Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1990 Nov;87(21):8545–8549. doi: 10.1073/pnas.87.21.8545

Dioxin-inducible, Ah receptor-dependent transcription in vitro.

L P Wen 1, N Koeiman 1, J P Whitlock Jr 1
PMCID: PMC54993  PMID: 2172990

Abstract

We have developed a homologous in vitro transcription system that requires (i) 2,3,7,8-tetrachlorodibenzo-p-dioxin (called TCDD or dioxin), (ii) the Ah receptor, and (iii) a dioxin-responsive enhancer for activity. Unfractionated nuclear extracts from mouse hepatoma cells contain an inhibitor and fail to direct transcription in vitro. However, following phosphocellulose chromatography and reconstitution, the fractionated nuclear extract directs accurate transcription in vitro, using as a template the promoter/enhancer region from the mouse cytochrome P1-450 gene (Cyp1a1) linked to a "G-free cassette" (which generates a transcript with no guanosine residues). Extracts from TCDD-treated cells exhibit higher activity than extracts from untreated cells when transcribing a template containing both the promoter and enhancer but not when transcribing a template containing the promoter alone. Extracts from Ah receptor-defective cells fail to direct in vitro transcription in a TCDD-inducible fashion. A regulatory element that contains two binding sites for the liganded Ah receptor plus a truncated Cyp1a1 promoter suffices to direct TCDD-inducible, Ah receptor-dependent transcription in vitro. The inducible, receptor-dependent, enhancer-dependent properties of this system make it appropriate for analyzing in vitro the mechanism of dioxin action and the function of the Ah receptor.

Full text

PDF
8545

Images in this article

8546Image
on p.8546
8547Image
on p.8547
8547Image
on p.8547
8547Image
on p.8547
8547Image
on p.8547
8548Image
on p.8548
8548Image
on p.8548

Selected References

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

  1. Benezra R., Davis R. L., Lockshon D., Turner D. L., Weintraub H. The protein Id: a negative regulator of helix-loop-helix DNA binding proteins. Cell. 1990 Apr 6;61(1):49–59. doi: 10.1016/0092-8674(90)90214-y. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Fisher J. M., Jones K. W., Whitlock J. P., Jr Activation of transcription as a general mechanism of 2,3,7,8-tetrachlorodibenzo-p-dioxin action. Mol Carcinog. 1989;1(4):216–221. doi: 10.1002/mc.2940010403. [DOI] [PubMed] [Google Scholar]
  4. Freedman L. P., Yoshinaga S. K., Vanderbilt J. N., Yamamoto K. R. In vitro transcription enhancement by purified derivatives of the glucocorticoid receptor. Science. 1989 Jul 21;245(4915):298–301. doi: 10.1126/science.2473529. [DOI] [PubMed] [Google Scholar]
  5. Gonzalez F. J. The molecular biology of cytochrome P450s. Pharmacol Rev. 1988 Dec;40(4):243–288. [PubMed] [Google Scholar]
  6. Gorski K., Carneiro M., Schibler U. Tissue-specific in vitro transcription from the mouse albumin promoter. Cell. 1986 Dec 5;47(5):767–776. doi: 10.1016/0092-8674(86)90519-2. [DOI] [PubMed] [Google Scholar]
  7. Hankinson O., Andersen R. D., Birren B. W., Sander F., Negishi M., Nebert D. W. Mutations affecting the regulation of transcription of the cytochrome P1-450 gene in the mouse Hepa-1 cell line. J Biol Chem. 1985 Feb 10;260(3):1790–1795. [PubMed] [Google Scholar]
  8. Hankinson O. Dominant and recessive aryl hydrocarbon hydroxylase-deficient mutants of mouse hepatoma line, Hepa-1, and assignment of recessive mutants to three complementation groups. Somatic Cell Genet. 1983 Jul;9(4):497–514. doi: 10.1007/BF01543050. [DOI] [PubMed] [Google Scholar]
  9. Hirst M. A., Jones K. W., Whitlock J. P., Jr Activation of cytochrome P450IA1 gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin in wild-type and high-activity variant mouse hepatoma cells. Mol Carcinog. 1989;2(1):40–46. doi: 10.1002/mc.2940020107. [DOI] [PubMed] [Google Scholar]
  10. Israel D. I., Whitlock J. P., Jr Regulation of cytochrome P1-450 gene transcription by 2,3,7, 8-tetrachlorodibenzo-p-dioxin in wild type and variant mouse hepatoma cells. J Biol Chem. 1984 May 10;259(9):5400–5402. [PubMed] [Google Scholar]
  11. Jones K. W., Whitlock J. P., Jr Functional analysis of the transcriptional promoter for the CYP1A1 gene. Mol Cell Biol. 1990 Oct;10(10):5098–5105. doi: 10.1128/mcb.10.10.5098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jones P. B., Galeazzi D. R., Fisher J. M., Whitlock J. P., Jr Control of cytochrome P1-450 gene expression by dioxin. Science. 1985 Mar 22;227(4693):1499–1502. doi: 10.1126/science.3856321. [DOI] [PubMed] [Google Scholar]
  13. Jones P. B., Miller A. G., Israel D. I., Galeazzi D. R., Whitlock J. P., Jr Biochemical and genetic analysis of variant mouse hepatoma cells which overtranscribe the cytochrome P1-450 gene in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin. J Biol Chem. 1984 Oct 25;259(20):12357–12363. [PubMed] [Google Scholar]
  14. Kalff M., Gross B., Beato M. Progesterone receptor stimulates transcription of mouse mammary tumour virus in a cell-free system. Nature. 1990 Mar 22;344(6264):360–362. doi: 10.1038/344360a0. [DOI] [PubMed] [Google Scholar]
  15. Klein-Hitpass L., Tsai S. Y., Weigel N. L., Allan G. F., Riley D., Rodriguez R., Schrader W. T., Tsai M. J., O'Malley B. W. The progesterone receptor stimulates cell-free transcription by enhancing the formation of a stable preinitiation complex. Cell. 1990 Jan 26;60(2):247–257. doi: 10.1016/0092-8674(90)90740-6. [DOI] [PubMed] [Google Scholar]
  16. Lilienfeld D. E., Gallo M. A. 2,4-D, 2,4,5-T, and 2,3,7,8-TCDD: an overview. Epidemiol Rev. 1989;11:28–58. doi: 10.1093/oxfordjournals.epirev.a036044. [DOI] [PubMed] [Google Scholar]
  17. Matsui T., Segall J., Weil P. A., Roeder R. G. Multiple factors required for accurate initiation of transcription by purified RNA polymerase II. J Biol Chem. 1980 Dec 25;255(24):11992–11996. [PubMed] [Google Scholar]
  18. Miller A. G., Israel D., Whitlock J. P., Jr Biochemical and genetic analysis of variant mouse hepatoma cells defective in the induction of benzo(a)pyrene-metabolizing enzyme activity. J Biol Chem. 1983 Mar 25;258(6):3523–3527. [PubMed] [Google Scholar]
  19. Nakajima N., Horikoshi M., Roeder R. G. Factors involved in specific transcription by mammalian RNA polymerase II: purification, genetic specificity, and TATA box-promoter interactions of TFIID. Mol Cell Biol. 1988 Oct;8(10):4028–4040. doi: 10.1128/mcb.8.10.4028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ohkuma Y., Horikoshi M., Roeder R. G., Desplan C. Binding site-dependent direct activation and repression of in vitro transcription by Drosophila homeodomain proteins. Cell. 1990 May 4;61(3):475–484. doi: 10.1016/0092-8674(90)90529-n. [DOI] [PubMed] [Google Scholar]
  21. Poland A., Knutson J. C. 2,3,7,8-tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Annu Rev Pharmacol Toxicol. 1982;22:517–554. doi: 10.1146/annurev.pa.22.040182.002505. [DOI] [PubMed] [Google Scholar]
  22. Sawadogo M., Roeder R. G. Factors involved in specific transcription by human RNA polymerase II: analysis by a rapid and quantitative in vitro assay. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4394–4398. doi: 10.1073/pnas.82.13.4394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Whitlock J. P., Jr, Galeazzi D. R. 2,3,7,8-Tetrachlorodibenzo-p-dioxin receptors in wild type and variant mouse hepatoma cells. Nuclear location and strength of nuclear binding. J Biol Chem. 1984 Jan 25;259(2):980–985. [PubMed] [Google Scholar]
  24. Whitlock J. P., Jr Genetic and molecular aspects of 2,3,7,8-tetrachlorodibenzo-p-dioxin action. Annu Rev Pharmacol Toxicol. 1990;30:251–277. doi: 10.1146/annurev.pa.30.040190.001343. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES