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. 1995 Mar;15(3):1254–1264. doi: 10.1128/mcb.15.3.1254

The embryonic enhancer-binding protein SSAP contains a novel DNA-binding domain which has homology to several RNA-binding proteins.

D J DeAngelo 1, J DeFalco 1, L Rybacki 1, G Childs 1
PMCID: PMC230348  PMID: 7862119

Abstract

Stage-specific activator protein (SSAP) is a 43-kDa polypeptide that binds to an enhancer element of the sea urchin late histone H1 gene. This enhancer element mediates the transcriptional activation of the late histone H1 gene in a temporally specific manner at the mid-blastula stage of embryogenesis. We have cloned cDNAs encoding SSAP by using polyclonal antibodies raised against purified SSAP to screen expression libraries. SSAP is unrelated to previously characterized transcription factors; however, it exhibits striking homology to a large family of proteins involved in RNA processing. The protein is a sequence-specific DNA-binding protein that recognizes both single- and double-stranded DNA. The DNA-binding domain of the protein was localized to the conserved RNA recognition motif (RRM). In addition to tandem copies of this conserved domain, SSAP contains a central domain that is rich in glutamine and glycine and a C-terminal domain that is enriched in serine, threonine, and basic amino acids. Overexpression of SSAP in sea urchin embryos by microinjection of either synthetic mRNA or an SSAP expression vector results in four- to eightfold transactivation of target reporter genes that contain the enhancer sequence. Transactivation occurs beginning only at the mid-blastula stage of development, suggesting that SSAP must be modified in a stage-specific manner in order to activate transcription. In addition, there are a number of other RRM-containing proteins that contain glutamine-rich regions which are postulated to function in the regulation of RNA processing. Instead, we suggest that SSAP is a member of a family of glutamine-rich RRM proteins which constitute a novel class of transcription factors.

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Selected References

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  1. Bellen H. J., Kooyer S., D'Evelyn D., Pearlman J. The Drosophila couch potato protein is expressed in nuclei of peripheral neuronal precursors and shows homology to RNA-binding proteins. Genes Dev. 1992 Nov;6(11):2125–2136. doi: 10.1101/gad.6.11.2125. [DOI] [PubMed] [Google Scholar]
  2. Boggs R. T., Gregor P., Idriss S., Belote J. M., McKeown M. Regulation of sexual differentiation in D. melanogaster via alternative splicing of RNA from the transformer gene. Cell. 1987 Aug 28;50(5):739–747. doi: 10.1016/0092-8674(87)90332-1. [DOI] [PubMed] [Google Scholar]
  3. Cameron R. A., Smith L. C., Britten R. J., Davidson E. H. Ligand-dependent stimulation of introduced mammalian brain receptors alters spicule symmetry and other morphogenetic events in sea urchin embryos. Mech Dev. 1994 Jan;45(1):31–47. doi: 10.1016/0925-4773(94)90051-5. [DOI] [PubMed] [Google Scholar]
  4. Cantor C. R. DNA choreography. Cell. 1981 Aug;25(2):293–295. doi: 10.1016/0092-8674(81)90045-3. [DOI] [PubMed] [Google Scholar]
  5. Childs G., Nocente-McGrath C., Lieber T., Holt C., Knowles J. A. Sea urchin (lytechinus pictus) late-stage histone H3 and H4 genes: characterization and mapping of a clustered but nontandemly linked multigene family. Cell. 1982 Dec;31(2 Pt 1):383–393. doi: 10.1016/0092-8674(82)90132-5. [DOI] [PubMed] [Google Scholar]
  6. Cohen L. H., Newrock K. M., Zweidler A. Stage-specific switches in histone synthesis during embryogenesis of the sea urchin. Science. 1975 Dec 5;190(4218):994–997. doi: 10.1126/science.1237932. [DOI] [PubMed] [Google Scholar]
  7. Colin A. M. Rapid repetitive microinjection. Methods Cell Biol. 1986;27:395–406. [PubMed] [Google Scholar]
  8. Courey A. J., Holtzman D. A., Jackson S. P., Tjian R. Synergistic activation by the glutamine-rich domains of human transcription factor Sp1. Cell. 1989 Dec 1;59(5):827–836. doi: 10.1016/0092-8674(89)90606-5. [DOI] [PubMed] [Google Scholar]
  9. Courey A. J., Tjian R. Analysis of Sp1 in vivo reveals multiple transcriptional domains, including a novel glutamine-rich activation motif. Cell. 1988 Dec 2;55(5):887–898. doi: 10.1016/0092-8674(88)90144-4. [DOI] [PubMed] [Google Scholar]
  10. Crozat A., Aman P., Mandahl N., Ron D. Fusion of CHOP to a novel RNA-binding protein in human myxoid liposarcoma. Nature. 1993 Jun 17;363(6430):640–644. doi: 10.1038/363640a0. [DOI] [PubMed] [Google Scholar]
  11. DeAngelo D. J., DeFalco J., Childs G. Purification and characterization of the stage-specific embryonic enhancer-binding protein SSAP-1. Mol Cell Biol. 1993 Mar;13(3):1746–1758. doi: 10.1128/mcb.13.3.1746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Delattre O., Zucman J., Plougastel B., Desmaze C., Melot T., Peter M., Kovar H., Joubert I., de Jong P., Rouleau G. Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours. Nature. 1992 Sep 10;359(6391):162–165. doi: 10.1038/359162a0. [DOI] [PubMed] [Google Scholar]
  13. DiLiberto M., Lai Z. C., Fei H., Childs G. Developmental control of promoter-specific factors responsible for the embryonic activation and inactivation of the sea urchin early histone H3 gene. Genes Dev. 1989 Jul;3(7):973–985. doi: 10.1101/gad.3.7.973. [DOI] [PubMed] [Google Scholar]
  14. Dreyfuss G., Matunis M. J., Piñol-Roma S., Burd C. G. hnRNP proteins and the biogenesis of mRNA. Annu Rev Biochem. 1993;62:289–321. doi: 10.1146/annurev.bi.62.070193.001445. [DOI] [PubMed] [Google Scholar]
  15. Duncan R., Bazar L., Michelotti G., Tomonaga T., Krutzsch H., Avigan M., Levens D. A sequence-specific, single-strand binding protein activates the far upstream element of c-myc and defines a new DNA-binding motif. Genes Dev. 1994 Feb 15;8(4):465–480. doi: 10.1101/gad.8.4.465. [DOI] [PubMed] [Google Scholar]
  16. Gaillard C., Strauss F. Sequence-specific single-strand-binding protein for the simian virus 40 early promoter stimulates transcription in vitro. J Mol Biol. 1990 Sep 20;215(2):245–255. doi: 10.1016/S0022-2836(05)80343-2. [DOI] [PubMed] [Google Scholar]
  17. Gan L., Wessel G. M., Klein W. H. Regulatory elements from the related spec genes of Strongylocentrotus purpuratus yield different spatial patterns with a lacZ reporter gene. Dev Biol. 1990 Dec;142(2):346–359. doi: 10.1016/0012-1606(90)90355-m. [DOI] [PubMed] [Google Scholar]
  18. Grange T., de Sa C. M., Oddos J., Pictet R. Human mRNA polyadenylate binding protein: evolutionary conservation of a nucleic acid binding motif. Nucleic Acids Res. 1987 Jun 25;15(12):4771–4787. doi: 10.1093/nar/15.12.4771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hentschel C. C. Homocopolymer sequences in the spacer of a sea urchin histone gene repeat are sensitive to S1 nuclease. Nature. 1982 Feb 25;295(5851):714–716. doi: 10.1038/295714a0. [DOI] [PubMed] [Google Scholar]
  20. Hoey T., Levine M. Divergent homeo box proteins recognize similar DNA sequences in Drosophila. Nature. 1988 Apr 28;332(6167):858–861. doi: 10.1038/332858a0. [DOI] [PubMed] [Google Scholar]
  21. Ito M., Bell J., Lyons G., Maxson R. Synthesis and turnover of late H2B histone mRNA in developing embryos of the sea urchin, Strongylocentrotus purpuratus. Dev Biol. 1988 Sep;129(1):147–158. doi: 10.1016/0012-1606(88)90169-8. [DOI] [PubMed] [Google Scholar]
  22. Jantzen H. M., Admon A., Bell S. P., Tjian R. Nucleolar transcription factor hUBF contains a DNA-binding motif with homology to HMG proteins. Nature. 1990 Apr 26;344(6269):830–836. doi: 10.1038/344830a0. [DOI] [PubMed] [Google Scholar]
  23. Kenan D. J., Query C. C., Keene J. D. RNA recognition: towards identifying determinants of specificity. Trends Biochem Sci. 1991 Jun;16(6):214–220. doi: 10.1016/0968-0004(91)90088-d. [DOI] [PubMed] [Google Scholar]
  24. Knowles J. A., Childs G. J. Comparison of the late H1 histone genes of the sea urchins Lytechinus pictus and Strongelocentrotus purpuratus. Nucleic Acids Res. 1986 Oct 24;14(20):8121–8133. doi: 10.1093/nar/14.20.8121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Knowles J. A., Childs G. J. Temporal expression of late histone messenger RNA in the sea urchin Lytechinus pictus. Proc Natl Acad Sci U S A. 1984 Apr;81(8):2411–2415. doi: 10.1073/pnas.81.8.2411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Knowles J. A., Lai Z. C., Childs G. J. Isolation, characterization, and expression of the gene encoding the late histone subtype H1-gamma of the sea urchin Strongylocentrotus purpuratus. Mol Cell Biol. 1987 Jan;7(1):478–485. doi: 10.1128/mcb.7.1.478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Krainer A. R., Mayeda A., Kozak D., Binns G. Functional expression of cloned human splicing factor SF2: homology to RNA-binding proteins, U1 70K, and Drosophila splicing regulators. Cell. 1991 Jul 26;66(2):383–394. doi: 10.1016/0092-8674(91)90627-b. [DOI] [PubMed] [Google Scholar]
  28. La Teana A., Brandi A., Falconi M., Spurio R., Pon C. L., Gualerzi C. O. Identification of a cold shock transcriptional enhancer of the Escherichia coli gene encoding nucleoid protein H-NS. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10907–10911. doi: 10.1073/pnas.88.23.10907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lai Z. C., Childs G. Characterization of the structure and transcriptional patterns of the gene encoding the late histone subtype H1-beta of the sea urchin Strongylocentrotus purpuratus. Mol Cell Biol. 1988 Apr;8(4):1842–1844. doi: 10.1128/mcb.8.4.1842. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lai Z. C., DeAngelo D. J., DiLiberto M., Childs G. An embryonic enhancer determines the temporal activation of a sea urchin late H1 gene. Mol Cell Biol. 1989 Jun;9(6):2315–2321. doi: 10.1128/mcb.9.6.2315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lai Z. C., Maxson R., Childs G. Both basal and ontogenic promoter elements affect the timing and level of expression of a sea urchin H1 gene during early embryogenesis. Genes Dev. 1988 Feb;2(2):173–183. doi: 10.1101/gad.2.2.173. [DOI] [PubMed] [Google Scholar]
  32. Larsen A., Weintraub H. An altered DNA conformation detected by S1 nuclease occurs at specific regions in active chick globin chromatin. Cell. 1982 Jun;29(2):609–622. doi: 10.1016/0092-8674(82)90177-5. [DOI] [PubMed] [Google Scholar]
  33. Mancebo R., Lo P. C., Mount S. M. Structure and expression of the Drosophila melanogaster gene for the U1 small nuclear ribonucleoprotein particle 70K protein. Mol Cell Biol. 1990 Jun;10(6):2492–2502. doi: 10.1128/mcb.10.6.2492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Maxson R., Cohn R., Kedes L., Mohun T. Expression and organization of histone genes. Annu Rev Genet. 1983;17:239–277. doi: 10.1146/annurev.ge.17.120183.001323. [DOI] [PubMed] [Google Scholar]
  35. May W. A., Lessnick S. L., Braun B. S., Klemsz M., Lewis B. C., Lunsford L. B., Hromas R., Denny C. T. The Ewing's sarcoma EWS/FLI-1 fusion gene encodes a more potent transcriptional activator and is a more powerful transforming gene than FLI-1. Mol Cell Biol. 1993 Dec;13(12):7393–7398. doi: 10.1128/mcb.13.12.7393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. McMahon A. P., Novak T. J., Britten R. J., Davidson E. H. Inducible expression of a cloned heat shock fusion gene in sea urchin embryos. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7490–7494. doi: 10.1073/pnas.81.23.7490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Merrill B. M., Stone K. L., Cobianchi F., Wilson S. H., Williams K. R. Phenylalanines that are conserved among several RNA-binding proteins form part of a nucleic acid-binding pocket in the A1 heterogeneous nuclear ribonucleoprotein. J Biol Chem. 1988 Mar 5;263(7):3307–3313. [PubMed] [Google Scholar]
  38. Nickol J. M., Felsenfeld G. DNA conformation at the 5' end of the chicken adult beta-globin gene. Cell. 1983 Dec;35(2 Pt 1):467–477. doi: 10.1016/0092-8674(83)90180-0. [DOI] [PubMed] [Google Scholar]
  39. Nordheim A., Rich A. Negatively supercoiled simian virus 40 DNA contains Z-DNA segments within transcriptional enhancer sequences. Nature. 1983 Jun 23;303(5919):674–679. doi: 10.1038/303674a0. [DOI] [PubMed] [Google Scholar]
  40. Olson M. O., Rivers Z. M., Thompson B. A., Kao W. Y., Case S. T. Interaction of nucleolar phosphoprotein C23 with cloned segments of rat ribosomal deoxyribonucleic acid. Biochemistry. 1983 Jul 5;22(14):3345–3351. doi: 10.1021/bi00283a007. [DOI] [PubMed] [Google Scholar]
  41. Pabo C. O., Sauer R. T. Transcription factors: structural families and principles of DNA recognition. Annu Rev Biochem. 1992;61:1053–1095. doi: 10.1146/annurev.bi.61.070192.005201. [DOI] [PubMed] [Google Scholar]
  42. Query C. C., Bentley R. C., Keene J. D. A common RNA recognition motif identified within a defined U1 RNA binding domain of the 70K U1 snRNP protein. Cell. 1989 Apr 7;57(1):89–101. doi: 10.1016/0092-8674(89)90175-x. [DOI] [PubMed] [Google Scholar]
  43. Ranjan M., Tafuri S. R., Wolffe A. P. Masking mRNA from translation in somatic cells. Genes Dev. 1993 Sep;7(9):1725–1736. doi: 10.1101/gad.7.9.1725. [DOI] [PubMed] [Google Scholar]
  44. Santoro I. M., Yi T. M., Walsh K. Identification of single-stranded-DNA-binding proteins that interact with muscle gene elements. Mol Cell Biol. 1991 Apr;11(4):1944–1953. doi: 10.1128/mcb.11.4.1944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sarkar G., Sommer S. S. Access to a messenger RNA sequence or its protein product is not limited by tissue or species specificity. Science. 1989 Apr 21;244(4902):331–334. doi: 10.1126/science.2565599. [DOI] [PubMed] [Google Scholar]
  46. Scherly D., Boelens W., Dathan N. A., van Venrooij W. J., Mattaj I. W. Major determinants of the specificity of interaction between small nuclear ribonucleoproteins U1A and U2B'' and their cognate RNAs. Nature. 1990 Jun 7;345(6275):502–506. doi: 10.1038/345502a0. [DOI] [PubMed] [Google Scholar]
  47. Schindelin H., Marahiel M. A., Heinemann U. Universal nucleic acid-binding domain revealed by crystal structure of the B. subtilis major cold-shock protein. Nature. 1993 Jul 8;364(6433):164–168. doi: 10.1038/364164a0. [DOI] [PubMed] [Google Scholar]
  48. Schon E., Evans T., Welsh J., Efstratiadis A. Conformation of promoter DNA: fine mapping of S1-hypersensitive sites. Cell. 1983 Dec;35(3 Pt 2):837–848. doi: 10.1016/0092-8674(83)90116-2. [DOI] [PubMed] [Google Scholar]
  49. Simpson R. T. Modulation of nucleosome structure by histone subtypes in sea urchin embryos. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6803–6807. doi: 10.1073/pnas.78.11.6803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  51. Sturm R. A., Das G., Herr W. The ubiquitous octamer-binding protein Oct-1 contains a POU domain with a homeo box subdomain. Genes Dev. 1988 Dec;2(12A):1582–1599. doi: 10.1101/gad.2.12a.1582. [DOI] [PubMed] [Google Scholar]
  52. Tada H., Khalili K. A novel sequence-specific DNA-binding protein, LCP-1, interacts with single-stranded DNA and differentially regulates early gene expression of the human neurotropic JC virus. J Virol. 1992 Dec;66(12):6885–6892. doi: 10.1128/jvi.66.12.6885-6892.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Tafuri S. R., Wolffe A. P. Xenopus Y-box transcription factors: molecular cloning, functional analysis and developmental regulation. Proc Natl Acad Sci U S A. 1990 Nov;87(22):9028–9032. doi: 10.1073/pnas.87.22.9028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Takimoto M., Tomonaga T., Matunis M., Avigan M., Krutzsch H., Dreyfuss G., Levens D. Specific binding of heterogeneous ribonucleoprotein particle protein K to the human c-myc promoter, in vitro. J Biol Chem. 1993 Aug 25;268(24):18249–18258. [PubMed] [Google Scholar]
  55. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Turner R., Tjian R. Leucine repeats and an adjacent DNA binding domain mediate the formation of functional cFos-cJun heterodimers. Science. 1989 Mar 31;243(4899):1689–1694. doi: 10.1126/science.2494701. [DOI] [PubMed] [Google Scholar]
  57. Weinberger C., Hollenberg S. M., Ong E. S., Harmon J. M., Brower S. T., Cidlowski J., Thompson E. B., Rosenfeld M. G., Evans R. M. Identification of human glucocorticoid receptor complementary DNA clones by epitope selection. Science. 1985 May 10;228(4700):740–742. doi: 10.1126/science.2581314. [DOI] [PubMed] [Google Scholar]
  58. Wu H. M., Crothers D. M. The locus of sequence-directed and protein-induced DNA bending. Nature. 1984 Apr 5;308(5959):509–513. doi: 10.1038/308509a0. [DOI] [PubMed] [Google Scholar]
  59. Young R. A., Davis R. W. Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1194–1198. doi: 10.1073/pnas.80.5.1194. [DOI] [PMC free article] [PubMed] [Google Scholar]

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