis of CNBP target genes tcf7l2 and ptk7 by whole-mount RNA in situ hybridization (WISH). 24 hpf-staged zebrafish embryos microinjected with mis-MO (A–B and E–F) or spl-MO (C–D and G–H) morpholinos were analyzed for the expression of tcf7l2 and ptk7 genes by WISH. Lateral and frontal–dorsal views are shown with the anterior regions pointing to the left. Scale bar (200 μm) is represented in C and G. Number and percentages of morphant phenotypes are indicated for each embryo stage. The expression territories more affected in morphants are marked with dashed-lines in control (mis-MO) embryos. bs, brain structures; dd, dorsal diencephalon; e, eye; mhb, midbrain–hindbrain border.
Fig. 8. Expression of β-catenin/TCF target genes in CNBP-depleted or overexpressing embryos. The relative abundance of axin2, ccnd1, sp5l and myca was determined by RT-qPCR using total RNA extractions from 24 hpf CNBP-depleted and CNBP-overexpressing embryos. Gene expression levels were normalized using ef1a and rpl13a and relativized to the corresponding controls. (A) Expression analysis on CNBP-depleted embryos. (B) Expression analysis on CNBP-overexpressing embryos. Bars represent mean of relative abundance and standard deviation (SD), n = 3. *p < 0.1, **p < 0.05 (ANOVA test).
