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. 1995 Jul;15(7):3840–3847. doi: 10.1128/mcb.15.7.3840

Pi 1 binding sites are negative regulators of bcl-2 expression in pre-B cells.

H M Chen 1, L M Boxer 1
PMCID: PMC230623  PMID: 7791791

Abstract

The bcl-2 gene is differentially regulated during B-cell development, with low-level expression in pre-B cells and higher-level expression in mature B cells. These changes correlate with susceptibility to cell death by apoptosis and suggest that the Bcl-2 protein may play a role in the control of cell death during B-cell development. We have identified two negative regulatory regions in the human bcl-2 5' flanking and 5' untranslated regions in pre-B cells; these regions have no significant function in mature B cells. Further investigation of these regions revealed two pre-B-cell-specific enhancer elements (pi 1 sites) in the 5' negative regulatory region and one in the 3' negative regulatory region. Mutational analysis confirmed that these three sites functioned as negative regulators of the bcl-2 promoter in the pre-B-cell line Nalm-6. Electrophoretic mobility shift assays with each of the three sites demonstrated a complex of identical mobility to that formed with the immunoglobulin heavy-chain enhancer pi 1 site. UV cross-linking experiments revealed that a protein with a molecular mass of 58 kDa bound to the three bcl-2 sites and to the immunoglobulin enhancer site. This protein reacted with an antibody against Ets family proteins. Constructs with the isolated pi 1 sites linked to the simian virus 40 promoter were used in transient transfection experiments in the pre-B-cell line. The bcl-2 sites decreased expression of the simian virus 40 promoter, while the immunoglobulin enhancer site increased its expression. The pi 1 sites in the bcl-2 gene may play a role in the developmental regulation of bcl-2 expression during B-cell differentiation.

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Selected References

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  1. Cleary M. L., Smith S. D., Sklar J. Cloning and structural analysis of cDNAs for bcl-2 and a hybrid bcl-2/immunoglobulin transcript resulting from the t(14;18) translocation. Cell. 1986 Oct 10;47(1):19–28. doi: 10.1016/0092-8674(86)90362-4. [DOI] [PubMed] [Google Scholar]
  2. Ehlich A., Schaal S., Gu H., Kitamura D., Müller W., Rajewsky K. Immunoglobulin heavy and light chain genes rearrange independently at early stages of B cell development. Cell. 1993 Mar 12;72(5):695–704. doi: 10.1016/0092-8674(93)90398-a. [DOI] [PubMed] [Google Scholar]
  3. Gauss G. H., Lieber M. R. DEAE-dextran enhances electroporation of mammalian cells. Nucleic Acids Res. 1992 Dec 25;20(24):6739–6740. doi: 10.1093/nar/20.24.6739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Goodnow C. C., Crosbie J., Adelstein S., Lavoie T. B., Smith-Gill S. J., Brink R. A., Pritchard-Briscoe H., Wotherspoon J. S., Loblay R. H., Raphael K. Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature. 1988 Aug 25;334(6184):676–682. doi: 10.1038/334676a0. [DOI] [PubMed] [Google Scholar]
  5. Graninger W. B., Seto M., Boutain B., Goldman P., Korsmeyer S. J. Expression of Bcl-2 and Bcl-2-Ig fusion transcripts in normal and neoplastic cells. J Clin Invest. 1987 Nov;80(5):1512–1515. doi: 10.1172/JCI113235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gurfinkel N., Unger T., Givol D., Mushinski J. F. Expression of the bcl-2 gene in mouse B lymphocytic cell lines is differentiation stage specific. Eur J Immunol. 1987 Apr;17(4):567–570. doi: 10.1002/eji.1830170421. [DOI] [PubMed] [Google Scholar]
  7. Hartley S. B., Cooke M. P., Fulcher D. A., Harris A. W., Cory S., Basten A., Goodnow C. C. Elimination of self-reactive B lymphocytes proceeds in two stages: arrested development and cell death. Cell. 1993 Feb 12;72(3):325–335. doi: 10.1016/0092-8674(93)90111-3. [DOI] [PubMed] [Google Scholar]
  8. Haury M., Freitas A., Hermitte V., Coutinho A., Hibner U. The physiology of bcl-2 expression in murine B lymphocytes. Oncogene. 1993 May;8(5):1257–1262. [PubMed] [Google Scholar]
  9. Hockenbery D. M., Zutter M., Hickey W., Nahm M., Korsmeyer S. J. BCL2 protein is topographically restricted in tissues characterized by apoptotic cell death. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):6961–6965. doi: 10.1073/pnas.88.16.6961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hockenbery D., Nuñez G., Milliman C., Schreiber R. D., Korsmeyer S. J. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature. 1990 Nov 22;348(6299):334–336. doi: 10.1038/348334a0. [DOI] [PubMed] [Google Scholar]
  11. Hsu J. C., Cressman D. E., Taub R. Promoter-specific trans-activation and inhibition mediated by JunB. Cancer Res. 1993 Aug 15;53(16):3789–3794. [PubMed] [Google Scholar]
  12. Johnson F. B., Krasnow M. A. Differential regulation of transcription preinitiation complex assembly by activator and repressor homeo domain proteins. Genes Dev. 1992 Nov;6(11):2177–2189. doi: 10.1101/gad.6.11.2177. [DOI] [PubMed] [Google Scholar]
  13. Kamachi Y., Kondoh H. Overlapping positive and negative regulatory elements determine lens-specific activity of the delta 1-crystallin enhancer. Mol Cell Biol. 1993 Sep;13(9):5206–5215. doi: 10.1128/mcb.13.9.5206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lansford R. D., McFadden H. J., Siu S. T., Cox J. S., Cann G. M., Koshland M. E. A promoter element that exerts positive and negative control of the interleukin 2-responsive J-chain gene. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5966–5970. doi: 10.1073/pnas.89.13.5966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lehming N., Thanos D., Brickman J. M., Ma J., Maniatis T., Ptashne M. An HMG-like protein that can switch a transcriptional activator to a repressor. Nature. 1994 Sep 8;371(6493):175–179. doi: 10.1038/371175a0. [DOI] [PubMed] [Google Scholar]
  16. Li Y. S., Hayakawa K., Hardy R. R. The regulated expression of B lineage associated genes during B cell differentiation in bone marrow and fetal liver. J Exp Med. 1993 Sep 1;178(3):951–960. doi: 10.1084/jem.178.3.951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Libermann T. A., Baltimore D. Pi, a pre-B-cell-specific enhancer element in the immunoglobulin heavy-chain enhancer. Mol Cell Biol. 1993 Oct;13(10):5957–5969. doi: 10.1128/mcb.13.10.5957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Libermann T. A., Lenardo M., Baltimore D. Involvement of a second lymphoid-specific enhancer element in the regulation of immunoglobulin heavy-chain gene expression. Mol Cell Biol. 1990 Jun;10(6):3155–3162. doi: 10.1128/mcb.10.6.3155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lopez M., Oettgen P., Akbarali Y., Dendorfer U., Libermann T. A. ERP, a new member of the ets transcription factor/oncoprotein family: cloning, characterization, and differential expression during B-lymphocyte development. Mol Cell Biol. 1994 May;14(5):3292–3309. doi: 10.1128/mcb.14.5.3292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. McDonnell T. J., Korsmeyer S. J. Progression from lymphoid hyperplasia to high-grade malignant lymphoma in mice transgenic for the t(14; 18). Nature. 1991 Jan 17;349(6306):254–256. doi: 10.1038/349254a0. [DOI] [PubMed] [Google Scholar]
  21. Merino R., Ding L., Veis D. J., Korsmeyer S. J., Nuñez G. Developmental regulation of the Bcl-2 protein and susceptibility to cell death in B lymphocytes. EMBO J. 1994 Feb 1;13(3):683–691. doi: 10.1002/j.1460-2075.1994.tb06307.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Miyashita T., Harigai M., Hanada M., Reed J. C. Identification of a p53-dependent negative response element in the bcl-2 gene. Cancer Res. 1994 Jun 15;54(12):3131–3135. [PubMed] [Google Scholar]
  23. Nelsen B., Tian G., Erman B., Gregoire J., Maki R., Graves B., Sen R. Regulation of lymphoid-specific immunoglobulin mu heavy chain gene enhancer by ETS-domain proteins. Science. 1993 Jul 2;261(5117):82–86. doi: 10.1126/science.8316859. [DOI] [PubMed] [Google Scholar]
  24. Nemazee D. A., Bürki K. Clonal deletion of B lymphocytes in a transgenic mouse bearing anti-MHC class I antibody genes. Nature. 1989 Feb 9;337(6207):562–566. doi: 10.1038/337562a0. [DOI] [PubMed] [Google Scholar]
  25. Nuñez G., London L., Hockenbery D., Alexander M., McKearn J. P., Korsmeyer S. J. Deregulated Bcl-2 gene expression selectively prolongs survival of growth factor-deprived hemopoietic cell lines. J Immunol. 1990 May 1;144(9):3602–3610. [PubMed] [Google Scholar]
  26. Osmond D. G., Kim N., Manoukian R., Phillips R. A., Rico-Vargas S. A., Jacobsen K. Dynamics and localization of early B-lymphocyte precursor cells (pro-B cells) in the bone marrow of scid mice. Blood. 1992 Apr 1;79(7):1695–1703. [PubMed] [Google Scholar]
  27. Pezzella F., Gatter K. C., Mason D. Y., Bastard C., Duval C., Krajewski A., Turner G. E., Ross F. M., Clark H., Jones D. B. Bcl-2 protein expression in follicular lymphomas in absence of 14;18 translocation. Lancet. 1990 Dec 15;336(8729):1510–1511. doi: 10.1016/0140-6736(90)93216-c. [DOI] [PubMed] [Google Scholar]
  28. Rivera R. R., Stuiver M. H., Steenbergen R., Murre C. Ets proteins: new factors that regulate immunoglobulin heavy-chain gene expression. Mol Cell Biol. 1993 Nov;13(11):7163–7169. doi: 10.1128/mcb.13.11.7163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Seto M., Jaeger U., Hockett R. D., Graninger W., Bennett S., Goldman P., Korsmeyer S. J. Alternative promoters and exons, somatic mutation and deregulation of the Bcl-2-Ig fusion gene in lymphoma. EMBO J. 1988 Jan;7(1):123–131. doi: 10.1002/j.1460-2075.1988.tb02791.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shrivastava A., Saleque S., Kalpana G. V., Artandi S., Goff S. P., Calame K. Inhibition of transcriptional regulator Yin-Yang-1 by association with c-Myc. Science. 1993 Dec 17;262(5141):1889–1892. doi: 10.1126/science.8266081. [DOI] [PubMed] [Google Scholar]
  31. Silverman G. A., Green E. D., Young R. L., Jockel J. I., Domer P. H., Korsmeyer S. J. Meiotic recombination between yeast artificial chromosomes yields a single clone containing the entire BCL2 protooncogene. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9913–9917. doi: 10.1073/pnas.87.24.9913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Vaux D. L., Cory S., Adams J. M. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature. 1988 Sep 29;335(6189):440–442. doi: 10.1038/335440a0. [DOI] [PubMed] [Google Scholar]
  33. Weintraub H., Genetta T., Kadesch T. Tissue-specific gene activation by MyoD: determination of specificity by cis-acting repression elements. Genes Dev. 1994 Sep 15;8(18):2203–2211. doi: 10.1101/gad.8.18.2203. [DOI] [PubMed] [Google Scholar]
  34. Young R. L., Korsmeyer S. J. A negative regulatory element in the bcl-2 5'-untranslated region inhibits expression from an upstream promoter. Mol Cell Biol. 1993 Jun;13(6):3686–3697. doi: 10.1128/mcb.13.6.3686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Yunis J. J. The chromosomal basis of human neoplasia. Science. 1983 Jul 15;221(4607):227–236. doi: 10.1126/science.6336310. [DOI] [PubMed] [Google Scholar]

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