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. 1994 Sep 11;22(18):3787–3792. doi: 10.1093/nar/22.18.3787

Three NF-kappa B sites in the I kappa B-alpha promoter are required for induction of gene expression by TNF alpha.

C Y Ito 1, A G Kazantsev 1, A S Baldwin Jr 1
PMCID: PMC308363  PMID: 7937093

Abstract

NF-kappa B was first identified as a postive regulator which bound to a 10 bp sequence in the first intron of the Ig kappa light chain gene. Further characterization of this transcription factor has revealed that NF-kappa B is kept from binding to its consensus sequence by its inhibitor, IkB-alpha, which retains NF-kappa B in the cytoplasm. Upon receiving various extra- and intracellular signals, I kappa B-alpha is rapidly degraded and NF-kappa B is induced to translocate into the nucleus. This process precedes the rapid induction of I kappa B-alpha mRNA and protein. To understand how I kappa B-alpha is replenished, we have cloned and sequenced the 5' flanking region of the I kappa B-alpha gene and have identified the transcription start site and three NF-kappa B sites in this region. Further characterization of these NF-kappa B sites show that they have different affinities for three specific protein complexes which we identify here to consist of various members of the Rel family. In transient assays, cotransfection with a p65 expression vector is able to activate an I kappa B-alpha promoter-CAT reporter construct and all three NF-kappa B sites are required for full activation of the I kappa B-alpha gene following stimulation with TNF-alpha. Our data confirm a transcriptional autoregulatory loop involved in maintaining appropriate NF-kappa B and I kappa B-alpha levels in the cell.

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Selected References

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  1. Baeuerle P. A., Baltimore D. Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-kappa B transcription factor. Cell. 1988 Apr 22;53(2):211–217. doi: 10.1016/0092-8674(88)90382-0. [DOI] [PubMed] [Google Scholar]
  2. Ballard D. W., Walker W. H., Doerre S., Sista P., Molitor J. A., Dixon E. P., Peffer N. J., Hannink M., Greene W. C. The v-rel oncogene encodes a kappa B enhancer binding protein that inhibits NF-kappa B function. Cell. 1990 Nov 16;63(4):803–814. doi: 10.1016/0092-8674(90)90146-6. [DOI] [PubMed] [Google Scholar]
  3. Beg A. A., Baldwin A. S., Jr The I kappa B proteins: multifunctional regulators of Rel/NF-kappa B transcription factors. Genes Dev. 1993 Nov;7(11):2064–2070. doi: 10.1101/gad.7.11.2064. [DOI] [PubMed] [Google Scholar]
  4. Beg A. A., Finco T. S., Nantermet P. V., Baldwin A. S., Jr Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I kappa B alpha: a mechanism for NF-kappa B activation. Mol Cell Biol. 1993 Jun;13(6):3301–3310. doi: 10.1128/mcb.13.6.3301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Beg A. A., Ruben S. M., Scheinman R. I., Haskill S., Rosen C. A., Baldwin A. S., Jr I kappa B interacts with the nuclear localization sequences of the subunits of NF-kappa B: a mechanism for cytoplasmic retention. Genes Dev. 1992 Oct;6(10):1899–1913. doi: 10.1101/gad.6.10.1899. [DOI] [PubMed] [Google Scholar]
  6. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  7. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  8. Cherrington J. M., Mocarski E. S. Human cytomegalovirus ie1 transactivates the alpha promoter-enhancer via an 18-base-pair repeat element. J Virol. 1989 Mar;63(3):1435–1440. doi: 10.1128/jvi.63.3.1435-1440.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chiao P. J., Miyamoto S., Verma I. M. Autoregulation of I kappa B alpha activity. Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):28–32. doi: 10.1073/pnas.91.1.28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cogswell P. C., Scheinman R. I., Baldwin A. S., Jr Promoter of the human NF-kappa B p50/p105 gene. Regulation by NF-kappa B subunits and by c-REL. J Immunol. 1993 Apr 1;150(7):2794–2804. [PubMed] [Google Scholar]
  11. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  12. Grilli M., Chiu J. J., Lenardo M. J. NF-kappa B and Rel: participants in a multiform transcriptional regulatory system. Int Rev Cytol. 1993;143:1–62. doi: 10.1016/s0074-7696(08)61873-2. [DOI] [PubMed] [Google Scholar]
  13. Haskill S., Beg A. A., Tompkins S. M., Morris J. S., Yurochko A. D., Sampson-Johannes A., Mondal K., Ralph P., Baldwin A. S., Jr Characterization of an immediate-early gene induced in adherent monocytes that encodes I kappa B-like activity. Cell. 1991 Jun 28;65(7):1281–1289. doi: 10.1016/0092-8674(91)90022-q. [DOI] [PubMed] [Google Scholar]
  14. Kieran M., Blank V., Logeat F., Vandekerckhove J., Lottspeich F., Le Bail O., Urban M. B., Kourilsky P., Baeuerle P. A., Israël A. The DNA binding subunit of NF-kappa B is identical to factor KBF1 and homologous to the rel oncogene product. Cell. 1990 Sep 7;62(5):1007–1018. doi: 10.1016/0092-8674(90)90275-j. [DOI] [PubMed] [Google Scholar]
  15. Le Bail O., Schmidt-Ullrich R., Israël A. Promoter analysis of the gene encoding the I kappa B-alpha/MAD3 inhibitor of NF-kappa B: positive regulation by members of the rel/NF-kappa B family. EMBO J. 1993 Dec 15;12(13):5043–5049. doi: 10.1002/j.1460-2075.1993.tb06197.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Luckow B., Schütz G. CAT constructions with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements. Nucleic Acids Res. 1987 Jul 10;15(13):5490–5490. doi: 10.1093/nar/15.13.5490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Molitor J. A., Walker W. H., Doerre S., Ballard D. W., Greene W. C. NF-kappa B: a family of inducible and differentially expressed enhancer-binding proteins in human T cells. Proc Natl Acad Sci U S A. 1990 Dec;87(24):10028–10032. doi: 10.1073/pnas.87.24.10028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Muesing M. A., Smith D. H., Capon D. J. Regulation of mRNA accumulation by a human immunodeficiency virus trans-activator protein. Cell. 1987 Feb 27;48(4):691–701. doi: 10.1016/0092-8674(87)90247-9. [DOI] [PubMed] [Google Scholar]
  19. Nabel G., Baltimore D. An inducible transcription factor activates expression of human immunodeficiency virus in T cells. Nature. 1987 Apr 16;326(6114):711–713. doi: 10.1038/326711a0. [DOI] [PubMed] [Google Scholar]
  20. Nolan G. P., Baltimore D. The inhibitory ankyrin and activator Rel proteins. Curr Opin Genet Dev. 1992 Apr;2(2):211–220. doi: 10.1016/s0959-437x(05)80276-x. [DOI] [PubMed] [Google Scholar]
  21. Nolan G. P., Ghosh S., Liou H. C., Tempst P., Baltimore D. DNA binding and I kappa B inhibition of the cloned p65 subunit of NF-kappa B, a rel-related polypeptide. Cell. 1991 Mar 8;64(5):961–969. doi: 10.1016/0092-8674(91)90320-x. [DOI] [PubMed] [Google Scholar]
  22. Perkins N. D., Edwards N. L., Duckett C. S., Agranoff A. B., Schmid R. M., Nabel G. J. A cooperative interaction between NF-kappa B and Sp1 is required for HIV-1 enhancer activation. EMBO J. 1993 Sep;12(9):3551–3558. doi: 10.1002/j.1460-2075.1993.tb06029.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Schmid R. M., Perkins N. D., Duckett C. S., Andrews P. C., Nabel G. J. Cloning of an NF-kappa B subunit which stimulates HIV transcription in synergy with p65. Nature. 1991 Aug 22;352(6337):733–736. doi: 10.1038/352733a0. [DOI] [PubMed] [Google Scholar]
  25. Scott M. L., Fujita T., Liou H. C., Nolan G. P., Baltimore D. The p65 subunit of NF-kappa B regulates I kappa B by two distinct mechanisms. Genes Dev. 1993 Jul;7(7A):1266–1276. doi: 10.1101/gad.7.7a.1266. [DOI] [PubMed] [Google Scholar]
  26. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  27. Stein B., Cogswell P. C., Baldwin A. S., Jr Functional and physical associations between NF-kappa B and C/EBP family members: a Rel domain-bZIP interaction. Mol Cell Biol. 1993 Jul;13(7):3964–3974. doi: 10.1128/mcb.13.7.3964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sun S. C., Ganchi P. A., Ballard D. W., Greene W. C. NF-kappa B controls expression of inhibitor I kappa B alpha: evidence for an inducible autoregulatory pathway. Science. 1993 Mar 26;259(5103):1912–1915. doi: 10.1126/science.8096091. [DOI] [PubMed] [Google Scholar]
  29. Ten R. M., Paya C. V., Israël N., Le Bail O., Mattei M. G., Virelizier J. L., Kourilsky P., Israël A. The characterization of the promoter of the gene encoding the p50 subunit of NF-kappa B indicates that it participates in its own regulation. EMBO J. 1992 Jan;11(1):195–203. doi: 10.1002/j.1460-2075.1992.tb05042.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. de Martin R., Vanhove B., Cheng Q., Hofer E., Csizmadia V., Winkler H., Bach F. H. Cytokine-inducible expression in endothelial cells of an I kappa B alpha-like gene is regulated by NF kappa B. EMBO J. 1993 Jul;12(7):2773–2779. doi: 10.1002/j.1460-2075.1993.tb05938.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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