Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1993 Mar;13(3):1746–1758. doi: 10.1128/mcb.13.3.1746

Purification and characterization of the stage-specific embryonic enhancer-binding protein SSAP-1.

D J DeAngelo 1, J DeFalco 1, G Childs 1
PMCID: PMC359487  PMID: 8441410

Abstract

We have demonstrated that a highly conserved segment of DNA between positions -288 and -317 (upstream sequence element IV [USE IV]) is largely responsible for the transcriptional activation of the sea urchin H1-beta histone gene during the blastula stage of embryogenesis. This sequence is capable of acting as an embryonic enhancer element, activating target genes in a stage-specific manner. Nuclear extracts prepared from developmentally-staged organisms before and after the gene is activated all contain a factor which specifically binds to the enhancer. We have purified a 43-kDa polypeptide which binds to and footprints the USE IV enhancer element. We refer to this protein as stage-specific activator protein 1 (SSAP-1). Early in development before the enhancer is active, SSAP appears as a 43-kDa monomer, but it undergoes a change in its molecular weight beginning at about 12 h postfertilization (early blastula) which precisely parallels the increase in H1-beta gene expression. Modified SSAP has an apparent molecular mass of approximately 90 to 100 kDa and contains at least one 43-kDa SSAP polypeptide. Thus, it is the disappearance of the 43-kDa species and the appearance of the 90- to 100-kDa species which coincide with the H1-beta gene activation. The correlation between the change in molecular weight of SSAP and the stage-specific activation of H1-beta gene expression strongly suggests that this higher-molecular-weight form of SSAP is directly responsible for the blastula stage-specific transcriptional activation of the late H1 gene.

Full text

PDF
1746

Images in this article

1749Image
on p.1749
1750Image
on p.1750
1750Image
on p.1750
1752Image
on p.1752
1753Image
on p.1753
1753Image
on p.1753
1753Image
on p.1753
1754Image
on p.1754
1755Image
on p.1755
1755Image
on p.1755

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ansorge W. Fast and sensitive detection of protein and DNA bands by treatment with potassium permanganate. J Biochem Biophys Methods. 1985 May;11(1):13–20. doi: 10.1016/0165-022x(85)90037-5. [DOI] [PubMed] [Google Scholar]
  2. Biggin M. D., Tjian R. Transcription factors that activate the Ultrabithorax promoter in developmentally staged extracts. Cell. 1988 Jun 3;53(5):699–711. doi: 10.1016/0092-8674(88)90088-8. [DOI] [PubMed] [Google Scholar]
  3. Briggs M. R., Kadonaga J. T., Bell S. P., Tjian R. Purification and biochemical characterization of the promoter-specific transcription factor, Sp1. Science. 1986 Oct 3;234(4772):47–52. doi: 10.1126/science.3529394. [DOI] [PubMed] [Google Scholar]
  4. Calzone F. J., Thézé N., Thiebaud P., Hill R. L., Britten R. J., Davidson E. H. Developmental appearance of factors that bind specifically to cis-regulatory sequences of a gene expressed in the sea urchin embryo. Genes Dev. 1988 Sep;2(9):1074–1088. doi: 10.1101/gad.2.9.1074. [DOI] [PubMed] [Google Scholar]
  5. Childs G., Maxson R., Kedes L. H. Histone gene expression during sea urchin embryogenesis: isolation and characterization of early and late messenger RNAs of Strongylocentrotus purpuratus by gene-specific hybridization and template activity. Dev Biol. 1979 Nov;73(1):153–173. doi: 10.1016/0012-1606(79)90144-1. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Colin A. M., Catlin T. L., Kidson S. H., Maxson R. Closely linked early and late histone H2B genes are differentially expressed after microinjection into sea urchin zygotes. Proc Natl Acad Sci U S A. 1988 Jan;85(2):507–510. doi: 10.1073/pnas.85.2.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Colin A. M. Rapid repetitive microinjection. Methods Cell Biol. 1986;27:395–406. [PubMed] [Google Scholar]
  9. Curran T., Franza B. R., Jr Fos and Jun: the AP-1 connection. Cell. 1988 Nov 4;55(3):395–397. doi: 10.1016/0092-8674(88)90024-4. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Flytzanis C. N., Britten R. J., Davidson E. H. Ontogenic activation of a fusion gene introduced into sea urchin eggs. Proc Natl Acad Sci U S A. 1987 Jan;84(1):151–155. doi: 10.1073/pnas.84.1.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Galas D. J., Schmitz A. DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 1978 Sep;5(9):3157–3170. doi: 10.1093/nar/5.9.3157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gan L., Zhang W., Klein W. H. Repetitive DNA sequences linked to the sea urchin spec genes contain transcriptional enhancer-like elements. Dev Biol. 1990 May;139(1):186–196. doi: 10.1016/0012-1606(90)90287-s. [DOI] [PubMed] [Google Scholar]
  14. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hai T. W., Liu F., Allegretto E. A., Karin M., Green M. R. A family of immunologically related transcription factors that includes multiple forms of ATF and AP-1. Genes Dev. 1988 Oct;2(10):1216–1226. doi: 10.1101/gad.2.10.1216. [DOI] [PubMed] [Google Scholar]
  16. Halazonetis T. D., Georgopoulos K., Greenberg M. E., Leder P. c-Jun dimerizes with itself and with c-Fos, forming complexes of different DNA binding affinities. Cell. 1988 Dec 2;55(5):917–924. doi: 10.1016/0092-8674(88)90147-x. [DOI] [PubMed] [Google Scholar]
  17. Heberlein U., Tjian R. Temporal pattern of alcohol dehydrogenase gene transcription reproduced by Drosophila stage-specific embryonic extracts. Nature. 1988 Feb 4;331(6155):410–415. doi: 10.1038/331410a0. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Hough-Evans B. R., Franks R. R., Cameron R. A., Britten R. J., Davidson E. H. Correct cell-type-specific expression of a fusion gene injected into sea urchin eggs. Dev Biol. 1987 Jun;121(2):576–579. doi: 10.1016/0012-1606(87)90193-x. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Kadonaga J. T., Tjian R. Affinity purification of sequence-specific DNA binding proteins. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5889–5893. doi: 10.1073/pnas.83.16.5889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Katula K. S., Hough-Evans B. R., Britten R. J., Davidson E. H. Ontogenic expression of a CyI actin fusion gene injected into sea urchin eggs. Development. 1987 Nov;101(3):437–447. doi: 10.1242/dev.101.3.437. [DOI] [PubMed] [Google Scholar]
  23. Kaumeyer J. F., Weinberg E. S. Sequence, organization and expression of late embryonic H3 and H4 histone genes from the sea urchin, Strongylocentrotus purpuratus. Nucleic Acids Res. 1986 Jun 11;14(11):4557–4576. doi: 10.1093/nar/14.11.4557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kemler I., Busslinger M. Characterization of two nonallelic pairs of late histone H2A and H2B genes of the sea urchin: differential regulation in the embryo and tissue-specific expression in the adult. Mol Cell Biol. 1986 Nov;6(11):3746–3754. doi: 10.1128/mcb.6.11.3746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. 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]
  28. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [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. Lieber T., Weisser K., Childs G. Analysis of histone gene expression in adult tissues of the sea urchins Strongylocentrotus purpuratus and Lytechinus pictus: tissue-specific expression of sperm histone genes. Mol Cell Biol. 1986 Jul;6(7):2602–2612. doi: 10.1128/mcb.6.7.2602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mauron A., Kedes L., Hough-Evans B. R., Davidson E. H. Accumulation of individual histone mRNAs during embryogenesis of the sea urchin Strongylocentrotus purpuratus. Dev Biol. 1982 Dec;94(2):425–434. doi: 10.1016/0012-1606(82)90359-1. [DOI] [PubMed] [Google Scholar]
  34. Maxson R. E., Jr, Wilt F. H. Accumulation of the early histone messenger RNAs during the development of Strongylocentrotus purpuratus. Dev Biol. 1982 Dec;94(2):435–440. doi: 10.1016/0012-1606(82)90360-8. [DOI] [PubMed] [Google Scholar]
  35. Maxson R. E., Jr, Wilt F. H. The rate of synthesis of histone mRNA during the development of sea urchin embryos (Strongylocentrotus purpuratus). Dev Biol. 1981 Apr 30;83(2):380–386. doi: 10.1016/0012-1606(81)90485-1. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Maxson R., Mohun T., Gormezano G., Childs G., Kedes L. Distinct organizations and patterns of expression of early and late histone gene sets in the sea urchin. Nature. 1983 Jan 13;301(5896):120–125. doi: 10.1038/301120a0. [DOI] [PubMed] [Google Scholar]
  38. McMahon A. P., Flytzanis C. N., Hough-Evans B. R., Katula K. S., Britten R. J., Davidson E. H. Introduction of cloned DNA into sea urchin egg cytoplasm: replication and persistence during embryogenesis. Dev Biol. 1985 Apr;108(2):420–430. doi: 10.1016/0012-1606(85)90045-4. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Murre C., McCaw P. S., Baltimore D. A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell. 1989 Mar 10;56(5):777–783. doi: 10.1016/0092-8674(89)90682-x. [DOI] [PubMed] [Google Scholar]
  41. Murre C., McCaw P. S., Vaessin H., Caudy M., Jan L. Y., Jan Y. N., Cabrera C. V., Buskin J. N., Hauschka S. D., Lassar A. B. Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence. Cell. 1989 Aug 11;58(3):537–544. doi: 10.1016/0092-8674(89)90434-0. [DOI] [PubMed] [Google Scholar]
  42. Peterson C. L., Calame K. Proteins binding to site C2 (muE3) in the immunoglobulin heavy-chain enhancer exist in multiple oligomeric forms. Mol Cell Biol. 1989 Feb;9(2):776–786. doi: 10.1128/mcb.9.2.776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Reith W., Herrero-Sanchez C., Kobr M., Silacci P., Berte C., Barras E., Fey S., Mach B. MHC class II regulatory factor RFX has a novel DNA-binding domain and a functionally independent dimerization domain. Genes Dev. 1990 Sep;4(9):1528–1540. doi: 10.1101/gad.4.9.1528. [DOI] [PubMed] [Google Scholar]
  44. Ricci F., Stein E. M. Oscillatory singular integrals and harmonic analysis on nilpotent groups. Proc Natl Acad Sci U S A. 1986 Jan;83(1):1–3. doi: 10.1073/pnas.83.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tomlinson C. R., Klein W. H. Temporal and spatial transcriptional regulation of the aboral ectoderm-specific Spec genes during sea urchin embryogenesis. Mol Reprod Dev. 1990 Apr;25(4):328–338. doi: 10.1002/mrd.1080250404. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Vitelli L., Kemler I., Lauber B., Birnstiel M. L., Busslinger M. Developmental regulation of micro-injected histone genes in sea urchin embryos. Dev Biol. 1988 May;127(1):54–63. doi: 10.1016/0012-1606(88)90188-1. [DOI] [PubMed] [Google Scholar]
  48. Weinberg E. S., Hendricks M. B., Hemminki K., Kuwabara P. E., Farrelly L. A. Timing and rates of synthesis of early histone mRNA in the embryo of Strongylocentrotus purpuratus. Dev Biol. 1983 Jul;98(1):117–129. doi: 10.1016/0012-1606(83)90340-8. [DOI] [PubMed] [Google Scholar]
  49. Yamamoto K. K., Gonzalez G. A., Biggs W. H., 3rd, Montminy M. R. Phosphorylation-induced binding and transcriptional efficacy of nuclear factor CREB. Nature. 1988 Aug 11;334(6182):494–498. doi: 10.1038/334494a0. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES