Molecular Biology Reports

, Volume 39, Issue 3, pp 2633–2644 | Cite as

In silico characterization of the neural alpha tubulin gene promoter of the sea urchin embryo Paracentrotus lividus by phylogenetic footprinting

Article

Abstract

During Paracentrotus lividus sea urchin embryo development one alpha and one beta tubulin genes are expressed specifically in the neural cells and they are early end output of the gene regulatory network that specifies the neural commitment. In this paper we have used a comparative genomics approach to identify conserved regulatory elements in the P. lividus neural alpha tubulin gene. To this purpose, we have first isolated a genomic clone containing the entire gene plus 4.5 Kb of 5′ upstream sequences. Then, we have shown by gene transfer experiments that its non-coding region drives the spatio-temporal gene expression corresponding substantially to that of the endogenous gene. In addition, we have identified by genome and EST sequence analysis the S. purpuratus alpha tubulin orthologous gene and we propose a revised annotation of some tubulin family members. Moreover, by computational techniques we delineate at least three putative regulatory regions located both in the upstream region and in the first intron containing putative binding sites for Forkhead and Nkx transcription factor families.

Keywords

Sea urchin Neural development Gene expression Phylogenetic footprint Cis-regulatory analysis 

Notes

Acknowledgments

This study was supported by the University of Palermo, Italy, grant from MIUR “ex 60%” to F.G.

Supplementary material

References

  1. 1.
    Rosenbaum J (2000) Cytoskeleton: functions for tubulin modifications at last. Curr Biol 10:R801–R803. doi:10.1016/S0960-9822(00)00767-3 PubMedCrossRefGoogle Scholar
  2. 2.
    Bonnet C, Boucher D, Lazereg S, Pedrotti B, Islam K, Denoulet P, Larcher JC (2001) Differential binding regulation of microtubule-associated proteins MAP1A, MAP1B, and MAP2 by tubulin polyglutamylation. J Biol Chem 276:12839–12848. doi:10.1074/jbc.M011380200 PubMedCrossRefGoogle Scholar
  3. 3.
    Calzone FJ, Höög C, Teplow DB, Cutting AE, Zeller RW, Britten RJ, Davidson EH (1991) Gene regulatory factors of the sea urchin embryo. I. Purification by affinity chromatography and cloning of P3A2, a novel DNA-binding protein. Development 112:335–350PubMedGoogle Scholar
  4. 4.
    Gianguzza F, Di Bernardo MG, Sollazzo M, Palla F, Ciaccio M, Carra E, Spinelli G (1989) DNA sequence and pattern of expression of the sea urchin (Paracentrotus lividus) alpha-tubulin genes. Mol Reprod Dev 1:170–181. doi:10.1002/mrd.1080010305 PubMedCrossRefGoogle Scholar
  5. 5.
    Gianguzza F, Casano C, Ragusa M (1995) Alpha-tubulin marker gene of neural territory of sea urchin embryos detected by whole-mount in situ hybridization. Int J Dev Biol 39:477–483PubMedGoogle Scholar
  6. 6.
    Sasaki H, Kominami T (2008) Specification process of animal plate in the sea urchin embryo. Dev Growth Differ 50:595–606. doi:10.1111/j.1440-169X.2008.01057.x PubMedCrossRefGoogle Scholar
  7. 7.
    Yaguchi S, Yaguchi J, Burke RD (2006) Specification of ectoderm restricts the size of the animal plate and patterns neurogenesis in sea urchin embryos. Development 133:2337–2346. doi:10.1242/dev.02396 PubMedCrossRefGoogle Scholar
  8. 8.
    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. doi:10.1016/S1534-5807(04)00056-5 PubMedCrossRefGoogle Scholar
  9. 9.
    Yaguchi S, Yaguchi J, Burke RD (2007) Sp-Smad2/3 mediates patterning of neurogenic ectoderm by nodal in the sea urchin embryo. Dev Biol 302:494–503. doi:10.1016/j.ydbio.2006.10.010 PubMedCrossRefGoogle Scholar
  10. 10.
    Ben-Tabou de-Leon S, Davidson EH (2006) Deciphering the underlying mechanism of specification and differentiation: the sea urchin gene regulatory network. Science Signaling STKE. pe47. doi: 10.1126/stke.3612006pe47
  11. 11.
    Su YH, Li E, Geiss GK, Longabaugh WJ, Krämer A, Davidson EH (2009) A perturbation model of the gene regulatory network for oral and aboral ectoderm specification in the sea urchin embryo. Dev Biol 329:410–421. doi:10.1016/j.ydbio.2009.02.029 PubMedCrossRefGoogle Scholar
  12. 12.
    Davidson EH (2009) Network design principles from the sea urchin embryo. Curr Opin Genet Dev 19:535–540. doi:10.1016/j.gde.2009.10.007 PubMedCrossRefGoogle Scholar
  13. 13.
    Cameron RA, Mahairas G, Rast JP et al (2000) A sea urchin genome project: sequence scan, virtual map, and additional resources. Proc Natl Acad Sci USA 97:9514–9518. doi:10.1073/pnas.160261897 PubMedCrossRefGoogle Scholar
  14. 14.
    Sea Urchin Genome Sequencing Consortium (2006) The genome of the sea urchin Strongylocentrotus purpuratus. Science 314(5801):941–952. doi:10.1126/science.1133609 CrossRefGoogle Scholar
  15. 15.
    Elnitski L, Jin VX, Farnham PJ, Jones SJ (2006) Locating mammalian transcription factor binding sites: a survey of computational and experimental techniques. Genome Res 16:1455–1464. doi:10.1101/gr.4140006 PubMedCrossRefGoogle Scholar
  16. 16.
    Smith AB (1988) Phylogenetic relationship, divergence times, and rates of molecular evolution for camarodont sea urchins. Mol Biol Evol 5:345–365Google Scholar
  17. 17.
    Costa S, Ragusa MA, Drago G, Casano C, Alaimo G, Guida N, Gianguzza F (2004) Sea urchin neural alpha2 tubulin gene: isolation and promoter analysis. Biochem Biophys Res Commun 316:446–453. doi:10.1016/j.bbrc.2004.02.070 PubMedCrossRefGoogle Scholar
  18. 18.
    Arnone MI, Dmochowski IJ, Gache C (2004) Using reporter genes to study cis-regulatory elements. Methods Cell Biol 74:621–652. doi:10.1016/S0091-679X(04)74025-X PubMedCrossRefGoogle Scholar
  19. 19.
    McMahon AP, Flytzanis CN, Hough-Evans BR, Katula KS, Britten RJ, Davidson EH (1985) Introduction of cloned DNA into sea urchin egg cytoplasm: replication and persistence during embryogenesis. Dev Biol 108:420–430. doi:10.1016/0012-1606(85)90045-4 PubMedCrossRefGoogle Scholar
  20. 20.
    Cameron RA, Samanta M, Yuan A, He D, Davidson E (2009) SpBase: the sea urchin genome database and web site. Nucleic Acids Res 37:D750–D754. doi:10.1093/nar/gkn887 PubMedCrossRefGoogle Scholar
  21. 21.
    Lassmann T, Sonnhammer EL (2005) Kalign–an accurate and fast multiple sequence alignment algorithm. BMC Bioinform 6:298. doi:10.1186/1471-2105-6-298 CrossRefGoogle Scholar
  22. 22.
    Poustka AJ, Groth D, Hennig S, Thamm S, Cameron A, Beck A, Reinhardt R, Herwig R, Panopoulou G, Lehrach H (2003) Generation, annotation, evolutionary analysis, and database integration of 20,000 unique sea urchin EST clusters. Genome Res 13:2736–2746. doi:10.1101/gr.1674103 PubMedCrossRefGoogle Scholar
  23. 23.
    Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948. doi:10.1093/bioinformatics/btm404 PubMedCrossRefGoogle Scholar
  24. 24.
    Huang X (1994) On global sequence alignment. Comput Appl Biosci 10:227–235. doi:10.1093/bioinformatics/10.3.227 PubMedGoogle Scholar
  25. 25.
    Rebeiz M, Posakony JW (2004) GenePalette: a universal software tool for genome sequence visualization and analysis. Dev Biol 271:431–438. doi:10.1016/j.ydbio.2004.04.011 PubMedCrossRefGoogle Scholar
  26. 26.
    Dubchak I, Ryaboy DV (2006) VISTA family of computational tools for comparative analysis of DNA sequences and whole genomes. Methods Mol Biol 338:69–89. doi:10.1385/1-59745-097-9:69 PubMedGoogle Scholar
  27. 27.
    Brown CT, Xie Y, Davidson EH, Cameron RA (2005) Paircomp, FamilyRelationsII and Cartwheel: tools for interspecific sequence comparison. BMC Bioinform 6:70. doi:10.1186/1471-2105-6-70 CrossRefGoogle Scholar
  28. 28.
    Berezikov E, Guryev V, Plasterk RH, Cuppen E (2004) CONREAL: conserved regulatory elements anchored alignment algorithm for identification of transcription factor binding sites by phylogenetic footprinting. Genome Res 14:170–178. doi:10.1101/gr.1642804 PubMedCrossRefGoogle Scholar
  29. 29.
    Khodiyar VK, Maltais LJ, Ruef BJ, Sneddon KM, Smith JR, Shimoyama M, Cabral F, Dumontet C, Dutcher SK, Harvey RJ, Lafanechère L, Murray JM, Nogales E, Piquemal D, Stanchi F, Povey S, Lovering RC (2007) A revised nomenclature for the human and rodent alpha-tubulin gene family. Genomics 90:285–289. doi:10.1016/j.ygeno.2007.04.008 PubMedCrossRefGoogle Scholar
  30. 30.
    Morris RL, Hoffman MP, Obar RA, McCafferty SS, Gibbons IR, Leone AD, Cool J, Allgood EL, Musante AM, Judkins KM, Rossetti BJ, Rawson AP, Burgess DR (2006) Analysis of cytoskeletal and motility proteins in the sea urchin genome assembly. Dev Biol 300:219–237. doi:10.1016/j.ydbio.2006.08.052 PubMedCrossRefGoogle Scholar
  31. 31.
    Duboc V, Röttinger E, Lapraz F, Besnardeau L, Lepage T (2005) Left-right asymmetry in the sea urchin embryo is regulated by nodal signaling on the right side. Dev Cell 9(1):147–158. doi:10.1016/j.devcel.2005.05.008 PubMedCrossRefGoogle Scholar
  32. 32.
    Dibb NJ, Newman AJ (1989) Evidence that introns arose at proto-splice sites. EMBO J 8:2015–2021PubMedGoogle Scholar
  33. 33.
    Perumal BS, Sakharkar KR, Chow VT, Pandjassarame K, Sakharkar MK (2005) Intron position conservation across eukaryotic lineages in tubulin genes. Front Biosci 10:2412–2419. doi:10.2741/1706 PubMedCrossRefGoogle Scholar
  34. 34.
    Grumbling G, Strelets V (2006) FlyBase: anatomical data, images and queries. Nucleic Acids Res 34:D484–D488. doi:10.1093/nar/gkj068 PubMedCrossRefGoogle Scholar
  35. 35.
    Yuh C, Brown CT, Livi CB, Rowen L, Clarke PJC, Davidson EH (2002) Patchy interspecific sequence similarities efficiently identify positive cis-regulatory elements in the sea urchin. Dev Biol 246(1):148–161. doi:10.1006/dbio.2002.0618 PubMedCrossRefGoogle Scholar
  36. 36.
    Frazer KA, Pachter L, Poliakov A, Rubin EM, Dubchak I (2004) VISTA: computational tools for comparative genomics. Nucleic Acids Res 32:W273–W279. doi:10.1093/nar/gkh458 PubMedCrossRefGoogle Scholar
  37. 37.
    Burke RD, Angerer LM, Elphick MR, Humphrey GW, Yaguchi S, Kiyama T, Liang S, Mu X, Agca C, Klein WH, Brandhorst BP, Rowe M, Wilson K, Churcher AM, Taylor JS, Chen N, Murray G, Wang D, Mellott D, Olinski R, Hallböök F, Thorndyke MC (2006) A genomic view of the sea urchin nervous system. Dev Biol 300:434–460. doi:10.1016/j.ydbio.2006.08.007 PubMedCrossRefGoogle Scholar
  38. 38.
    Coffman JA, Kirchhamer CV, Harrington MG, Davidson EH (1997) SpMyb functions as an intramodular repressor to regulate spatial expression of CyIIIa in sea urchin embryos. Development 124:4717–4727PubMedGoogle Scholar
  39. 39.
    Phillips K, Luisi B (2000) The virtuoso of versatility: POU proteins that flex to fit. J Mol Biol 302:1023–1039. doi:10.1006/jmbi.2000.4107 PubMedCrossRefGoogle Scholar
  40. 40.
    Poustka AJ, Kühn A, Groth D, Weise V, Yaguchi S, Burke RD, Herwig R, Lehrach H, Panopoulou G (2007) A global view of gene expression in lithium and zinc treated sea urchin embryos: new components of gene regulatory networks. Genome Biol 8:R85. doi:10.1186/gb-2007-8-5-r85 PubMedCrossRefGoogle Scholar
  41. 41.
    Tu Q, Brown CT, Davidson EH, Oliveri P (2006) Sea urchin Forkhead gene family: phylogeny and embryonic expression. Dev Biol 300:49–62. doi:10.1016/j.ydbio.2006.09.031 PubMedCrossRefGoogle Scholar
  42. 42.
    Cameron RA, Rowen L, Nesbitt R, Bloom S, Rast JP, Berney K, Arenas-Mena C, Martinez P, Lucas S, Richardson PM, Davidson EH, Peterson KJ, Hood L (2006) Unusual gene order and organization of the sea urchin hox cluster. J Exp Zool B Mol Dev Evol 306:45–58. doi:10.1002/jez.b.21070 PubMedCrossRefGoogle Scholar
  43. 43.
    Garcia-Fernàndez J (2005) Hox, ParaHox, ProtoHox: facts and guesses. Heredity 94:145–152. doi:10.1038/sj.hdy.6800621 PubMedCrossRefGoogle Scholar
  44. 44.
    Arnone MI, Rizzo F, Annunciata R, Cameron RA, Peterson KJ, Martínez P (2006) Genetic organization and embryonic expression of the ParaHox genes in the sea urchin S. purpuratus: insights into the relationship between clustering and colinearity. Dev Biol 300:63–73. doi:10.1016/j.ydbio.2006.07.037 PubMedCrossRefGoogle Scholar
  45. 45.
    Brooke NM, Garcia-Fernàndez J, Holland PW (1998) The ParaHox gene cluster is an evolutionary sister of the Hox gene cluster. Nature 392:920–922. doi:10.1038/31933 PubMedCrossRefGoogle Scholar
  46. 46.
    Hatano M, Iitsuka Y, Yamamoto H, Dezawa M, Yusa S, Kohno Y, Tokuhisa T (1997) Ncx, a Hox11 related gene, is expressed in a variety of tissues derived from neural crest cells. Anat Embryol 195:419–425. doi:10.1007/s004290050061 PubMedCrossRefGoogle Scholar
  47. 47.
    Shirasawa S, Yunker AMR, Roth KA, Brown GA, Horning S, Korsmeyer SJ (1997) Enx (Hox11L1)-deficient mice develop myenteric neuronal hyperplasia and megacolon. Nat Med 3:646–650. doi:10.1038/nm0697-646 PubMedCrossRefGoogle Scholar
  48. 48.
    McMahon AP (2000) Neural patterning: the role of Nkx genes in the ventral spinal cord. Genes Dev 14:2261–2264. doi:10.1101/gad.840800 PubMedCrossRefGoogle Scholar
  49. 49.
    Cheesman SE, Layden MJ, Von Ohlen T, Doe CQ, Eisen JS (2004) Zebrafish and fly Nkx6 proteins have similar CNS expression patterns and regulate motoneuron formation. Development 131:5221–5232. doi:10.1242/dev.01397 PubMedCrossRefGoogle Scholar
  50. 50.
    Pattyn A, Vallstedt A, Dias JM, Sander M, Ericson J (2003) Complementary roles for Nkx6 and Nkx2 class proteins in the establishment of motoneuron identity in the hindbrain. Development 130:4149–4159. doi:10.1242/dev.00641 PubMedCrossRefGoogle Scholar
  51. 51.
    Takacs CM, Amore G, Oliveri P, Poustka AJ, Wang D, Burke RD, Peterson KJ (2004) Expression of an NK2 homeodomain gene in the apical ectoderm defines a new territory in the early sea urchin embryo. Dev Biol 269:152–164. doi:10.1016/j.ydbio.2004.01.023 PubMedCrossRefGoogle Scholar
  52. 52.
    Smolenicka Z, Pani F, Hwang KJ, Gruschus JM, Ferretti JA (2003) Sequence of a conserved region of a new sea urchin homeobox gene from the NK family. Cell Biol Int 27:81–87PubMedCrossRefGoogle Scholar
  53. 53.
    Dunn EF, Moy VN, Angerer LM, Angerer RC, Morris RL, Peterson KJ (2007) Molecular paleoecology: using gene regulatory analysis to address the origins of complex life cycles in the late Precambrian. Evol Dev 9:10–24. doi:10.1111/j.1525-142X.2006.00134.x PubMedCrossRefGoogle Scholar
  54. 54.
    Ludueña RF, Banerjee A (2008) The isotypes of tubulin. Distribution and functional significance. In: Fojo T (ed) The role of microtubules in cell biology neurobiology and oncology. Humana Press, Totowa, pp 123–175. doi:10.1007/978-1-59745-336-3 CrossRefGoogle Scholar
  55. 55.
    Casano C, Ragusa M, Cutrera M, Costa S, Gianguzza F (1996) Spatial expression of alpha and beta tubulin genes in the late embryogenesis of the sea urchin P. lividus. Int J Dev Biol 40:1033–1041Google Scholar
  56. 56.
    Otim O, Amore G, Minokawa T, McClay DR, Davidson EH (2004) SpHnf6, a transcription factor that executes multiple functions in sea urchin embryogenesis. Dev Biol 273:226–243. doi:10.1016/j.ydbio.2004.05.033 PubMedCrossRefGoogle Scholar
  57. 57.
    Stöbe P, Stein MAS, Habring-Müller A, Bezdan D, Fuchs AL, Hueber SD, Wu H, Lohmann I (2009) Multifactorial regulation of a hox target gene. PLoS Genet 5:e1000412. doi:10.1371/journal.pgen.1000412 PubMedCrossRefGoogle Scholar

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© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  1. 1.Dipartimento di Scienze e Tecnologie Molecolari e BiomolecolariUniversità degli Studi di PalermoPalermoItaly

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