Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1995 Nov 25;23(22):4649–4656. doi: 10.1093/nar/23.22.4649

Regulation of mouse thymidylate synthase gene expression in growth-stimulated cells: upstream S phase control elements are indistinguishable from the essential promoter elements.

J Ash 1, W C Liao 1, Y Ke 1, L F Johnson 1
PMCID: PMC307439  PMID: 8524656

Abstract

Expression of the mammalian thymidylate synthase (TS) gene in growth-stimulated cells is closely coordinated with entry into S phase. Previous studies with transfected TS minigenes have shown that sequences upstream of the coding region as well as an intron in the transcribed region are both necessary for proper regulation of TS mRNA content in growth-stimulated cells. The goal of the present study was to identify the upstream regulatory elements. Minigenes consisting of TS 5' flanking sequences linked to the TS coding region (interrupted by introns 1 and 2) were stably transfected into mouse 3T6 cells. Deletion and site-directed mutagenesis of the 5' flanking region revealed that there is a close correspondence between the upstream sequences that are necessary for S phase regulation and the 30 nucleotide region that is essential for promoter activity. These observations raised the possibility that regulation of the TS gene occurs at the transcriptional level. However, nuclear run-on assays showed that the rate of transcription of the TS gene changed very little during the G1-S phase transition. Furthermore, when the TS promoter was linked to an intron-less luciferase indicator gene, there was no change in expression following growth-stimulation. Therefore it appears that the TS gene is controlled primarily at the posttranscriptional level, and that the TS essential promoter region is necessary (although not sufficient) for proper S phase regulation.

Full text

PDF
4649

Images in this article

4652Image
on p.4652
4653Image
on p.4653
4654Image
on p.4654

Selected References

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

  1. Almasan A., Yin Y., Kelly R. E., Lee E. Y., Bradley A., Li W., Bertino J. R., Wahl G. M. Deficiency of retinoblastoma protein leads to inappropriate S-phase entry, activation of E2F-responsive genes, and apoptosis. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5436–5440. doi: 10.1073/pnas.92.12.5436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ash J., Ke Y., Korb M., Johnson L. F. Introns are essential for growth-regulated expression of the mouse thymidylate synthase gene. Mol Cell Biol. 1993 Mar;13(3):1565–1571. doi: 10.1128/mcb.13.3.1565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ayusawa D., Shimizu K., Koyama H., Kaneda S., Takeishi K., Seno T. Cell-cycle-directed regulation of thymidylate synthase messenger RNA in human diploid fibroblasts stimulated to proliferate. J Mol Biol. 1986 Aug 20;190(4):559–567. doi: 10.1016/0022-2836(86)90241-x. [DOI] [PubMed] [Google Scholar]
  4. Chu E., Koeller D. M., Casey J. L., Drake J. C., Chabner B. A., Elwood P. C., Zinn S., Allegra C. J. Autoregulation of human thymidylate synthase messenger RNA translation by thymidylate synthase. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8977–8981. doi: 10.1073/pnas.88.20.8977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chung S., Perry R. P. Importance of introns for expression of mouse ribosomal protein gene rpL32. Mol Cell Biol. 1989 May;9(5):2075–2082. doi: 10.1128/mcb.9.5.2075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cieśla J., Weiner K. X., Weiner R. S., Reston J. T., Maley G. F., Maley F. Isolation and expression of rat thymidylate synthase cDNA: phylogenetic comparison with human and mouse thymidylate synthases. Biochim Biophys Acta. 1995 Apr 4;1261(2):233–242. doi: 10.1016/0167-4781(95)00008-5. [DOI] [PubMed] [Google Scholar]
  7. Conrad A. H. Thymidylate synthetase activity in cultured mammalian cells. J Biol Chem. 1971 Mar 10;246(5):1318–1323. [PubMed] [Google Scholar]
  8. DeGregori J., Kowalik T., Nevins J. R. Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes. Mol Cell Biol. 1995 Aug;15(8):4215–4224. doi: 10.1128/mcb.15.8.4215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Deng T. L., Li D. W., Jenh C. H., Johnson L. F. Structure of the gene for mouse thymidylate synthase. Locations of introns and multiple transcriptional start sites. J Biol Chem. 1986 Dec 5;261(34):16000–16005. [PubMed] [Google Scholar]
  10. Deng T., Li Y., Jolliff K., Johnson L. F. The mouse thymidylate synthase promoter: essential elements are in close proximity to the transcriptional initiation sites. Mol Cell Biol. 1989 Sep;9(9):4079–4082. doi: 10.1128/mcb.9.9.4079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dudov K. P., Perry R. P. The gene family encoding the mouse ribosomal protein L32 contains a uniquely expressed intron-containing gene and an unmutated processed gene. Cell. 1984 Jun;37(2):457–468. doi: 10.1016/0092-8674(84)90376-3. [DOI] [PubMed] [Google Scholar]
  12. Farnham P. J., Slansky J. E., Kollmar R. The role of E2F in the mammalian cell cycle. Biochim Biophys Acta. 1993 Aug 23;1155(2):125–131. doi: 10.1016/0304-419x(93)90001-s. [DOI] [PubMed] [Google Scholar]
  13. Geng Y., Johnson L. F. Lack of an initiator element is responsible for multiple transcriptional initiation sites of the TATA-less mouse thymidylate synthase promoter. Mol Cell Biol. 1993 Aug;13(8):4894–4903. doi: 10.1128/mcb.13.8.4894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Geyer P. K., Meyuhas O., Perry R. P., Johnson L. F. Regulation of ribosomal protein mRNA content and translation in growth-stimulated mouse fibroblasts. Mol Cell Biol. 1982 Jun;2(6):685–693. doi: 10.1128/mcb.2.6.685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hariharan N., Kelley D. E., Perry R. P. Equipotent mouse ribosomal protein promoters have a similar architecture that includes internal sequence elements. Genes Dev. 1989 Nov;3(11):1789–1800. doi: 10.1101/gad.3.11.1789. [DOI] [PubMed] [Google Scholar]
  16. Hernandez N., Lucito R. Elements required for transcription initiation of the human U2 snRNA gene coincide with elements required for snRNA 3' end formation. EMBO J. 1988 Oct;7(10):3125–3134. doi: 10.1002/j.1460-2075.1988.tb03179.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hernandez N., Weiner A. M. Formation of the 3' end of U1 snRNA requires compatible snRNA promoter elements. Cell. 1986 Oct 24;47(2):249–258. doi: 10.1016/0092-8674(86)90447-2. [DOI] [PubMed] [Google Scholar]
  18. Jenh C. H., Geyer P. K., Baskin F., Johnson L. F. Thymidylate synthase gene amplification in fluorodeoxyuridine-resistant mouse cell lines. Mol Pharmacol. 1985 Jul;28(1):80–85. [PubMed] [Google Scholar]
  19. Jenh C. H., Geyer P. K., Johnson L. F. Control of thymidylate synthase mRNA content and gene transcription in an overproducing mouse cell line. Mol Cell Biol. 1985 Oct;5(10):2527–2532. doi: 10.1128/mcb.5.10.2527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jenh C. H., Rao L. G., Johnson L. F. Regulation of thymidylate synthase enzyme synthesis in 5-fluorodeoxyuridine-resistant mouse fibroblasts during the transition from the resting to growing state. J Cell Physiol. 1985 Jan;122(1):149–154. doi: 10.1002/jcp.1041220122. [DOI] [PubMed] [Google Scholar]
  21. Jolliff K., Li Y., Johnson L. F. Multiple protein-DNA interactions in the TATAA-less mouse thymidylate synthase promoter. Nucleic Acids Res. 1991 May 11;19(9):2267–2274. doi: 10.1093/nar/19.9.2267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kelley D. E., Perry R. P. Transcriptional and posttranscriptional control of immunoglobulin mRNA production during B lymphocyte development. Nucleic Acids Res. 1986 Jul 11;14(13):5431–5447. doi: 10.1093/nar/14.13.5431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kim Y. K., Wells S., Lau Y. F., Lee A. S. Sequences contained within the promoter of the human thymidine kinase gene can direct cell-cycle regulation of heterologous fusion genes. Proc Natl Acad Sci U S A. 1988 Aug;85(16):5894–5898. doi: 10.1073/pnas.85.16.5894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Li Y., Li D., Osborn K., Johnson L. F. The 5'-flanking region of the mouse thymidylate synthase gene is necessary but not sufficient for normal regulation in growth-stimulated cells. Mol Cell Biol. 1991 Feb;11(2):1023–1029. doi: 10.1128/mcb.11.2.1023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Li Y., Slansky J. E., Myers D. J., Drinkwater N. R., Kaelin W. G., Farnham P. J. Cloning, chromosomal location, and characterization of mouse E2F1. Mol Cell Biol. 1994 Mar;14(3):1861–1869. doi: 10.1128/mcb.14.3.1861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Liao W. C., Ash J., Johnson L. F. Bidirectional promoter of the mouse thymidylate synthase gene. Nucleic Acids Res. 1994 Oct 11;22(20):4044–4049. doi: 10.1093/nar/22.20.4044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. McIntosh E. M., Ord R. W., Storms R. K. Transcriptional regulation of the cell cycle-dependent thymidylate synthase gene of Saccharomyces cerevisiae. Mol Cell Biol. 1988 Nov;8(11):4616–4624. doi: 10.1128/mcb.8.11.4616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Means A. L., Slansky J. E., McMahon S. L., Knuth M. W., Farnham P. J. The HIP1 binding site is required for growth regulation of the dihydrofolate reductase gene promoter. Mol Cell Biol. 1992 Mar;12(3):1054–1063. doi: 10.1128/mcb.12.3.1054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Navalgund L. G., Rossana C., Muench A. J., Johnson L. F. Cell cycle regulation of thymidylate synthetase gene expression in cultured mouse fibroblasts. J Biol Chem. 1980 Aug 10;255(15):7386–7390. [PubMed] [Google Scholar]
  30. Neuberger M. S., Williams G. T. The intron requirement for immunoglobulin gene expression is dependent upon the promoter. Nucleic Acids Res. 1988 Jul 25;16(14B):6713–6724. doi: 10.1093/nar/16.14.6713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pearson B. E., Nasheuer H. P., Wang T. S. Human DNA polymerase alpha gene: sequences controlling expression in cycling and serum-stimulated cells. Mol Cell Biol. 1991 Apr;11(4):2081–2095. doi: 10.1128/mcb.11.4.2081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rossana C., Gollakota Rao L., Johnson L. F. Thymidylate synthetase overproduction in 5-fluorodeoxyuridine-resistant mouse fibroblasts. Mol Cell Biol. 1982 Sep;2(9):1118–1125. doi: 10.1128/mcb.2.9.1118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Schilling L. J., Farnham P. J. Inappropriate transcription from the 5' end of the murine dihydrofolate reductase gene masks transcriptional regulation. Nucleic Acids Res. 1994 Aug 11;22(15):3061–3068. doi: 10.1093/nar/22.15.3061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sisodia S. S., Sollner-Webb B., Cleveland D. W. Specificity of RNA maturation pathways: RNAs transcribed by RNA polymerase III are not substrates for splicing or polyadenylation. Mol Cell Biol. 1987 Oct;7(10):3602–3612. doi: 10.1128/mcb.7.10.3602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Smale S. T., Tjian R. Transcription of herpes simplex virus tk sequences under the control of wild-type and mutant human RNA polymerase I promoters. Mol Cell Biol. 1985 Feb;5(2):352–362. doi: 10.1128/mcb.5.2.352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Storms R. K., Ord R. W., Greenwood M. T., Mirdamadi B., Chu F. K., Belfort M. Cell cycle-dependent expression of thymidylate synthase in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Dec;4(12):2858–2864. doi: 10.1128/mcb.4.12.2858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. TODARO G. J., GREEN H. Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J Cell Biol. 1963 May;17:299–313. doi: 10.1083/jcb.17.2.299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Takeishi K., Kaneda S., Ayusawa D., Shimizu K., Gotoh O., Seno T. Human thymidylate synthase gene: isolation of phage clones which cover a functionally active gene and structural analysis of the region upstream from the translation initiation codon. J Biochem. 1989 Oct;106(4):575–583. doi: 10.1093/oxfordjournals.jbchem.a122898. [DOI] [PubMed] [Google Scholar]
  40. Wasylyk B., Hahn S. L., Giovane A. The Ets family of transcription factors. Eur J Biochem. 1993 Jan 15;211(1-2):7–18. doi: 10.1007/978-3-642-78757-7_2. [DOI] [PubMed] [Google Scholar]
  41. de Vegvar H. E., Lund E., Dahlberg J. E. 3' end formation of U1 snRNA precursors is coupled to transcription from snRNA promoters. Cell. 1986 Oct 24;47(2):259–266. doi: 10.1016/0092-8674(86)90448-4. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES