Perturbation of Developmental Regulatory Gene Expression by a G

Communication Cite This: Biochemistry 2018, 57, 4391−4394

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Perturbation of Developmental Regulatory Gene Expression by a G‑Quadruplex DNA Inducer in the Sea Urchin Embryo Giuseppina Turturici, Veronica La Fiora, Alessio Terenzi,† Giampaolo Barone,* and Vincenzo Cavalieri*

Biochemistry 2018.57:4391-4394. Downloaded from pubs.acs.org by UNIV OF LOUISIANA AT LAFAYETTE on 10/11/18. For personal use only.

Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, Viale delle Scienze Edificio 16, 90128 Palermo, Italy fundamental laws directing embryogenesis lie in the genomic DNA and are accomplished by gene regulatory networks (GRNs). 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 dorsalspecific transcription repressor functioning at the top of the dorsal/ventral GRN circuitry13−15 and identified a compact cisregulatory module that is 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 hbox12a for the proper embryonic development.

ABSTRACT: The 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 G4targeting ligands could potentially regulate multiple cellular processes via either stabilizing or disruptive effects on G4 motifs. Research in this field aims to prove the direct role of G4 ligands and/or 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. It is worth mentioning that 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.

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G-Quadruplex (G4) DNA is a noncanonical 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 vivo,2,3 and transcriptional roles for promoter-specific G4s have been highlighted by several studies using G4-targeting ligands.4,5 Several square planar salen-like metal complexes have been recently reported as selective G4 binders,6,7 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 and/or 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 behaviors 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 © 2018 American Chemical Society

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) at high-performance liquid chromatography 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) and 100 mM KCl. Folding was afforded by heating the solutions to 95 °C for 5 min and then by slowly cooling them at room temperature overnight. Nisaln was dissolved in DMSO and diluted using 50 mM Tris-HCl (pH 7.4) and 100 mM KCl to the desired concentration (final DMSO percentage of 70%) of embryos enduring Nisaln exposure instead contained blastomeres of disproportionate sizes, suggesting that uneven cleavage occurred in these specimens (Figure 2a,c,c′). Moreover, ∼10% of Nisaln-treated blastulae displayed a degenerating phenotype characterized by a blastocoel abnormally filled with cells (Figure 2a,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,e). On this basis, 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

Figure 4. Changes in the gene expression level of territorial marker genes assessed by qPCR during the development of Nisaln-treated embryos. Data are shown as the percentage of the mRNA level normalized with respect to control embryos at the indicated stages. The error bars are standard errors for the qPCR replicates.

necessary for the establishment of the dorsal/ventral polarity in P. lividus,13,14 this finding suggests that dorsal/ventral polarization was impaired in Nisaln-treated embryos. As a first piece of evidence supporting this hypothesis, Nisaln exerted an almost identical transcriptional outcome on the dorsal-specific marker tbx2/323 and concurrent downregulation of the two ventral-specific markers nodal24 and gsc25 at the blastula stage (Figure 4). Accordingly, the level of 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, the ventro-lateral 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 almost double the amount of mRNA of the primary-mesenchyme-specific marker sm50 compared to unperturbed embryos (Figure 4). Moreover, qPCR analysis of the secondary-mesenchymespecific 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, because it is known that Nodal signaling antagonizes the specification of pigment cells28 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). 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

Figure 3. Developmental effects of Nisaln exposure during morphogenesis of P. lividus. (A) Colored 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 presence of Nisaln and observed at the prism stage.

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 approximately half of the latter contained supernumerary mesenchyme cells (Figure 3). We also noticed that the swimming behavior of Nisalntreated embryos changed markedly, ranging from abnormal locomotion to immobilization. In particular, while all of the control larvae exhibited forward movements throughout the water column, ∼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 behavior progressed to almost 75% at a higher concentration, either 2.5 or 5 μM, of Nisaln. 4393

DOI: 10.1021/acs.biochem.8b00551 Biochemistry 2018, 57, 4391−4394

Communication

Biochemistry

(7) Vilar, R. (2018) Nucleic acid quadruplexes and metallo-drugs. In Metallo-drugs: Development and action of anticancer agents (Sigel, A., Sigel, H., Freisinger, E., and Sigel, R. K. O., Eds.) pp 325−349, De Gruyter. (8) Terenzi, A., Bonsignore, R., Spinello, A., Gentile, C., Martorana, A., Ducani, C., Högberg, B., Almerico, A. M., Lauria, A., and Barone, G. (2014) RSC Adv. 4, 33245−33256. (9) Terenzi, A., Lötsch, D., van Schoonhoven, S., Roller, A., Kowol, C. R., Berger, W., Keppler, B. K., and Barone, G. (2016) Dalton Trans. 45, 7758−7767. (10) Pellicanò, M., Picone, P., Cavalieri, V., Carrotta, R., Spinelli, G., and Di Carlo, M. (2009) Arch. Biochem. Biophys. 483, 120−126. (11) 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., and Bolouri, H. (2002) Science 295, 1669−1678. (12) Martik, M. L., Lyons, D. C., and McClay, D. R. (2016) F1000Research 5, 203. (13) Cavalieri, V., and Spinelli, G. (2014) eLife 3, No. e04664. (14) Cavalieri, V., and Spinelli, G. (2015) PLoS One 10, No. e0143860. (15) Cavalieri, V., and Spinelli, G. (2015) Symmetry 7, 1721−1733. (16) Cavalieri, V., Di Bernardo, M., Anello, L., and Spinelli, G. (2008) Dev. Biol. 321, 455−469. (17) Cavalieri, V., Melfi, R., and Spinelli, G. (2013) PLoS Genet. 9, No. e1003847. (18) Cavalieri, V., Melfi, R., and Spinelli, G. (2009) Nucleic Acids Res. 37, 7407−7415. (19) Cavalieri, V., Geraci, F., and Spinelli, G. (2017) PLoS One 12, No. e0174404. (20) Cavalieri, V., Guarcello, R., and Spinelli, G. (2011) Development 138, 4279−4290. (21) Kikin, O., D’Antonio, L., and Bagga, P. S. (2006) Nucleic Acids Res. 34, W676−682. (22) 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., and Stephens, R. M. (2012) Nucleic Acids Res. 41, D94−100. (23) Croce, J., Lhomond, G., and Gache, C. (2003) Mech. Dev. 120, 561−572. (24) Duboc, V., Röttinger, E., Besnardeau, L., and Lepage, T. (2004) Dev. Cell 6, 397−410. (25) Angerer, L. M., Oleksyn, D. W., Levine, A. M., Li, X., Klein, W. H., and Angerer, R. C. (2001) Development 128, 4393−4404. (26) Cavalieri, V., Di Bernardo, M., and Spinelli, G. (2007) Gene Expression Patterns 7, 124−130. (27) Röttinger, E., Saudemont, A., Duboc, V., Besnardeau, L., McClay, D., and Lepage, T. (2007) Development 135, 353−365. (28) Duboc, V., Lapraz, F., Saudemont, A., Bessodes, N., Mekpoh, F., Haillot, E., Quirin, M., and Lepage, T. (2010) Development 137, 223−235. (29) Bochman, M. L., Paeschke, K., and Zakian, V. A. (2012) Nat. Rev. Genet. 13, 770−780. (30) Du, Z., Zhao, Y., and Li, N. (2008) Genome Res. 18, 233−241.

Nisaln. With 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 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 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 the explanation, our findings will be a guide for the design of new compounds with improved selectivity for G4, also representing a first step toward further characterization of Nisaln and derivatives in vertebrate animal models for preclinical purposes.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Alessio Terenzi: 0000-0001-9751-1373 Giampaolo Barone: 0000-0001-8773-2359 Vincenzo Cavalieri: 0000-0003-0906-9963 Present Address †

A.T.: Donostia International Physics Center, Paseo Manuel de Lardizabal 4, Donostia 20018, Spain. Author Contributions

V.C. conceived the experiments, analyzed the 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. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Many thanks are due to Francesca Faillaci and Chiara Reina for technical assistance. ABBREVIATIONS DMSO, dimethyl sulfoxide; G4, G-quadruplex; GRN, gene regulatory network; Hpf, hours postfertilization; Salen, N,N′bis(salicylidene)-ethylenediamine.

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REFERENCES

(1) Rhodes, D., and Lipps, H. J. (2015) Nucleic Acids Res. 43, 8627− 8637. (2) Lipps, H. J., and Rhodes, D. (2009) Trends Cell Biol. 19, 414− 422. (3) Biffi, G., Tannahill, D., McCafferty, J., and Balasubramanian, S. (2013) Nat. Chem. 5, 182−186. (4) Kaulage, M., Maji, B., Bhat, J., Iwasaki, Y., Chatterjee, S., Bhattacharya, S., and Muniyappa, K. (2016) J. Med. Chem. 59, 5035− 5050. (5) Renčiuk, D., Ryneš, J., Kejnovská, I., Foldynová-Trantírková, S., Andäng, M., Trantírek, L., and Vorlíčková, M. (2017) Biochim. Biophys. Acta, Gene Regul. Mech. 1860, 175−183. (6) Ali, A., Kamra, M., Roy, S., Muniyappa, K., and Bhattacharya, S. (2016) Chem. - Asian J. 11, 2542−2554. 4394

DOI: 10.1021/acs.biochem.8b00551 Biochemistry 2018, 57, 4391−4394