Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

Nucleic Acids Research logoLink to Nucleic Acids Research
. 1988 Jul 11;16(13):5771–5781. doi: 10.1093/nar/16.13.5771

Heavy metal ions in transcription factors from HeLa cells: Sp1, but not octamer transcription factor requires zinc for DNA binding and for activator function.

G Westin 1, W Schaffner 1
PMCID: PMC336828  PMID: 3135532

Abstract

Zinc is an important cofactor for many enzymes involved in nucleic acid metabolism such as DNA and RNA polymerases, reverse transcriptase and tRNA synthetases. We have developed an inducible in vitro transcription system using metal-depleted nuclear extracts to reveal the presence and functional relevance of heavy metal ions in transcription factors. Using protein-DNA binding assays (band shift and DNAase I footprint) we show that Sp1, a promoter-specific vertebrate transcription factor that binds to the "GC box" (Sequence in text), is reversibly inactivated by metal-depletion. Zinc is required for specific DNA binding in vitro and is also essential for Sp1 factor-directed transcription. In contrast, another factor from HeLa cells, the so-called octamer transcription factor (OTF) that binds to the sequence 5'-ATGCAAATNA, is not affected by metal-depletion and thus seems not to be a zinc metalloprotein.

Full text

PDF
5771

Images in this article

Selected References

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

  1. Ballard D. W., Bothwell A. Mutational analysis of the immunoglobulin heavy chain promoter region. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9626–9630. doi: 10.1073/pnas.83.24.9626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barberis A., Superti-Furga G., Busslinger M. Mutually exclusive interaction of the CCAAT-binding factor and of a displacement protein with overlapping sequences of a histone gene promoter. Cell. 1987 Jul 31;50(3):347–359. doi: 10.1016/0092-8674(87)90489-2. [DOI] [PubMed] [Google Scholar]
  3. Berg J. M. Potential metal-binding domains in nucleic acid binding proteins. Science. 1986 Apr 25;232(4749):485–487. doi: 10.1126/science.2421409. [DOI] [PubMed] [Google Scholar]
  4. Blumberg H., Eisen A., Sledziewski A., Bader D., Young E. T. Two zinc fingers of a yeast regulatory protein shown by genetic evidence to be essential for its function. 1987 Jul 30-Aug 5Nature. 328(6129):443–445. doi: 10.1038/328443a0. [DOI] [PubMed] [Google Scholar]
  5. Bohmann D., Keller W., Dale T., Schöler H. R., Tebb G., Mattaj I. W. A transcription factor which binds to the enhancers of SV40, immunoglobulin heavy chain and U2 snRNA genes. Nature. 1987 Jan 15;325(6101):268–272. doi: 10.1038/325268a0. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Carbon P., Murgo S., Ebel J. P., Krol A., Tebb G., Mattaj L. W. A common octamer motif binding protein is involved in the transcription of U6 snRNA by RNA polymerase III and U2 snRNA by RNA polymerase II. Cell. 1987 Oct 9;51(1):71–79. doi: 10.1016/0092-8674(87)90011-0. [DOI] [PubMed] [Google Scholar]
  8. Chavrier P., Lemaire P., Revelant O., Bravo R., Charnay P. Characterization of a mouse multigene family that encodes zinc finger structures. Mol Cell Biol. 1988 Mar;8(3):1319–1326. doi: 10.1128/mcb.8.3.1319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chowdhury K., Deutsch U., Gruss P. A multigene family encoding several "finger" structures is present and differentially active in mammalian genomes. Cell. 1987 Mar 13;48(5):771–778. doi: 10.1016/0092-8674(87)90074-2. [DOI] [PubMed] [Google Scholar]
  10. Dierks P., van Ooyen A., Cochran M. D., Dobkin C., Reiser J., Weissmann C. Three regions upstream from the cap site are required for efficient and accurate transcription of the rabbit beta-globin gene in mouse 3T6 cells. Cell. 1983 Mar;32(3):695–706. doi: 10.1016/0092-8674(83)90055-7. [DOI] [PubMed] [Google Scholar]
  11. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dynan W. S., Tjian R. The promoter-specific transcription factor Sp1 binds to upstream sequences in the SV40 early promoter. Cell. 1983 Nov;35(1):79–87. doi: 10.1016/0092-8674(83)90210-6. [DOI] [PubMed] [Google Scholar]
  13. Falkner F. G., Mocikat R., Zachau H. G. Sequences closely related to an immunoglobulin gene promoter/enhancer element occur also upstream of other eukaryotic and of prokaryotic genes. Nucleic Acids Res. 1986 Nov 25;14(22):8819–8827. doi: 10.1093/nar/14.22.8819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Falkner F. G., Neumann E., Zachau H. G. Tissue specificity of the initiation of immunoglobulin kappa gene transcription. Hoppe Seylers Z Physiol Chem. 1984 Nov;365(11):1331–1343. doi: 10.1515/bchm2.1984.365.2.1331. [DOI] [PubMed] [Google Scholar]
  15. Fletcher C., Heintz N., Roeder R. G. Purification and characterization of OTF-1, a transcription factor regulating cell cycle expression of a human histone H2b gene. Cell. 1987 Dec 4;51(5):773–781. doi: 10.1016/0092-8674(87)90100-0. [DOI] [PubMed] [Google Scholar]
  16. Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Garner M. M., Revzin A. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 1981 Jul 10;9(13):3047–3060. doi: 10.1093/nar/9.13.3047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gerster T., Matthias P., Thali M., Jiricny J., Schaffner W. Cell type-specificity elements of the immunoglobulin heavy chain gene enhancer. EMBO J. 1987 May;6(5):1323–1330. doi: 10.1002/j.1460-2075.1987.tb02371.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hanas J. S., Hazuda D. J., Bogenhagen D. F., Wu F. Y., Wu C. W. Xenopus transcription factor A requires zinc for binding to the 5 S RNA gene. J Biol Chem. 1983 Dec 10;258(23):14120–14125. [PubMed] [Google Scholar]
  21. Janson L., Bark C., Pettersson U. Identification of proteins interacting with the enhancer of human U2 small nuclear RNA genes. Nucleic Acids Res. 1987 Jul 10;15(13):4997–5016. doi: 10.1093/nar/15.13.4997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Johnston M. Genetic evidence that zinc is an essential co-factor in the DNA binding domain of GAL4 protein. Nature. 1987 Jul 23;328(6128):353–355. doi: 10.1038/328353a0. [DOI] [PubMed] [Google Scholar]
  23. Jones K. A., Tjian R. Sp1 binds to promoter sequences and activates herpes simplex virus 'immediate-early' gene transcription in vitro. Nature. 1985 Sep 12;317(6033):179–182. doi: 10.1038/317179a0. [DOI] [PubMed] [Google Scholar]
  24. Kadonaga J. T., Carner K. R., Masiarz F. R., Tjian R. Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. Cell. 1987 Dec 24;51(6):1079–1090. doi: 10.1016/0092-8674(87)90594-0. [DOI] [PubMed] [Google Scholar]
  25. Miller J., McLachlan A. D., Klug A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J. 1985 Jun;4(6):1609–1614. doi: 10.1002/j.1460-2075.1985.tb03825.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Muriel W. J., Cole J., Lehmann A. R. Molecular analysis of ouabain-resistant mutants of the mouse lymphoma cell line L5178Y. Mutagenesis. 1987 Sep;2(5):383–389. doi: 10.1093/mutage/2.5.383. [DOI] [PubMed] [Google Scholar]
  27. Page D. C., Mosher R., Simpson E. M., Fisher E. M., Mardon G., Pollack J., McGillivray B., de la Chapelle A., Brown L. G. The sex-determining region of the human Y chromosome encodes a finger protein. Cell. 1987 Dec 24;51(6):1091–1104. doi: 10.1016/0092-8674(87)90595-2. [DOI] [PubMed] [Google Scholar]
  28. Parslow T. G., Blair D. L., Murphy W. J., Granner D. K. Structure of the 5' ends of immunoglobulin genes: a novel conserved sequence. Proc Natl Acad Sci U S A. 1984 May;81(9):2650–2654. doi: 10.1073/pnas.81.9.2650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Posorske L. H., Cohn M., Yanagisawa N., Auld D. S. Methionyl-tRNA synthetase of Escherichia coli. A zinc metalloprotein. Biochim Biophys Acta. 1979 Jan 25;576(1):128–133. doi: 10.1016/0005-2795(79)90491-4. [DOI] [PubMed] [Google Scholar]
  30. Pruijn G. J., van Driel W., van Miltenburg R. T., van der Vliet P. C. Promoter and enhancer elements containing a conserved sequence motif are recognized by nuclear factor III, a protein stimulating adenovirus DNA replication. EMBO J. 1987 Dec 1;6(12):3771–3778. doi: 10.1002/j.1460-2075.1987.tb02712.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pruijn G. J., van Driel W., van der Vliet P. C. Nuclear factor III, a novel sequence-specific DNA-binding protein from HeLa cells stimulating adenovirus DNA replication. Nature. 1986 Aug 14;322(6080):656–659. doi: 10.1038/322656a0. [DOI] [PubMed] [Google Scholar]
  32. Rosales R., Vigneron M., Macchi M., Davidson I., Xiao J. H., Chambon P. In vitro binding of cell-specific and ubiquitous nuclear proteins to the octamer motif of the SV40 enhancer and related motifs present in other promoters and enhancers. EMBO J. 1987 Oct;6(10):3015–3025. doi: 10.1002/j.1460-2075.1987.tb02607.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Scheidereit C., Heguy A., Roeder R. G. Identification and purification of a human lymphoid-specific octamer-binding protein (OTF-2) that activates transcription of an immunoglobulin promoter in vitro. Cell. 1987 Dec 4;51(5):783–793. doi: 10.1016/0092-8674(87)90101-2. [DOI] [PubMed] [Google Scholar]
  34. Staudt L. M., Singh H., Sen R., Wirth T., Sharp P. A., Baltimore D. A lymphoid-specific protein binding to the octamer motif of immunoglobulin genes. Nature. 1986 Oct 16;323(6089):640–643. doi: 10.1038/323640a0. [DOI] [PubMed] [Google Scholar]
  35. Sturm R., Baumruker T., Franza B. R., Jr, Herr W. A 100-kD HeLa cell octamer binding protein (OBP100) interacts differently with two separate octamer-related sequences within the SV40 enhancer. Genes Dev. 1987 Dec;1(10):1147–1160. doi: 10.1101/gad.1.10.1147. [DOI] [PubMed] [Google Scholar]
  36. Weber F., de Villiers J., Schaffner W. An SV40 "enhancer trap" incorporates exogenous enhancers or generates enhancers from its own sequences. Cell. 1984 Apr;36(4):983–992. doi: 10.1016/0092-8674(84)90048-5. [DOI] [PubMed] [Google Scholar]
  37. Westin G., Gerster T., Müller M. M., Schaffner G., Schaffner W. OVEC, a versatile system to study transcription in mammalian cells and cell-free extracts. Nucleic Acids Res. 1987 Sep 11;15(17):6787–6798. doi: 10.1093/nar/15.17.6787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wirth T., Staudt L., Baltimore D. An octamer oligonucleotide upstream of a TATA motif is sufficient for lymphoid-specific promoter activity. Nature. 1987 Sep 10;329(6135):174–178. doi: 10.1038/329174a0. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES