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Perturbation of developmental regulatory gene expression by G-quadruplex DNA inducer in the sea urchin embryo. Giuseppina Turturici, Veronica La Fiora, Alessio Terenzi, Giampaolo Barone, and Vincenzo Cavalieri Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00551 • Publication Date (Web): 16 Jul 2018 Downloaded from http://pubs.acs.org on July 17, 2018
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Biochemistry
Perturbation of developmental regulatory gene expression by Gquadruplex DNA inducer in the sea urchin embryo. Giuseppina Turturici, Veronica La Fiora, Alessio Terenzi†, Giampaolo Barone*, and Vincenzo Cavalieri* Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, Viale delle Scienze Edificio 16, 90128 Palermo, Italy KEYWORDS: G-quadruplex, developmental gene regulatory network, sea urchin embryo ABSTRACT: G-quadruplex (G4) is a four-stranded DNA structure identified in vivo in Guanine-rich regions located in the promoter of a number of genes. Intriguing evidence suggested that small molecules acting as G4-targeting ligands could potentially regulate multiple cellular processes either by stabilizing or disruptive effects on G4 motifs. Research in this field aims to prove the direct role of G4 ligands/structures on a specific biological process in a complex living organism. In this study, we evaluate in vivo the effects of a nickel(II)-salnaphen-like complex, named Nisaln, a potent G4 binder and stabilizer, during embryogenesis of the sea urchin embryo. We describe developmental defects inflicted by Nisaln and correlate them with variation in the expression of several regulatory genes. Worth mentioning, we show that Nisaln binds a G4 structure in the promoter of hbox12-a, a gene lying at the top of the developmental regulatory hierarchy, inducing overexpression of this gene.
Introduction G-quadruplex (G4) DNA is a non-canonical dynamic structure folded in Guanine-rich regions. The G4 basic unit is stabilized by potassium cations and arises following the association of Hoogsteen-paired coplanar guanine tetrads, which stack and connect with one another by looping the sugar-phosphate backbone.1 The occurrence of this four-stranded structural motif has been demonstrated in vivo2,3, and transcriptional roles for promoterspecific G4s have been highlighted by several studies using G4targeting ligands.4,5 Several square planar salen-like metal complexes have been recently reported as selective G4 binders6,7, whereas a recent example from our group is the nickel(II)salnaphen-like compound Nisaln.8,9 A weakness of these studies is that nonspecific or pleiotropic effects of G4 ligands cannot be ruled out, because eukaryote genomes possess numerous genes potentially containing G4 in their promoter. It follows that the primary challenge is to prove the direct role of G4 ligands/structures on a specific biological process in a complex living organism. The sea urchin embryo is a particularly suitable model for this purpose. It has a relatively simple morphology and follows a stereotypically invariant embryogenesis, giving rise to few cell types. Embryos are transparent, allowing detailed observation of cell behaviours in vivo after exposure to different kinds of molecules.10 Sea urchins are not subjected to animal welfare concerns, and large numbers of synchronously developing embryos can be obtained easily. Most of the developmental program and the fundamental laws directing embryogenesis lie in the genomic
DNA and are accomplished by gene regulatory networks (GRN). The interconnections constituting the GRN of the sea urchin embryo have been elucidated in detail.11,12 In this regard, we showed that the hbox12-a gene encodes a dorsal-specific transcription repressor functioning at the top of the dorsal/ventral GRN circuitry,13-15 and identified a compact cis-regulatory module responsible for the restricted spatiotemporal expression of the gene.16 In this study, we focus on a G-rich tract located in the hbox12-a promoter, assessing that this sequence forms a G4 structure contributing to the transcriptional control of hbox12-a for the proper embryonic development. Materials and methods For circular dichroism (CD) experiments, an oligonucleotide recreating the hbox12-a G4-forming sequence (5’GGGACGAAGGGAGGGAGGG-3’), was purchased from Eurofins Genomics (https://www.eurofinsgenomics.eu) in HPLC grade. It was dissolved in 1 mM Tris-HCl pH 7.4 to yield a 100 µM solution, which was then diluted to the desired concentration using the same buffer or 50 mM Tris-HCl pH 7.4/100 mM KCl. Folding was afforded by heating the solutions up to 95 °C for 5 min and then by slowly cooling at room temperature overnight. Nisaln was dissolved in DMSO and diluted using 50 mM TrisHCl pH 7.4/100 mM KCl to the desired concentration (final DMSO percentage was 70%) of embryos enduring Nisaln exposure instead contained blastomeres of disproportionate sizes, suggesting that uneven cleavage occurred in these specimens (Figure 2a and c-c’). Moreover, about 10% of Nisaln-treated blastulae displayed a degenerating phenotype characterized by blastocoel abnormally filled with cells (Figure 2a and d). When administered at 7.5 and 10 µM, Nisaln inflicted embryo death before hatching in approximately 70% and 100% of embryos, respectively (Figure 2a and e).
Figure 1. (a) Promoter structure of the hbox12-a gene showing positions of cis-regulatory elements on sense (+) and antisense (-) DNA strand, and highlighting in inset the potential G4-forming
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Biochemistry Figure 2. Developmental effects of Nisaln exposure during early embryogenesis of P. lividus. (a) Coloured bars in the histograms show the percentages of the observed phenotypes. Standard deviation values ranged from 0.1 to 1.5 for all samples. (b-e) Representative embryos cultured in the absence or in the presence of Nisaln and observed at the early-blastula stage. The embryos shown in (b,c) and (b’,c’) are observed from a surface and equatorial view, respectively. Based on this, we focused the analysis of further developmental effects solely on embryos treated with 1, 2.5, and 5 µM solutions of Nisaln, observing them when the controls were at the gastrula and prism stages (Figure 3). At the lowest dosage, almost 80% of embryos were able to hatch and further form mesenchyme cells, archenteron and skeletal elements similar to control embryos (Figure 3a), indicating that both morphogenetic movements and specification of main tissues were not impaired by Nisaln. By contrast, while 20-25% of the embryos exposed to both the higher concentrations of Nisaln had died, almost 40% exhibited delayed development, and about half of the latter contained supernumerary mesenchyme cells (Figure 3).
Figure 3. Developmental effects of Nisaln exposure during morphogenesis of P. lividus. (A) Coloured bars in the histograms show the percentages of the observed phenotypes. Standard deviation values ranged from 0.1 to 1.5 for all samples. (B) Representative embryos cultured in the absence or in the presence of Nisaln and observed at the prism stage. We also noticed that the swimming behaviour of Nisaln-treated embryos changed markedly, ranging from abnormal locomotion to immobilization. In particular, while all of the control larvae exhibited forward movements throughout the water column, about 40% of embryos exposed to 1 µM Nisaln settled to the bottom of the culture plate and spun slowly around an axis. The fraction of embryos displaying perturbed swimming behaviour progressed to almost 75% at higher concentration, either 2.5 or 5 µM, of Nisaln. Next, we ascertained the specification of different cell types in embryos exposed to 5 µM Nisaln, by measuring the mRNA abundance of a set of territorial marker genes. We found that exposure to Nisaln caused upregulation of hbox12-a at early-blastula stage (Figure 4). Since hbox12-a is normally necessary for establishment of the dorsal/ventral polarity in P. lividus,13,14 this finding suggests that dorsal/ventral polarization was impaired in Nisalntreated embryos. As a first piece of evidence supporting this hypothesis, Nisaln exerted an almost identical transcriptional outcome on the dorsal-specific marker tbx2/3,23 and concurrent down-regulation of the two ventral-specific markers nodal24 and gsc25 at the blastula stage (Figure 4). Accordingly, the expression of the two ventro-lateral ectoderm-specific markers otp26 and
pax2/5/820 was significantly reduced in Nisaln-treated gastrulae (Figure 4), again indicating that axial patterning was impaired in these embryos. The mRNA abundance of fgf did not appear to be affected by exposure to Nisaln (Figure 4). However, at the gastrula stage fgf is expressed in two distinct territories, namely ventrolateral ectoderm and the adjoining skeletogenic cell clusters,27 and differential effects on these territories cannot be distinguished by qPCR. We appraised that both primary- and secondary-mesenchyme cell populations increased in Nisaln-treated gastrulae. Accordingly, Nisaln-treated gastrulae contained an almost double mRNA amount of the primary-mesenchyme-specific marker sm50 compared to unperturbed embryos (Figure 4). Moreover, qPCR analysis of the secondary-mesenchyme-specific markers papss and gcm revealed that the supernumerary secondary-mesechyme cells were most probably a cohort of pigment-cell precursors (Figure 4). This hypothesis is congruent with the observed downregulation of nodal expression, since it is known that Nodal signaling antagonizes the specification of pigment cells,28 and is supported by the observation that the expression level of the blastocoelar-specific marker gata1/2/328 did not vary following exposure to Nisaln (Figure 4).
Figure 4. Changes in gene expression level of territorial marker genes assessed by qPCR during development of Nisaln-treated embryos. Data are shown as the percentage of mRNA level normalized with respect to control embryos at the indicated stages. The error bars are standard errors for the qPCR replicates. In conclusion, we highlighted that the transcriptional outcome of several genes constituting the developmental GRN of the sea urchin is differentially affected by exposure to Nisaln. Being hbox12-a at the top of the regulatory hierarchy, most probably the observed effects depend upon overexpression of this gene, induced by binding of Nisaln to the G4 structure in the hbox12-a. The mechanism by which such a G4 contributes to hbox12-a gene transcription remains an open question. The G4 structure might recruit specific transcription activators or it could hold the DNA molecule in a transcriptionally permissive configuration, as reported by studies on distinct genes.29,30 Alternatively, G4-folding might remove a repressive process normally involved in limiting the transcriptional outcome and/or restricting the spatial expression domain of hbox12-a. Whatever is the explanation, our findings will be a guide for the design of new compounds with improved selectivity for G4, also representing a first step towards further characterization of Nisaln and derivatives in vertebrate animal models for preclinical purposes.
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AUTHOR INFORMATION
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Corresponding Author *
[email protected],
[email protected] (11)
Present Addresses † Donostia International Physics Center, Paseo Manuel de Lardizabal 4, Donostia, 20018, Spain
Author Contributions V.C. conceived the experiments, analyzed data and wrote the manuscript. A.T. and G.B. synthesized the Nisaln compound, G.T., A.T. and G.B. performed the spectroscopic studies, while G.T., V.L.F., and V.C. performed all the other experiments. All authors have given approval to the final version of the manuscript.
Funding Sources
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This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. (15)
Notes The authors declare no competing financial interests.
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ACKNOWLEDGMENT Many thanks are due to Francesca Faillaci and Chiara Reina for technical assistance. (17)
ABBREVIATIONS DMSO, dimethyl sulfoxide; G4, G-quadruplex; GRN, gene regulatory network; hpf, hours post-fertilization; Salen, N,N’-bis(salicylidene)-ethylenediamine.
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Another step toward DNA selective targeting: Ni(II) and Cu(II) complexes of a Schiff base ligand able to bind gene promoter G-quadruplexes. Dalton Trans. 45, 7758-7767. Pellicanò, M., Picone, P., Cavalieri, V., Carrotta, R., Spinelli, G., Di Carlo, M. (2009) The sea urchin embryo: a model to study Alzheimer's beta amyloid induced toxicity. Arch. Biochem. Biophys. 483, 120-126. Davidson, E. H., Rast, J. P., Oliveri, P., Ransick, A., Calestani, C., Yuh, C. H., Minokawa, T., Amore, G., Hinman, V., Arenas-Mena, C., Otim, O., Brown, C. T., Livi, C. B., Lee, P. Y., Revilla, R., Rust, A. G., Pan, Z., Schilstra, M. J., Clarke, P. J., Arnone, M. I., Rowen, L., Cameron, R. A., McClay, D. R., Hood, L., Bolouri, H. (2002) A genomic regulatory network for development. Science 295, 1669–1678. Martik, M.L., Lyons, D. C., McClay, D. R. (2016) Developmental gene regulatory networks in sea urchins and what we can learn from them. F1000Res. 5, F1000 Faculty Rev-203. Cavalieri, V., Spinelli, G. (2014) Early asymmetric cues triggering the dorsal/ventral gene regulatory network of the sea urchin embryo. Elife 3, e04664. Cavalieri, V., Spinelli, G. (2015) Ectopic hbox12 Expression Evoked by Histone Deacetylase Inhibition Disrupts Axial Specification of the Sea Urchin Embryo. PLoS One 10, e0143860. Cavalieri, V., Spinelli, G. (2015) Symmetry Breaking and Establishment of Dorsal/Ventral Polarity in the Early Sea Urchin Embryo. Symmetry 7, 1721-1733. Cavalieri, V., Di Bernardo, M., Anello, L., Spinelli, G. (2008) cis-Regulatory sequences driving the expression of the Hbox12 homeobox-containing gene in the presumptive aboral ectoderm territory of the Paracentrotus lividus sea urchin embryo. Dev. Biol. 321, 455-469. Cavalieri, V., Melfi, R., Spinelli, G. (2013) The Compass-like locus, exclusive to the ambulacrarians, encodes a chromatin insulator binding protein in the sea urchin embryo. PLoS Genet. 9, e1003847. Cavalieri, V., Melfi, R., Spinelli, G. (2009) Promoter activity of the sea urchin (Paracentrotus lividus) nucleosomal H3 and H2A and linker H1 α-histone genes is modulated by enhancer and chromatin insulator. Nucleic Acids Res. 37, 7407-7415. Cavalieri, V., Geraci, F., Spinelli, G. (2017) Diversification of spatiotemporal expression and copy number variation of the echinoid hbox12/pmar1/micro1 multigene family. PLoS One 12, e0174404. Cavalieri, V., Guarcello, R., Spinelli, G. (2011) Specific expression of a TRIM-containing factor in ectoderm cells affects the skeletal morphogenetic program of the sea urchin embryo. Development 138, 4279-4290. Kikin, O., D'Antonio, L., Bagga, P. S. (2006) QGRS Mapper: a web-based server for predicting G-quadruplexes in nucleotide sequences. Nucleic Acids Res. 34, W676-682. Cer, R. Z., Donohue, D. E., Mudunuri, U. S., Temiz, N. A., Loss, M. A., Starner, N. J., Halusa, G. N., Volfovsky, N., Yi, M., Luke, B. T., Bacolla, A., Collins, J. R., Stephens, R. M. (2013) Non-B DB v2.0: a database of predicted non-B DNAforming motifs and its associated tools. Nucleic Acids Res. 41, D94-100. Croce, J., Lhomond, G., Gache, C. (2003) Coquillette, a sea urchin T-box gene of the Tbx2 subfamily, is expressed asymmetrically along the oral-aboral axis of the embryo and is involved in skeletogenesis. Mech. Dev. 120, 561-572. Duboc, V., Röttinger, E., Besnardeau, L., Lepage, T. (2004) Nodal and BMP2/4 signaling organizes the oral-aboral axis of the sea urchin embryo. Dev. Cell 6, 397-410. Angerer, L. M., Oleksyn, D. W., Levine, A. M., Li, X., Klein, W. H., Angerer, R. C. (2001) Sea urchin goosecoid function links fate specification along the animal-vegetal and oralaboral embryonic axes. Development 128, 4393-4404. Cavalieri, V., Di Bernardo, M., Spinelli, G. (2007) Regulatory sequences driving expression of the sea urchin Otp homeobox gene in oral ectoderm cells. Gene Expr. Patterns 7, 1241-30. Röttinger, E., Saudemont, A., Duboc, V., Besnardeau, L., McClay, D., Lepage, T. (2008) FGF signals guide migration of mesenchymal cells, control skeletal morphogenesis and regu-
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(a) Promoter structure of the hbox12-a gene showing positions of cis-regulatory elements on sense (+) and antisense (-) DNA strand, and highlighting in inset the potential G4-forming sequence predicted by QGRS Mapper. Runs of Guanines are un-derlined; (b) QGRS scores indicating the likelihood of G4 for-mation referred to the sequences located in the promoter of the indi-cated genes; the sequences shown, except for hbox12-a, were re-trieved from the public G4IPDB database (http://bsbe.iiti.ac.in/bsbe/ipdb/index.php); (c) CD spectra of 4.5 µM hbox12-a sequence annealed in the indicated conditions; (d), Chemical structure of Nisaln. 99x110mm (300 x 300 DPI)
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Biochemistry
Developmental effects of Nisaln exposure during early embryogen-esis of P. lividus. (a) Coloured bars in the histograms show the per-centages of the observed phenotypes. Standard deviation values ranged from 0.1 to 1.5 for all samples. (b-e) Representative embry-os cultured in the absence or in the presence of Nisaln and observed at the early-blastula stage. The embryos shown in (b,c) and (b’,c’) are observed from a surface and equatorial view, respectively. 140x125mm (300 x 300 DPI)
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Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Developmental effects of Nisaln exposure during morphogenesis of P. lividus. (A) Coloured bars in the histograms show the percent-ages of the observed phenotypes. Standard deviation values ranged from 0.1 to 1.5 for all samples. (B) Representative embryos cul-tured in the absence or in the presence of Nisaln and observed at the prism stage. 110x63mm (300 x 300 DPI)
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Biochemistry
Changes in gene expression level of territorial marker genes assessed by qPCR during development of Nisaln-treated embryos. Data are shown as the percentage of mRNA level (left ordinate) normalized with respect to control embryos at the indicated stages. The error bars are standard errors for the qPCR replicates. 66x67mm (300 x 300 DPI)
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