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. 1995 Dec;15(12):6999–7009. doi: 10.1128/mcb.15.12.6999

Do products of the myc proto-oncogene play a role in transcriptional regulation of the prothymosin alpha gene?

P C Mol 1, R H Wang 1, D W Batey 1, L A Lee 1, C V Dang 1, S L Berger 1
PMCID: PMC230955  PMID: 8524267

Abstract

The Myc protein has been reported to activate transcription of the rat prothymosin alpha gene by binding to an enhancer element or E box (CACGTG) located in the first intron (S. Gaubatz et al., Mol. Cell. Biol. 14:3853-3862, 1994). The human prothymosin alpha gene contains two such motifs: in the promoter region at kb -1.2 and in intron 1, approximately 2 kb downstream of the transcriptional start site in a region which otherwise bears little homology to the rat gene. Using chloramphenicol acetyltransferase (CAT) reporter constructs driven either by the 5-kb human prothymosin alpha promoter or by a series of truncated promoters, we showed that removal of the E-box sequence had no effect on transient expression of CAT activity in mouse L cells. When intron 1 of the prothymosin alpha gene was inserted into the most extensive promoter construct downstream of the CAT coding region, a diminution in transcription, which remained virtually unchanged upon disruption of the E boxes, was observed. CAT constructs driven by the native prothymosin alpha promoter or the native promoter and intron were indifferent to Myc; equivalent CAT activity was observed in the presence of ectopic normal or mutant Myc genes. Similarly, expression of a transiently transfected wild-type prothymosin alpha gene as the reporter was not affected by a repertoire of myc-derived genes, including myc itself and dominant or recessive negative myc mutants. In COS-1 cells, equivalent amounts of the protein were produced from transfected prothymosin alpha genes regardless of whether genomic E boxes were disrupted, intron 1 was removed, or a repertoire of myc-derived genes was included in the transfection cocktail. More importantly, cotransfection of a dominant negative Max gene failed to reduce transcription of the endogenous prothymosin alpha gene in COS cells or the wild-type transfected gene in COS or L cells. Taken together, the data do not support the idea that Myc activates transcription of the intact human prothymosin alpha gene or reporter constructs that mimic its structure. Rather, they suggest that the human prothymosin alpha promoter and downstream elements are buffered so as to respond poorly, if at all, to transient fluctuations in transcription factors which regulate other genes.

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Selected References

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  1. Abate C., Luk D., Gentz R., Rauscher F. J., 3rd, Curran T. Expression and purification of the leucine zipper and DNA-binding domains of Fos and Jun: both Fos and Jun contact DNA directly. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1032–1036. doi: 10.1073/pnas.87.3.1032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adkins B., Mueller C., Okada C. Y., Reichert R. A., Weissman I. L., Spangrude G. J. Early events in T-cell maturation. Annu Rev Immunol. 1987;5:325–365. doi: 10.1146/annurev.iy.05.040187.001545. [DOI] [PubMed] [Google Scholar]
  3. Amati B., Land H. Myc-Max-Mad: a transcription factor network controlling cell cycle progression, differentiation and death. Curr Opin Genet Dev. 1994 Feb;4(1):102–108. doi: 10.1016/0959-437x(94)90098-1. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Barcia M. G., Castro J. M., Jullien C. D., Freire M. Prothymosin alpha is phosphorylated in proliferating stimulated cells. J Biol Chem. 1993 Mar 5;268(7):4704–4708. [PubMed] [Google Scholar]
  6. Beato M. Gene regulation by steroid hormones. Cell. 1989 Feb 10;56(3):335–344. doi: 10.1016/0092-8674(89)90237-7. [DOI] [PubMed] [Google Scholar]
  7. Beckmann H., Su L. K., Kadesch T. TFE3: a helix-loop-helix protein that activates transcription through the immunoglobulin enhancer muE3 motif. Genes Dev. 1990 Feb;4(2):167–179. doi: 10.1101/gad.4.2.167. [DOI] [PubMed] [Google Scholar]
  8. Bello-Fernandez C., Packham G., Cleveland J. L. The ornithine decarboxylase gene is a transcriptional target of c-Myc. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7804–7808. doi: 10.1073/pnas.90.16.7804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Benvenisty N., Leder A., Kuo A., Leder P. An embryonically expressed gene is a target for c-Myc regulation via the c-Myc-binding sequence. Genes Dev. 1992 Dec;6(12B):2513–2523. doi: 10.1101/gad.6.12b.2513. [DOI] [PubMed] [Google Scholar]
  10. Berger S. L., Manrow R. E., Lee H. Y. Phoenix mutagenesis: one-step reassembly of multiply cleaved plasmids with mixtures of mutant and wild-type fragments. Anal Biochem. 1993 Nov 1;214(2):571–579. doi: 10.1006/abio.1993.1540. [DOI] [PubMed] [Google Scholar]
  11. Billaud M., Isselbacher K. J., Bernards R. A dominant-negative mutant of Max that inhibits sequence-specific DNA binding by Myc proteins. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2739–2743. doi: 10.1073/pnas.90.7.2739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Blackwell T. K., Kretzner L., Blackwood E. M., Eisenman R. N., Weintraub H. Sequence-specific DNA binding by the c-Myc protein. Science. 1990 Nov 23;250(4984):1149–1151. doi: 10.1126/science.2251503. [DOI] [PubMed] [Google Scholar]
  13. Blackwood E. M., Eisenman R. N. Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc. Science. 1991 Mar 8;251(4998):1211–1217. doi: 10.1126/science.2006410. [DOI] [PubMed] [Google Scholar]
  14. Blackwood E. M., Lüscher B., Eisenman R. N. Myc and Max associate in vivo. Genes Dev. 1992 Jan;6(1):71–80. doi: 10.1101/gad.6.1.71. [DOI] [PubMed] [Google Scholar]
  15. Bürglin T. R., Mattaj I. W., Newmeyer D. D., Zeller R., De Robertis E. M. Cloning of nucleoplasmin from Xenopus laevis oocytes and analysis of its developmental expression. Genes Dev. 1987 Mar;1(1):97–107. doi: 10.1101/gad.1.1.97. [DOI] [PubMed] [Google Scholar]
  16. Clinton M., Frangou-Lazaridis M., Panneerselvam C., Horecker B. L. Prothymosin alpha and parathymosin: mRNA and polypeptide levels in rodent tissues. Arch Biochem Biophys. 1989 Feb 15;269(1):256–263. doi: 10.1016/0003-9861(89)90107-0. [DOI] [PubMed] [Google Scholar]
  17. Clinton M., Graeve L., el-Dorry H., Rodriguez-Boulan E., Horecker B. L. Evidence for nuclear targeting of prothymosin and parathymosin synthesized in situ. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6608–6612. doi: 10.1073/pnas.88.15.6608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Cobianchi F., Wilson S. H. Enzymes for modifying and labeling DNA and RNA. Methods Enzymol. 1987;152:94–110. doi: 10.1016/0076-6879(87)52013-4. [DOI] [PubMed] [Google Scholar]
  19. Cullen B. R. Use of eukaryotic expression technology in the functional analysis of cloned genes. Methods Enzymol. 1987;152:684–704. doi: 10.1016/0076-6879(87)52074-2. [DOI] [PubMed] [Google Scholar]
  20. Dang C. V. c-myc oncoprotein function. Biochim Biophys Acta. 1991 Dec 10;1072(2-3):103–113. doi: 10.1016/0304-419x(91)90009-a. [DOI] [PubMed] [Google Scholar]
  21. Dingwall C., Dilworth S. M., Black S. J., Kearsey S. E., Cox L. S., Laskey R. A. Nucleoplasmin cDNA sequence reveals polyglutamic acid tracts and a cluster of sequences homologous to putative nuclear localization signals. EMBO J. 1987 Jan;6(1):69–74. doi: 10.1002/j.1460-2075.1987.tb04720.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Eilers M., Picard D., Yamamoto K. R., Bishop J. M. Chimaeras of myc oncoprotein and steroid receptors cause hormone-dependent transformation of cells. Nature. 1989 Jul 6;340(6228):66–68. doi: 10.1038/340066a0. [DOI] [PubMed] [Google Scholar]
  23. Eilers M., Schirm S., Bishop J. M. The MYC protein activates transcription of the alpha-prothymosin gene. EMBO J. 1991 Jan;10(1):133–141. doi: 10.1002/j.1460-2075.1991.tb07929.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Eschenfeldt W. H., Berger S. L. The human prothymosin alpha gene is polymorphic and induced upon growth stimulation: evidence using a cloned cDNA. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9403–9407. doi: 10.1073/pnas.83.24.9403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Eschenfeldt W. H., Manrow R. E., Krug M. S., Berger S. L. Isolation and partial sequencing of the human prothymosin alpha gene family. Evidence against export of the gene products. J Biol Chem. 1989 May 5;264(13):7546–7555. [PubMed] [Google Scholar]
  26. Gaubatz S., Meichle A., Eilers M. An E-box element localized in the first intron mediates regulation of the prothymosin alpha gene by c-myc. Mol Cell Biol. 1994 Jun;14(6):3853–3862. doi: 10.1128/mcb.14.6.3853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Goodall G. J., Dominguez F., Horecker B. L. Molecular cloning of cDNA for human prothymosin alpha. Proc Natl Acad Sci U S A. 1986 Dec;83(23):8926–8928. doi: 10.1073/pnas.83.23.8926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Gregor P. D., Sawadogo M., Roeder R. G. The adenovirus major late transcription factor USF is a member of the helix-loop-helix group of regulatory proteins and binds to DNA as a dimer. Genes Dev. 1990 Oct;4(10):1730–1740. doi: 10.1101/gad.4.10.1730. [DOI] [PubMed] [Google Scholar]
  29. Gómez-Márquez J., Segade F., Dosil M., Pichel J. G., Bustelo X. R., Freire M. The expression of prothymosin alpha gene in T lymphocytes and leukemic lymphoid cells is tied to lymphocyte proliferation. J Biol Chem. 1989 May 25;264(15):8451–8454. [PubMed] [Google Scholar]
  30. Halazonetis T. D., Kandil A. N. Determination of the c-MYC DNA-binding site. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6162–6166. doi: 10.1073/pnas.88.14.6162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Haritos A. A., Horecker B. L. A radioimmunoassay for thymosin alpha 1 that detects the native polypeptide, prothymosin alpha. J Immunol Methods. 1985 Aug 2;81(2):199–205. doi: 10.1016/0022-1759(85)90204-2. [DOI] [PubMed] [Google Scholar]
  32. Haritos A. A., Tsolas O., Horecker B. L. Distribution of prothymosin alpha in rat tissues. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1391–1393. doi: 10.1073/pnas.81.5.1391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Hochuli E., Döbeli H., Schacher A. New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J Chromatogr. 1987 Dec 18;411:177–184. doi: 10.1016/s0021-9673(00)93969-4. [DOI] [PubMed] [Google Scholar]
  34. Kato G. J., Lee W. M., Chen L. L., Dang C. V. Max: functional domains and interaction with c-Myc. Genes Dev. 1992 Jan;6(1):81–92. doi: 10.1101/gad.6.1.81. [DOI] [PubMed] [Google Scholar]
  35. Krug M. S., Berger S. L. First-strand cDNA synthesis primed with oligo(dT). Methods Enzymol. 1987;152:316–325. doi: 10.1016/0076-6879(87)52036-5. [DOI] [PubMed] [Google Scholar]
  36. Lee L. A., Resar L. M., Dang C. V. Cell density and paradoxical transcriptional properties of c-Myc and Max in cultured mouse fibroblasts. J Clin Invest. 1995 Feb;95(2):900–904. doi: 10.1172/JCI117741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Manrow R. E., Berger S. L. GAG triplets as splice acceptors of last resort. An unusual form of alternative splicing in prothymosin alpha pre-mRNA. J Mol Biol. 1993 Nov 20;234(2):281–288. doi: 10.1006/jmbi.1993.1583. [DOI] [PubMed] [Google Scholar]
  38. Manrow R. E., Leone A., Krug M. S., Eschenfeldt W. H., Berger S. L. The human prothymosin alpha gene family contains several processed pseudogenes lacking deleterious lesions. Genomics. 1992 Jun;13(2):319–331. doi: 10.1016/0888-7543(92)90248-q. [DOI] [PubMed] [Google Scholar]
  39. Manrow R. E., Sburlati A. R., Hanover J. A., Berger S. L. Nuclear targeting of prothymosin alpha. J Biol Chem. 1991 Feb 25;266(6):3916–3924. [PubMed] [Google Scholar]
  40. Marcu K. B., Bossone S. A., Patel A. J. myc function and regulation. Annu Rev Biochem. 1992;61:809–860. doi: 10.1146/annurev.bi.61.070192.004113. [DOI] [PubMed] [Google Scholar]
  41. Meichle A., Philipp A., Eilers M. The functions of Myc proteins. Biochim Biophys Acta. 1992 Dec 16;1114(2-3):129–146. doi: 10.1016/0304-419x(92)90011-m. [DOI] [PubMed] [Google Scholar]
  42. Mori M., Barnard G. F., Staniunas R. J., Jessup J. M., Steele G. D., Jr, Chen L. B. Prothymosin-alpha mRNA expression correlates with that of c-myc in human colon cancer. Oncogene. 1993 Oct;8(10):2821–2826. [PubMed] [Google Scholar]
  43. Palvimo J., Linnala-Kankkunen A. Identification of a low-Mr acidic nuclear protein as prothymosin alpha. FEBS Lett. 1990 Dec 17;277(1-2):257–260. doi: 10.1016/0014-5793(90)80860-l. [DOI] [PubMed] [Google Scholar]
  44. Pan L. X., Haritos A. A., Wideman J., Komiyama T., Chang M., Stein S., Salvin S. B., Horecker B. L. Human prothymosin alpha: amino acid sequence and immunologic properties. Arch Biochem Biophys. 1986 Oct;250(1):197–201. doi: 10.1016/0003-9861(86)90717-4. [DOI] [PubMed] [Google Scholar]
  45. Prendergast G. C., Lawe D., Ziff E. B. Association of Myn, the murine homolog of max, with c-Myc stimulates methylation-sensitive DNA binding and ras cotransformation. Cell. 1991 May 3;65(3):395–407. doi: 10.1016/0092-8674(91)90457-a. [DOI] [PubMed] [Google Scholar]
  46. Prendergast G. C., Ziff E. B. A new bind for Myc. Trends Genet. 1992 Mar;8(3):91–96. doi: 10.1016/0168-9525(92)90196-b. [DOI] [PubMed] [Google Scholar]
  47. Reisman D., Elkind N. B., Roy B., Beamon J., Rotter V. c-Myc trans-activates the p53 promoter through a required downstream CACGTG motif. Cell Growth Differ. 1993 Feb;4(2):57–65. [PubMed] [Google Scholar]
  48. Sburlati A. R., De La Rosa A., Batey D. W., Kurys G. L., Manrow R. E., Pannell L. K., Martin B. M., Sheeley D. M., Berger S. L. Phosphorylation of human and bovine prothymosin alpha in vivo. Biochemistry. 1993 May 4;32(17):4587–4596. doi: 10.1021/bi00068a015. [DOI] [PubMed] [Google Scholar]
  49. Sburlati A. R., Manrow R. E., Berger S. L. Human prothymosin alpha: purification of a highly acidic nuclear protein by means of a phenol extraction. Protein Expr Purif. 1990 Nov;1(2):184–190. doi: 10.1016/1046-5928(90)90014-p. [DOI] [PubMed] [Google Scholar]
  50. Sburlati A. R., Manrow R. E., Berger S. L. Prothymosin alpha antisense oligomers inhibit myeloma cell division. Proc Natl Acad Sci U S A. 1991 Jan 1;88(1):253–257. doi: 10.1073/pnas.88.1.253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Schuermann M., Hennig G., Müller R. Transcriptional activation and transformation by chimaeric Fos-estrogen receptor proteins: altered properties as a consequence of gene fusion. Oncogene. 1993 Oct;8(10):2781–2790. [PubMed] [Google Scholar]
  52. Smith M. R., al-Katib A., Mohammad R., Silverman A., Szabo P., Khilnani S., Kohler W., Nath R., Mutchnick M. G. Prothymosin alpha gene expression correlates with proliferation, not differentiation, of HL-60 cells. Blood. 1993 Aug 15;82(4):1127–1132. [PubMed] [Google Scholar]
  53. Stone J., de Lange T., Ramsay G., Jakobovits E., Bishop J. M., Varmus H., Lee W. Definition of regions in human c-myc that are involved in transformation and nuclear localization. Mol Cell Biol. 1987 May;7(5):1697–1709. doi: 10.1128/mcb.7.5.1697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Szabo P., Panneerselvam C., Clinton M., Frangou-Lazaridis M., Weksler D., Whittington E., Macera M. J., Grzeschik K. H., Selvakumar A., Horecker B. L. Prothymosin alpha gene in humans: organization of its promoter region and localization to chromosome 2. Hum Genet. 1993 Feb;90(6):629–634. doi: 10.1007/BF00202480. [DOI] [PubMed] [Google Scholar]
  55. Tavtigian S. V., Zabludoff S. D., Wold B. J. Cloning of mid-G1 serum response genes and identification of a subset regulated by conditional myc expression. Mol Biol Cell. 1994 Mar;5(3):375–388. doi: 10.1091/mbc.5.3.375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Watts J. D., Cary P. D., Crane-Robinson C. Prothymosin alpha is a nuclear protein. FEBS Lett. 1989 Mar 13;245(1-2):17–20. doi: 10.1016/0014-5793(89)80182-6. [DOI] [PubMed] [Google Scholar]
  57. Watts J. D., Cary P. D., Sautiere P., Crane-Robinson C. Thymosins: both nuclear and cytoplasmic proteins. Eur J Biochem. 1990 Sep 24;192(3):643–651. doi: 10.1111/j.1432-1033.1990.tb19271.x. [DOI] [PubMed] [Google Scholar]

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