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. 1991 Dec;11(12):6166–6176. doi: 10.1128/mcb.11.12.6166

Positive autoregulation of c-myb expression via Myb binding sites in the 5' flanking region of the human c-myb gene.

N C Nicolaides 1, R Gualdi 1, C Casadevall 1, L Manzella 1, B Calabretta 1
PMCID: PMC361795  PMID: 1944282

Abstract

The nuclear proto-oncogene c-myb is preferentially expressed in lymphohematopoietic cells, in which it plays an important role in the processes of differentiation and proliferation. The mechanism(s) that regulates c-myb expression is not fully understood, although in mouse cells a regulatory mechanism involves a transcriptional block in the first intron. To analyze the contribution of the 5' flanking sequences in regulating the expression of the human c-myb gene, we isolated a genomic clone containing extensive 5' flanking sequences, the first exon, and a large portion of the first intron. Sequence analysis of a subcloned 1.3-kb BamHI insert corresponding to 687 nucleotides of the 5' flanking sequence, the entire first exon, and 300 nucleotides of the first intron revealed the presence of closely spaced putative Myb binding sites within a segment extending from nucleotides -616 to -575 upstream from the cap site. A 165-bp segment containing these putative Myb binding sites was linked to a human thymidine kinase (TK) cDNA driven by a low-activity proliferating cell nuclear antigen promoter and cotransfected into TK- ts13 cells with a plasmid in which a full-length human c-myb cDNA is driven by the early simian virus 40 promoter; Myb inducibility of TK mRNA expression was observed both in transient expression assays and in stable transformants. The highest level of inducibility was detected when the 165-bp fragment was placed 138 bp upstream of the proliferating cell nuclear antigen promoter-TK cDNA reporter unit or 3' of the TK cDNA. Mutation of the putative Myb binding sites greatly reduced c-myb transactivation of TK mRNA expression and specifically reduced the binding of in vitro-translated Myb protein at those sites. Finally, c-myb transactivated TK mRNA expression driven by a segment of the authentic c-myb 5' flanking region containing the Myb binding sites. These data suggest that human c-myb maintains high levels of Myb protein in cells that require this gene product for proliferation and/or differentiation by an autoregulatory mechanism involving Myb binding sites in the 5' flanking region.

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Selected References

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

  1. Alitalo K., Winqvist R., Lin C. C., de la Chapelle A., Schwab M., Bishop J. M. Aberrant expression of an amplified c-myb oncogene in two cell lines from a colon carcinoma. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4534–4538. doi: 10.1073/pnas.81.14.4534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anfossi G., Gewirtz A. M., Calabretta B. An oligomer complementary to c-myb-encoded mRNA inhibits proliferation of human myeloid leukemia cell lines. Proc Natl Acad Sci U S A. 1989 May;86(9):3379–3383. doi: 10.1073/pnas.86.9.3379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Angel P., Allegretto E. A., Okino S. T., Hattori K., Boyle W. J., Hunter T., Karin M. Oncogene jun encodes a sequence-specific trans-activator similar to AP-1. Nature. 1988 Mar 10;332(6160):166–171. doi: 10.1038/332166a0. [DOI] [PubMed] [Google Scholar]
  4. Angel P., Hattori K., Smeal T., Karin M. The jun proto-oncogene is positively autoregulated by its product, Jun/AP-1. Cell. 1988 Dec 2;55(5):875–885. doi: 10.1016/0092-8674(88)90143-2. [DOI] [PubMed] [Google Scholar]
  5. Bender T. P., Kuehl W. M. Murine myb protooncogene mRNA: cDNA sequence and evidence for 5' heterogeneity. Proc Natl Acad Sci U S A. 1986 May;83(10):3204–3208. doi: 10.1073/pnas.83.10.3204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bender T. P., Thompson C. B., Kuehl W. M. Differential expression of c-myb mRNA in murine B lymphomas by a block to transcription elongation. Science. 1987 Sep 18;237(4821):1473–1476. doi: 10.1126/science.3498214. [DOI] [PubMed] [Google Scholar]
  7. Biedenkapp H., Borgmeyer U., Sippel A. E., Klempnauer K. H. Viral myb oncogene encodes a sequence-specific DNA-binding activity. Nature. 1988 Oct 27;335(6193):835–837. doi: 10.1038/335835a0. [DOI] [PubMed] [Google Scholar]
  8. Bird A. P. CpG-rich islands and the function of DNA methylation. Nature. 1986 May 15;321(6067):209–213. doi: 10.1038/321209a0. [DOI] [PubMed] [Google Scholar]
  9. Bohmann D., Bos T. J., Admon A., Nishimura T., Vogt P. K., Tjian R. Human proto-oncogene c-jun encodes a DNA binding protein with structural and functional properties of transcription factor AP-1. Science. 1987 Dec 4;238(4832):1386–1392. doi: 10.1126/science.2825349. [DOI] [PubMed] [Google Scholar]
  10. Boyle W. J., Lampert M. A., Lipsick J. S., Baluda M. A. Avian myeloblastosis virus and E26 virus oncogene products are nuclear proteins. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4265–4269. doi: 10.1073/pnas.81.14.4265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Caracciolo D., Venturelli D., Valtieri M., Peschle C., Gewirtz A. M., Calabretta B. Stage-related proliferative activity determines c-myb functional requirements during normal human hematopoiesis. J Clin Invest. 1990 Jan;85(1):55–61. doi: 10.1172/JCI114433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chang C. D., Ottavio L., Travali S., Lipson K. E., Baserga R. Transcriptional and posttranscriptional regulation of the proliferating cell nuclear antigen gene. Mol Cell Biol. 1990 Jul;10(7):3289–3296. doi: 10.1128/mcb.10.7.3289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  14. Clarke M. F., Kukowska-Latallo J. F., Westin E., Smith M., Prochownik E. V. Constitutive expression of a c-myb cDNA blocks Friend murine erythroleukemia cell differentiation. Mol Cell Biol. 1988 Feb;8(2):884–892. doi: 10.1128/mcb.8.2.884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dasgupta P., Saikumar P., Reddy C. D., Reddy E. P. Myb protein binds to human immunodeficiency virus 1 long terminal repeat (LTR) sequences and transactivates LTR-mediated transcription. Proc Natl Acad Sci U S A. 1990 Oct;87(20):8090–8094. doi: 10.1073/pnas.87.20.8090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Duprey S. P., Boettiger D. Developmental regulation of c-myb in normal myeloid progenitor cells. Proc Natl Acad Sci U S A. 1985 Oct;82(20):6937–6941. doi: 10.1073/pnas.82.20.6937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Furukawa Y., Piwnica-Worms H., Ernst T. J., Kanakura Y., Griffin J. D. cdc2 gene expression at the G1 to S transition in human T lymphocytes. Science. 1990 Nov 9;250(4982):805–808. doi: 10.1126/science.2237430. [DOI] [PubMed] [Google Scholar]
  18. Gerondakis S., Bishop J. M. Structure of the protein encoded by the chicken proto-oncogene c-myb. Mol Cell Biol. 1986 Nov;6(11):3677–3684. doi: 10.1128/mcb.6.11.3677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gewirtz A. M., Anfossi G., Venturelli D., Valpreda S., Sims R., Calabretta B. G1/S transition in normal human T-lymphocytes requires the nuclear protein encoded by c-myb. Science. 1989 Jul 14;245(4914):180–183. doi: 10.1126/science.2665077. [DOI] [PubMed] [Google Scholar]
  20. Gewirtz A. M., Calabretta B. A c-myb antisense oligodeoxynucleotide inhibits normal human hematopoiesis in vitro. Science. 1988 Dec 2;242(4883):1303–1306. doi: 10.1126/science.2461588. [DOI] [PubMed] [Google Scholar]
  21. Gonda T. J., Gough N. M., Dunn A. R., de Blaquiere J. Nucleotide sequence of cDNA clones of the murine myb proto-oncogene. EMBO J. 1985 Aug;4(8):2003–2008. doi: 10.1002/j.1460-2075.1985.tb03884.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Griffin C. A., Baylin S. B. Expression of the c-myb oncogene in human small cell lung carcinoma. Cancer Res. 1985 Jan;45(1):272–275. [PubMed] [Google Scholar]
  23. Gritz L., Davies J. Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae. Gene. 1983 Nov;25(2-3):179–188. doi: 10.1016/0378-1119(83)90223-8. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Huttner K. M., Barbosa J. A., Scangos G. A., Pratcheva D. D., Ruddle F. H. DNA-mediated gene transfer without carrier DNA. J Cell Biol. 1981 Oct;91(1):153–156. doi: 10.1083/jcb.91.1.153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ibanez C. E., Lipsick J. S. trans activation of gene expression by v-myb. Mol Cell Biol. 1990 May;10(5):2285–2293. doi: 10.1128/mcb.10.5.2285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Katzen A. L., Kornberg T. B., Bishop J. M. Isolation of the proto-oncogene c-myb from D. melanogaster. Cell. 1985 Jun;41(2):449–456. doi: 10.1016/s0092-8674(85)80018-0. [DOI] [PubMed] [Google Scholar]
  28. Klempnauer K. H., Ramsay G., Bishop J. M., Moscovici M. G., Moscovici C., McGrath J. P., Levinson A. D. The product of the retroviral transforming gene v-myb is a truncated version of the protein encoded by the cellular oncogene c-myb. Cell. 1983 Jun;33(2):345–355. doi: 10.1016/0092-8674(83)90416-6. [DOI] [PubMed] [Google Scholar]
  29. Klempnauer K. H., Sippel A. E. The highly conserved amino-terminal region of the protein encoded by the v-myb oncogene functions as a DNA-binding domain. EMBO J. 1987 Sep;6(9):2719–2725. doi: 10.1002/j.1460-2075.1987.tb02565.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Klempnauer K. H., Symonds G., Evan G. I., Bishop J. M. Subcellular localization of proteins encoded by oncogenes of avian myeloblastosis virus and avian leukemia virus E26 and by chicken c-myb gene. Cell. 1984 Jun;37(2):537–547. doi: 10.1016/0092-8674(84)90384-2. [DOI] [PubMed] [Google Scholar]
  31. Krieg P. A., Melton D. A. In vitro RNA synthesis with SP6 RNA polymerase. Methods Enzymol. 1987;155:397–415. doi: 10.1016/0076-6879(87)55027-3. [DOI] [PubMed] [Google Scholar]
  32. Lipson K. E., Baserga R. Transcriptional activity of the human thymidine kinase gene determined by a method using the polymerase chain reaction and an intron-specific probe. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9774–9777. doi: 10.1073/pnas.86.24.9774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Majello B., Kenyon L. C., Dalla-Favera R. Human c-myb protooncogene: nucleotide sequence of cDNA and organization of the genomic locus. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9636–9640. doi: 10.1073/pnas.83.24.9636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. McClinton D., Stafford J., Brents L., Bender T. P., Kuehl W. M. Differentiation of mouse erythroleukemia cells is blocked by late up-regulation of a c-myb transgene. Mol Cell Biol. 1990 Feb;10(2):705–710. doi: 10.1128/mcb.10.2.705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. McMahon J., Howe K. M., Watson R. J. The induction of Friend erythroleukaemia differentiation is markedly affected by expression of a transfected c-myb cDNA. Oncogene. 1988 Dec;3(6):717–720. [PubMed] [Google Scholar]
  36. Mizuguchi G., Nakagoshi H., Nagase T., Nomura N., Date T., Ueno Y., Ishii S. DNA binding activity and transcriptional activator function of the human B-myb protein compared with c-MYB. J Biol Chem. 1990 Jun 5;265(16):9280–9284. [PubMed] [Google Scholar]
  37. Moelling K., Pfaff E., Beug H., Beimling P., Bunte T., Schaller H. E., Graf T. DNA-binding activity is associated with purified myb proteins from AMV and E26 viruses and is temperature-sensitive for E26 ts mutants. Cell. 1985 Apr;40(4):983–990. doi: 10.1016/0092-8674(85)90358-7. [DOI] [PubMed] [Google Scholar]
  38. Moscovici C. Leukemic transformation with avian myeloblastosis virus: present status. Curr Top Microbiol Immunol. 1975;71:79–101. doi: 10.1007/978-3-642-66193-8_2. [DOI] [PubMed] [Google Scholar]
  39. Nakabeppu Y., Ryder K., Nathans D. DNA binding activities of three murine Jun proteins: stimulation by Fos. Cell. 1988 Dec 2;55(5):907–915. doi: 10.1016/0092-8674(88)90146-8. [DOI] [PubMed] [Google Scholar]
  40. Nakagoshi H., Nagase T., Kanei-Ishii C., Ueno Y., Ishii S. Binding of the c-myb proto-oncogene product to the simian virus 40 enhancer stimulates transcription. J Biol Chem. 1990 Feb 25;265(6):3479–3483. [PubMed] [Google Scholar]
  41. Ness S. A., Marknell A., Graf T. The v-myb oncogene product binds to and activates the promyelocyte-specific mim-1 gene. Cell. 1989 Dec 22;59(6):1115–1125. doi: 10.1016/0092-8674(89)90767-8. [DOI] [PubMed] [Google Scholar]
  42. Paz-Ares J., Ghosal D., Wienand U., Peterson P. A., Saedler H. The regulatory c1 locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators. EMBO J. 1987 Dec 1;6(12):3553–3558. doi: 10.1002/j.1460-2075.1987.tb02684.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Reddy C. D., Reddy E. P. Differential binding of nuclear factors to the intron 1 sequences containing the transcriptional pause site correlates with c-myb expression. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7326–7330. doi: 10.1073/pnas.86.19.7326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Rosson D., Reddy E. P. Nucleotide sequence of chicken c-myb complementary DNA and implications for myb oncogene activation. Nature. 1986 Feb 13;319(6054):604–606. doi: 10.1038/319604a0. [DOI] [PubMed] [Google Scholar]
  45. Sakura H., Kanei-Ishii C., Nagase T., Nakagoshi H., Gonda T. J., Ishii S. Delineation of three functional domains of the transcriptional activator encoded by the c-myb protooncogene. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5758–5762. doi: 10.1073/pnas.86.15.5758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sobieszczuk P. W., Gonda T. J., Dunn A. R. Structure and biological activity of the transcriptional initiation sequences of the murine c-myb oncogene. Nucleic Acids Res. 1989 Dec 11;17(23):9593–9611. doi: 10.1093/nar/17.23.9593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Talavera A., Basilico C. Temperature sensitive mutants of BHK cells affected in cell cycle progression. J Cell Physiol. 1977 Sep;92(3):425–436. doi: 10.1002/jcp.1040920310. [DOI] [PubMed] [Google Scholar]
  48. Thiele C. J., Cohen P. S., Israel M. A. Regulation of c-myb expression in human neuroblastoma cells during retinoic acid-induced differentiation. Mol Cell Biol. 1988 Apr;8(4):1677–1683. doi: 10.1128/mcb.8.4.1677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Todokoro K., Watson R. J., Higo H., Amanuma H., Kuramochi S., Yanagisawa H., Ikawa Y. Down-regulation of c-myb gene expression is a prerequisite for erythropoietin-induced erythroid differentiation. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8900–8904. doi: 10.1073/pnas.85.23.8900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tokunaga K., Taniguchi H., Yoda K., Shimizu M., Sakiyama S. Nucleotide sequence of a full-length cDNA for mouse cytoskeletal beta-actin mRNA. Nucleic Acids Res. 1986 Mar 25;14(6):2829–2829. doi: 10.1093/nar/14.6.2829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Torelli G., Venturelli D., Coló A., Zanni C., Selleri L., Moretti L., Calabretta B., Torelli U. Expression of c-myb protooncogene and other cell cycle-related genes in normal and neoplastic human colonic mucosa. Cancer Res. 1987 Oct 15;47(20):5266–5269. [PubMed] [Google Scholar]
  53. Travali S., Reiss K., Ferber A., Petralia S., Mercer W. E., Calabretta B., Baserga R. Constitutively expressed c-myb abrogates the requirement for insulinlike growth factor 1 in 3T3 fibroblasts. Mol Cell Biol. 1991 Feb;11(2):731–736. doi: 10.1128/mcb.11.2.731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Watson R. J. A transcriptional arrest mechanism involved in controlling constitutive levels of mouse c-myb mRNA. Oncogene. 1988 Mar;2(3):267–272. [PubMed] [Google Scholar]
  55. Weston K., Bishop J. M. Transcriptional activation by the v-myb oncogene and its cellular progenitor, c-myb. Cell. 1989 Jul 14;58(1):85–93. doi: 10.1016/0092-8674(89)90405-4. [DOI] [PubMed] [Google Scholar]

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