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. 1997 May 15;25(10):1984–1990. doi: 10.1093/nar/25.10.1984

A novel genetic system to isolate a dominant negative effector on DNA-binding activity of Oct-2.

A Terunuma 1, K Shiba 1, T Noda 1
PMCID: PMC146677  PMID: 9115366

Abstract

Recent studies have revealed that interactions between transcription factors play an important role in regulation of gene expression in eukaryotic cells. To isolate cDNA clones that dominantly inhibit the DNA-binding activity of Oct-2, chosen as a representative factor, we have developed a novel screening system. This employs an Escherichia coli tester strain carrying a modified lac operon as a reporter gene, with the lac operator sequence replaced by an octamer sequence. Oct-2 expressed in this tester strain represses the expression of the reporter gene and changes the phenotype of the cell from Lac+to Lac-. Introduction of a cDNA expression library prepared from a human T-cell line into the Oct-2-harboring tester strain allowed selection of three Lac+clones out of 1 x 10(5) transformants. One of them, hT86, encoding a putative zinc finger protein was found to derepress beta-galactosidase activity in the Oct-2-harboring tester strain at the transcriptional level. In gel mobility shift assays, hT86 attenuated the intensity of the retarded band composed of the octamer probe and Oct-2, suggesting a dominant negative effect on the DNA-binding activity of Oct-2. The strategy described here provides a new approach for studying protein-protein interactions that govern the complex regulation of gene expression.

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Selected References

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

  1. Andersen B., Schonemann M. D., Flynn S. E., Pearse R. V., 2nd, Singh H., Rosenfeld M. G. Skn-1a and Skn-1i: two functionally distinct Oct-2-related factors expressed in epidermis. Science. 1993 Apr 2;260(5104):78–82. doi: 10.1126/science.7682011. [DOI] [PubMed] [Google Scholar]
  2. Ayer D. E., Kretzner L., Eisenman R. N. Mad: a heterodimeric partner for Max that antagonizes Myc transcriptional activity. Cell. 1993 Jan 29;72(2):211–222. doi: 10.1016/0092-8674(93)90661-9. [DOI] [PubMed] [Google Scholar]
  3. Baeuerle P. A., Baltimore D. I kappa B: a specific inhibitor of the NF-kappa B transcription factor. Science. 1988 Oct 28;242(4878):540–546. doi: 10.1126/science.3140380. [DOI] [PubMed] [Google Scholar]
  4. Benezra R., Davis R. L., Lockshon D., Turner D. L., Weintraub H. The protein Id: a negative regulator of helix-loop-helix DNA binding proteins. Cell. 1990 Apr 6;61(1):49–59. doi: 10.1016/0092-8674(90)90214-y. [DOI] [PubMed] [Google Scholar]
  5. Berg J. M., Shi Y. The galvanization of biology: a growing appreciation for the roles of zinc. Science. 1996 Feb 23;271(5252):1081–1085. doi: 10.1126/science.271.5252.1081. [DOI] [PubMed] [Google Scholar]
  6. Berg J. M. Zinc finger domains: hypotheses and current knowledge. Annu Rev Biophys Biophys Chem. 1990;19:405–421. doi: 10.1146/annurev.bb.19.060190.002201. [DOI] [PubMed] [Google Scholar]
  7. Botfield M. C., Jancso A., Weiss M. A. Mapping critical residues in eukaryotic DNA-binding proteins: a plasmid-based genetic selection strategy with application to the Oct-2 POU motif. Biochemistry. 1994 May 24;33(20):6177–6185. doi: 10.1021/bi00186a017. [DOI] [PubMed] [Google Scholar]
  8. Casadaban M. J., Chou J., Cohen S. N. In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol. 1980 Aug;143(2):971–980. doi: 10.1128/jb.143.2.971-980.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chien C. T., Bartel P. L., Sternglanz R., Fields S. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9578–9582. doi: 10.1073/pnas.88.21.9578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clerc R. G., Corcoran L. M., LeBowitz J. H., Baltimore D., Sharp P. A. The B-cell-specific Oct-2 protein contains POU box- and homeo box-type domains. Genes Dev. 1988 Dec;2(12A):1570–1581. doi: 10.1101/gad.2.12a.1570. [DOI] [PubMed] [Google Scholar]
  11. Corneliussen B., Holm M., Waltersson Y., Onions J., Hallberg B., Thornell A., Grundström T. Calcium/calmodulin inhibition of basic-helix-loop-helix transcription factor domains. Nature. 1994 Apr 21;368(6473):760–764. doi: 10.1038/368760a0. [DOI] [PubMed] [Google Scholar]
  12. Elledge S. J., Sugiono P., Guarente L., Davis R. W. Genetic selection for genes encoding sequence-specific DNA-binding proteins. Proc Natl Acad Sci U S A. 1989 May;86(10):3689–3693. doi: 10.1073/pnas.86.10.3689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  14. Folks T., Benn S., Rabson A., Theodore T., Hoggan M. D., Martin M., Lightfoote M., Sell K. Characterization of a continuous T-cell line susceptible to the cytopathic effects of the acquired immunodeficiency syndrome (AIDS)-associated retrovirus. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4539–4543. doi: 10.1073/pnas.82.13.4539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gazdar A. F., Carney D. N., Bunn P. A., Russell E. K., Jaffe E. S., Schechter G. P., Guccion J. G. Mitogen requirements for the in vitro propagation of cutaneous T-cell lymphomas. Blood. 1980 Mar;55(3):409–417. [PubMed] [Google Scholar]
  16. Gerster T., Balmaceda C. G., Roeder R. G. The cell type-specific octamer transcription factor OTF-2 has two domains required for the activation of transcription. EMBO J. 1990 May;9(5):1635–1643. doi: 10.1002/j.1460-2075.1990.tb08283.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hirano M., Shigesada K., Imai M. Construction and characterization of plasmid and lambda phage vector systems for study of transcriptional control in Escherichia coli. Gene. 1987;57(1):89–99. doi: 10.1016/0378-1119(87)90180-6. [DOI] [PubMed] [Google Scholar]
  18. Imagawa M., Miyamoto A., Shirakawa M., Hamada H., Muramatsu M. Stringent integrity requirements for both trans-activation and DNA-binding in a trans-activator, Oct3. Nucleic Acids Res. 1991 Aug 25;19(16):4503–4508. doi: 10.1093/nar/19.16.4503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ingraham H. A., Flynn S. E., Voss J. W., Albert V. R., Kapiloff M. S., Wilson L., Rosenfeld M. G. The POU-specific domain of Pit-1 is essential for sequence-specific, high affinity DNA binding and DNA-dependent Pit-1-Pit-1 interactions. Cell. 1990 Jun 15;61(6):1021–1033. doi: 10.1016/0092-8674(90)90067-o. [DOI] [PubMed] [Google Scholar]
  20. Jaenicke R., Rudolph R. Refolding and association of oligomeric proteins. Methods Enzymol. 1986;131:218–250. doi: 10.1016/0076-6879(86)31043-7. [DOI] [PubMed] [Google Scholar]
  21. Johnson W. A., Hirsh J. Binding of a Drosophila POU-domain protein to a sequence element regulating gene expression in specific dopaminergic neurons. Nature. 1990 Feb 1;343(6257):467–470. doi: 10.1038/343467a0. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Ko H. S., Fast P., McBride W., Staudt L. M. A human protein specific for the immunoglobulin octamer DNA motif contains a functional homeobox domain. Cell. 1988 Oct 7;55(1):135–144. doi: 10.1016/0092-8674(88)90016-5. [DOI] [PubMed] [Google Scholar]
  24. Lassar A. B., Davis R. L., Wright W. E., Kadesch T., Murre C., Voronova A., Baltimore D., Weintraub H. Functional activity of myogenic HLH proteins requires hetero-oligomerization with E12/E47-like proteins in vivo. Cell. 1991 Jul 26;66(2):305–315. doi: 10.1016/0092-8674(91)90620-e. [DOI] [PubMed] [Google Scholar]
  25. Luo Y., Fujii H., Gerster T., Roeder R. G. A novel B cell-derived coactivator potentiates the activation of immunoglobulin promoters by octamer-binding transcription factors. Cell. 1992 Oct 16;71(2):231–241. doi: 10.1016/0092-8674(92)90352-d. [DOI] [PubMed] [Google Scholar]
  26. Manly S. P., Matthews K. S. lac operator DNA modification in the presence of proteolytic fragments of the repressor protein. J Mol Biol. 1984 Nov 5;179(3):315–333. doi: 10.1016/0022-2836(84)90068-8. [DOI] [PubMed] [Google Scholar]
  27. Mead D. A., Szczesna-Skorupa E., Kemper B. Single-stranded DNA 'blue' T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. Protein Eng. 1986 Oct-Nov;1(1):67–74. doi: 10.1093/protein/1.1.67. [DOI] [PubMed] [Google Scholar]
  28. Müller M. M., Ruppert S., Schaffner W., Matthias P. A cloned octamer transcription factor stimulates transcription from lymphoid-specific promoters in non-B cells. Nature. 1988 Dec 8;336(6199):544–551. doi: 10.1038/336544a0. [DOI] [PubMed] [Google Scholar]
  29. Okamoto K., Okazawa H., Okuda A., Sakai M., Muramatsu M., Hamada H. A novel octamer binding transcription factor is differentially expressed in mouse embryonic cells. Cell. 1990 Feb 9;60(3):461–472. doi: 10.1016/0092-8674(90)90597-8. [DOI] [PubMed] [Google Scholar]
  30. Okubo H., Kaji Y., Yamasaki K., Kudo J., Inoue T., Niho Y., Sakai T., Hanada M. A case of IgM lambda pyroglobulinemia caused by lymphoma probably of ovarian origin. Nihon Ketsueki Gakkai Zasshi. 1985 Feb;48(1):124–133. [PubMed] [Google Scholar]
  31. Paulmier N., Yaniv M., von Wilcken-Bergmann B., Müller-Hill B. gal4 transcription activator protein of yeast can function as a repressor in Escherichia coli. EMBO J. 1987 Nov;6(11):3539–3542. doi: 10.1002/j.1460-2075.1987.tb02680.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Poellinger L., Yoza B. K., Roeder R. G. Functional cooperativity between protein molecules bound at two distinct sequence elements of the immunoglobulin heavy-chain promoter. Nature. 1989 Feb 9;337(6207):573–576. doi: 10.1038/337573a0. [DOI] [PubMed] [Google Scholar]
  33. Popovic M., Sarngadharan M. G., Read E., Gallo R. C. Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science. 1984 May 4;224(4648):497–500. doi: 10.1126/science.6200935. [DOI] [PubMed] [Google Scholar]
  34. Rose R. E. The nucleotide sequence of pACYC184. Nucleic Acids Res. 1988 Jan 11;16(1):355–355. doi: 10.1093/nar/16.1.355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rosenfeld M. G. POU-domain transcription factors: pou-er-ful developmental regulators. Genes Dev. 1991 Jun;5(6):897–907. doi: 10.1101/gad.5.6.897. [DOI] [PubMed] [Google Scholar]
  36. Scheidereit C., Cromlish J. A., Gerster T., Kawakami K., Balmaceda C. G., Currie R. A., Roeder R. G. A human lymphoid-specific transcription factor that activates immunoglobulin genes is a homoeobox protein. Nature. 1988 Dec 8;336(6199):551–557. doi: 10.1038/336551a0. [DOI] [PubMed] [Google Scholar]
  37. Schüle R., Evans R. M. Cross-coupling of signal transduction pathways: zinc finger meets leucine zipper. Trends Genet. 1991 Nov-Dec;7(11-12):377–381. doi: 10.1016/0168-9525(91)90259-s. [DOI] [PubMed] [Google Scholar]
  38. Shiba K., Mizobuchi K. Posttranscriptional control of plasmid ColIb-P9 repZ gene expression by a small RNA. J Bacteriol. 1990 Apr;172(4):1992–1997. doi: 10.1128/jb.172.4.1992-1997.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shigesada K., Itamura S., Kato M., Hatanaka M., Imai M., Tanaka M., Masuda N., Nagai J., Nakashima K. Construction of a new plasmid vector that can express cloned cDNA in all translational reading frames. Gene. 1987;53(2-3):163–172. doi: 10.1016/0378-1119(87)90004-7. [DOI] [PubMed] [Google Scholar]
  40. Sundström C., Nilsson K. Establishment and characterization of a human histiocytic lymphoma cell line (U-937). Int J Cancer. 1976 May 15;17(5):565–577. doi: 10.1002/ijc.2910170504. [DOI] [PubMed] [Google Scholar]
  41. Takada A., Takada Y., Minowada J. Immunological functions of human T-lymphoid cell line (MOLT). I. Release of immunosuppressive factors from the mixture of MOLT-4 cells and sheep red blood cells. J Exp Med. 1974 Aug 1;140(2):538–548. doi: 10.1084/jem.140.2.538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Treacy M. N., He X., Rosenfeld M. G. I-POU: a POU-domain protein that inhibits neuron-specific gene activation. Nature. 1991 Apr 18;350(6319):577–584. doi: 10.1038/350577a0. [DOI] [PubMed] [Google Scholar]
  43. Turner E. E. Similar DNA recognition properties of alternatively spliced Drosophila POU factors. Proc Natl Acad Sci U S A. 1996 Dec 24;93(26):15097–15101. doi: 10.1073/pnas.93.26.15097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Voss J. W., Wilson L., Rosenfeld M. G. POU-domain proteins Pit-1 and Oct-1 interact to form a heteromeric complex and can cooperate to induce expression of the prolactin promoter. Genes Dev. 1991 Jul;5(7):1309–1320. doi: 10.1101/gad.5.7.1309. [DOI] [PubMed] [Google Scholar]
  45. Webster L. C., Ricciardi R. P. trans-dominant mutants of E1A provide genetic evidence that the zinc finger of the trans-activating domain binds a transcription factor. Mol Cell Biol. 1991 Sep;11(9):4287–4296. doi: 10.1128/mcb.11.9.4287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Xue D., Tu Y., Chalfie M. Cooperative interactions between the Caenorhabditis elegans homeoproteins UNC-86 and MEC-3. Science. 1993 Sep 3;261(5126):1324–1328. doi: 10.1126/science.8103239. [DOI] [PubMed] [Google Scholar]
  47. Zervos A. S., Gyuris J., Brent R. Mxi1, a protein that specifically interacts with Max to bind Myc-Max recognition sites. Cell. 1993 Jan 29;72(2):223–232. doi: 10.1016/0092-8674(93)90662-a. [DOI] [PubMed] [Google Scholar]

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