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. 1989 Nov 25;17(22):9291–9304. doi: 10.1093/nar/17.22.9291

Functional analysis of GC element binding and transcription in the hamster dihydrofolate reductase gene promoter.

A G Swick 1, M C Blake 1, J W Kahn 1, J C Azizkhan 1
PMCID: PMC335132  PMID: 2587257

Abstract

Dihydrofolate reductase (DHFR) gene expression is required for cell growth. The DHFR gene promoter contains several GC elements capable of binding the transcription factor Sp1. In this report we have characterized the effect of protein(s) binding to these sequence elements in the Chinese hamster DHFR promoter on transcription. We have constructed a series of deletions containing from 896 to 103 bp 5' to the start of translation. The protein binding domains have been mapped by DNAse I footprint analysis using HeLa nuclear extract, and the function of the protein-binding elements has been assessed by in vitro transcription and transient CAT expression. Maximal transcription in vitro and CAT expression is obtained with a construct containing 3 GC elements extending to position -184. Removal of GC element binding factor(s), by competition with an oligonucleotide containing an Sp1 binding site, completely abolishes transcription in vitro and significantly diminishes CAT expression. Ten-fold higher molar excess of competitor is required to abolish SV40 early transcription, suggesting that the GC element interactions in the DHFR promoter are different from those in the SV40 early region. Co-transfection of a DHFR CAT construct with an expressor of Sp1 dramatically increased CAT expression in Drosophila cells.

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Selected References

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

  1. Ares M., Jr, Chung J. S., Giglio L., Weiner A. M. Distinct factors with Sp1 and NF-A specificities bind to adjacent functional elements of the human U2 snRNA gene enhancer. Genes Dev. 1987 Oct;1(8):808–817. doi: 10.1101/gad.1.8.808. [DOI] [PubMed] [Google Scholar]
  2. Azizkhan J. C., Vaughn J. P., Christy R. J., Hamlin J. L. Nucleotide sequence and nuclease hypersensitivity of the Chinese hamster dihydrofolate reductase gene promoter region. Biochemistry. 1986 Oct 7;25(20):6228–6236. doi: 10.1021/bi00368a059. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Carthew R. W., Chodosh L. A., Sharp P. A. An RNA polymerase II transcription factor binds to an upstream element in the adenovirus major late promoter. Cell. 1985 Dec;43(2 Pt 1):439–448. doi: 10.1016/0092-8674(85)90174-6. [DOI] [PubMed] [Google Scholar]
  5. Chen M. J., Shimada T., Moulton A. D., Cline A., Humphries R. K., Maizel J., Nienhuis A. W. The functional human dihydrofolate reductase gene. J Biol Chem. 1984 Mar 25;259(6):3933–3943. [PubMed] [Google Scholar]
  6. Ciudad C. J., Urlaub G., Chasin L. A. Deletion analysis of the Chinese hamster dihydrofolate reductase gene promoter. J Biol Chem. 1988 Nov 5;263(31):16274–16282. [PubMed] [Google Scholar]
  7. Courey A. J., Tjian R. Analysis of Sp1 in vivo reveals multiple transcriptional domains, including a novel glutamine-rich activation motif. Cell. 1988 Dec 2;55(5):887–898. doi: 10.1016/0092-8674(88)90144-4. [DOI] [PubMed] [Google Scholar]
  8. Crouse G. F., Stivaletta L. A., Smith M. L. Analysis of gene expression using episomal mouse dihydrofolate reductase minigenes. Nucleic Acids Res. 1988 Jul 25;16(14B):7025–7042. doi: 10.1093/nar/16.14.7025. [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. Di Nocera P. P., Dawid I. B. Transient expression of genes introduced into cultured cells of Drosophila. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7095–7098. doi: 10.1073/pnas.80.23.7095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dynan W. S., Sazer S., Tjian R., Schimke R. T. Transcription factor Sp1 recognizes a DNA sequence in the mouse dihydrofolate reductase promoter. Nature. 1986 Jan 16;319(6050):246–248. doi: 10.1038/319246a0. [DOI] [PubMed] [Google Scholar]
  13. Farnham P. J., Abrams J. M., Schimke R. T. Opposite-strand RNAs from the 5' flanking region of the mouse dihydrofolate reductase gene. Proc Natl Acad Sci U S A. 1985 Jun;82(12):3978–3982. doi: 10.1073/pnas.82.12.3978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Farnham P. J., Schimke R. T. In vitro transcription and delimitation of promoter elements of the murine dihydrofolate reductase gene. Mol Cell Biol. 1986 Jul;6(7):2392–2401. doi: 10.1128/mcb.6.7.2392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Farnham P. J., Schimke R. T. Transcriptional regulation of mouse dihydrofolate reductase in the cell cycle. J Biol Chem. 1985 Jun 25;260(12):7675–7680. [PubMed] [Google Scholar]
  16. Galas D. J., Schmitz A. DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 1978 Sep;5(9):3157–3170. doi: 10.1093/nar/5.9.3157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gidoni D., Kadonaga J. T., Barrera-Saldaña H., Takahashi K., Chambon P., Tjian R. Bidirectional SV40 transcription mediated by tandem Sp1 binding interactions. Science. 1985 Nov 1;230(4725):511–517. doi: 10.1126/science.2996137. [DOI] [PubMed] [Google Scholar]
  18. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Hayashi S., Kondoh H. In vivo competition of delta-crystallin gene expression by DNA fragments containing a GC box. Mol Cell Biol. 1986 Nov;6(11):4130–4132. doi: 10.1128/mcb.6.11.4130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ishii S., Kadonaga J. T., Tjian R., Brady J. N., Merlino G. T., Pastan I. Binding of the Sp1 transcription factor by the human Harvey ras1 proto-oncogene promoter. Science. 1986 Jun 13;232(4756):1410–1413. doi: 10.1126/science.3012774. [DOI] [PubMed] [Google Scholar]
  22. Jakobovits E. B., Schlokat U., Vannice J. L., Derynck R., Levinson A. D. The human transforming growth factor alpha promoter directs transcription initiation from a single site in the absence of a TATA sequence. Mol Cell Biol. 1988 Dec;8(12):5549–5554. doi: 10.1128/mcb.8.12.5549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jones K. A., Yamamoto K. R., Tjian R. Two distinct transcription factors bind to the HSV thymidine kinase promoter in vitro. Cell. 1985 Sep;42(2):559–572. doi: 10.1016/0092-8674(85)90113-8. [DOI] [PubMed] [Google Scholar]
  24. Kadonaga J. T., Tjian R. Affinity purification of sequence-specific DNA binding proteins. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5889–5893. doi: 10.1073/pnas.83.16.5889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lee W., Haslinger A., Karin M., Tjian R. Activation of transcription by two factors that bind promoter and enhancer sequences of the human metallothionein gene and SV40. Nature. 1987 Jan 22;325(6102):368–372. doi: 10.1038/325368a0. [DOI] [PubMed] [Google Scholar]
  26. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  27. Melton D. W., Konecki D. S., Brennand J., Caskey C. T. Structure, expression, and mutation of the hypoxanthine phosphoribosyltransferase gene. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2147–2151. doi: 10.1073/pnas.81.7.2147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mitchell P. J., Carothers A. M., Han J. H., Harding J. D., Kas E., Venolia L., Chasin L. A. Multiple transcription start sites, DNase I-hypersensitive sites, and an opposite-strand exon in the 5' region of the CHO dhfr gene. Mol Cell Biol. 1986 Feb;6(2):425–440. doi: 10.1128/mcb.6.2.425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Morioka H., Tennyson G. E., Nikodem V. M. Structural and functional analysis of the rat malic enzyme gene promoter. Mol Cell Biol. 1988 Aug;8(8):3542–3545. doi: 10.1128/mcb.8.8.3542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rauscher F. J., 3rd, Cohen D. R., Curran T., Bos T. J., Vogt P. K., Bohmann D., Tjian R., Franza B. R., Jr Fos-associated protein p39 is the product of the jun proto-oncogene. Science. 1988 May 20;240(4855):1010–1016. doi: 10.1126/science.3130660. [DOI] [PubMed] [Google Scholar]
  31. Rauscher F. J., 3rd, Sambucetti L. C., Curran T., Distel R. J., Spiegelman B. M. Common DNA binding site for Fos protein complexes and transcription factor AP-1. Cell. 1988 Feb 12;52(3):471–480. doi: 10.1016/s0092-8674(88)80039-4. [DOI] [PubMed] [Google Scholar]
  32. Reynolds G. A., Basu S. K., Osborne T. F., Chin D. J., Gil G., Brown M. S., Goldstein J. L., Luskey K. L. HMG CoA reductase: a negatively regulated gene with unusual promoter and 5' untranslated regions. Cell. 1984 Aug;38(1):275–285. doi: 10.1016/0092-8674(84)90549-x. [DOI] [PubMed] [Google Scholar]
  33. Santiago C., Collins M., Johnson L. F. In vitro and in vivo analysis of the control of dihydrofolate reductase gene transcription in serum-stimulated mouse fibroblasts. J Cell Physiol. 1984 Jan;118(1):79–86. doi: 10.1002/jcp.1041180114. [DOI] [PubMed] [Google Scholar]
  34. Sehgal A., Patil N., Chao M. A constitutive promoter directs expression of the nerve growth factor receptor gene. Mol Cell Biol. 1988 Aug;8(8):3160–3167. doi: 10.1128/mcb.8.8.3160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ucla E., Triffard F., Choquenet C., Chrétien Y., Dufour B. Récidive sur une néo-vessie iléale d'un carcinome urothélial. A propos d'un cas. Ann Urol (Paris) 1988;22(2):107–110. [PubMed] [Google Scholar]
  36. Valerio D., Duyvesteyn M. G., Dekker B. M., Weeda G., Berkvens T. M., van der Voorn L., van Ormondt H., van der Eb A. J. Adenosine deaminase: characterization and expression of a gene with a remarkable promoter. EMBO J. 1985 Feb;4(2):437–443. doi: 10.1002/j.1460-2075.1985.tb03648.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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