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Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1993 Apr;13(4):2504–2514. doi: 10.1128/mcb.13.4.2504

Ligand-dependent recruitment of the Arnt coregulator determines DNA recognition by the dioxin receptor.

M Whitelaw 1, I Pongratz 1, A Wilhelmsson 1, J A Gustafsson 1, L Poellinger 1
PMCID: PMC359572  PMID: 8384309

Abstract

The intracellular basic region/helix-loop-helix (bHLH) dioxin receptor mediates signal transduction by dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) and functions as a ligand-activated DNA binding protein directly interacting with target genes by binding to dioxin response elements. Here we show that the partially purified, ligand-bound receptor alone could not bind target DNA. In contrast, DNA binding by the receptor could be induced by addition of a cytosolic auxiliary activity which functionally and biochemically corresponded to the bHLH factor Arnt. While Arnt exhibited no detectable affinity for the dioxin response element in the absence of the dioxin receptor, it strongly promoted the DNA binding function of the ligand-activated but not the ligand-free receptor forms. Arnt also functionally reconstituted in vitro the DNA binding activity of a mutant, nuclear translocation-deficient dioxin receptor phenotype in cytosolic extracts from a dioxin-resistant hepatoma cell line. Importantly, coimmunoprecipitation experiments showed that Arnt physically interacted in solution with the ligand-activated dioxin receptor but failed to heterodimerize with the ligand-free, hsp90-associated receptor form. Mutational analysis suggested that the functional interaction between these two factors occurred via the bHLH motif of Arnt. These data suggest that dioxin receptor activity is governed by a complex pattern of combinatorial regulation involving repression by hsp90 and then by ligand-dependent recruitment of the positive coregulator Arnt. The dioxin receptor system also provides the first example of signal-controlled dimerization of bHLH factors.

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Selected References

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  1. Alksnis M., Barkhem T., Strömstedt P. E., Ahola H., Kutoh E., Gustafsson J. A., Poellinger L., Nilsson S. High level expression of functional full length and truncated glucocorticoid receptor in Chinese hamster ovary cells. Demonstration of ligand-induced down-regulation of expressed receptor mRNA and protein. J Biol Chem. 1991 Jun 5;266(16):10078–10085. [PubMed] [Google Scholar]
  2. Andersson S., Davis D. L., Dahlbäck H., Jörnvall H., Russell D. W. Cloning, structure, and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme. J Biol Chem. 1989 May 15;264(14):8222–8229. [PubMed] [Google Scholar]
  3. 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]
  4. Bengal E., Ransone L., Scharfmann R., Dwarki V. J., Tapscott S. J., Weintraub H., Verma I. M. Functional antagonism between c-Jun and MyoD proteins: a direct physical association. Cell. 1992 Feb 7;68(3):507–519. doi: 10.1016/0092-8674(92)90187-h. [DOI] [PubMed] [Google Scholar]
  5. Berghard A., Gradin K., Pongratz I., Whitelaw M., Poellinger L. Cross-coupling of signal transduction pathways: the dioxin receptor mediates induction of cytochrome P-450IA1 expression via a protein kinase C-dependent mechanism. Mol Cell Biol. 1993 Jan;13(1):677–689. doi: 10.1128/mcb.13.1.677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Bugge T. H., Pohl J., Lonnoy O., Stunnenberg H. G. RXR alpha, a promiscuous partner of retinoic acid and thyroid hormone receptors. EMBO J. 1992 Apr;11(4):1409–1418. doi: 10.1002/j.1460-2075.1992.tb05186.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Burbach K. M., Poland A., Bradfield C. A. Cloning of the Ah-receptor cDNA reveals a distinctive ligand-activated transcription factor. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8185–8189. doi: 10.1073/pnas.89.17.8185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Carrier F., Owens R. A., Nebert D. W., Puga A. Dioxin-dependent activation of murine Cyp1a-1 gene transcription requires protein kinase C-dependent phosphorylation. Mol Cell Biol. 1992 Apr;12(4):1856–1863. doi: 10.1128/mcb.12.4.1856. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cuthill S., Wilhelmsson A., Poellinger L. Role of the ligand in intracellular receptor function: receptor affinity determines activation in vitro of the latent dioxin receptor to a DNA-binding form. Mol Cell Biol. 1991 Jan;11(1):401–411. doi: 10.1128/mcb.11.1.401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Denis M., Cuthill S., Wikström A. C., Poellinger L., Gustafsson J. A. Association of the dioxin receptor with the Mr 90,000 heat shock protein: a structural kinship with the glucocorticoid receptor. Biochem Biophys Res Commun. 1988 Sep 15;155(2):801–807. doi: 10.1016/s0006-291x(88)80566-7. [DOI] [PubMed] [Google Scholar]
  13. Denis M., Poellinger L., Wikstöm A. C., Gustafsson J. A. Requirement of hormone for thermal conversion of the glucocorticoid receptor to a DNA-binding state. Nature. 1988 Jun 16;333(6174):686–688. doi: 10.1038/333686a0. [DOI] [PubMed] [Google Scholar]
  14. Denison M. S., Fisher J. M., Whitlock J. P., Jr Inducible, receptor-dependent protein-DNA interactions at a dioxin-responsive transcriptional enhancer. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2528–2532. doi: 10.1073/pnas.85.8.2528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Elferink C. J., Gasiewicz T. A., Whitlock J. P., Jr Protein-DNA interactions at a dioxin-responsive enhancer. Evidence that the transformed Ah receptor is heteromeric. J Biol Chem. 1990 Nov 25;265(33):20708–20712. [PubMed] [Google Scholar]
  16. Ema M., Sogawa K., Watanabe N., Chujoh Y., Matsushita N., Gotoh O., Funae Y., Fujii-Kuriyama Y. cDNA cloning and structure of mouse putative Ah receptor. Biochem Biophys Res Commun. 1992 Apr 15;184(1):246–253. doi: 10.1016/0006-291x(92)91185-s. [DOI] [PubMed] [Google Scholar]
  17. Favreau L. V., Pickett C. B. Transcriptional regulation of the rat NAD(P)H:quinone reductase gene. Identification of regulatory elements controlling basal level expression and inducible expression by planar aromatic compounds and phenolic antioxidants. J Biol Chem. 1991 Mar 5;266(7):4556–4561. [PubMed] [Google Scholar]
  18. Fisher D. E., Carr C. S., Parent L. A., Sharp P. A. TFEB has DNA-binding and oligomerization properties of a unique helix-loop-helix/leucine-zipper family. Genes Dev. 1991 Dec;5(12A):2342–2352. doi: 10.1101/gad.5.12a.2342. [DOI] [PubMed] [Google Scholar]
  19. Fujisawa-Sehara A., Yamane M., Fujii-Kuriyama Y. A DNA-binding factor specific for xenobiotic responsive elements of P-450c gene exists as a cryptic form in cytoplasm: its possible translocation to nucleus. Proc Natl Acad Sci U S A. 1988 Aug;85(16):5859–5863. doi: 10.1073/pnas.85.16.5859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gasiewicz T. A., Neal R. A. The examination and quantitation of tissue cytosolic receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin using hydroxylapatite. Anal Biochem. 1982 Jul 15;124(1):1–11. doi: 10.1016/0003-2697(82)90212-3. [DOI] [PubMed] [Google Scholar]
  21. Green S. Nuclear receptors and chemical carcinogenesis. Trends Pharmacol Sci. 1992 Jun;13(6):251–255. doi: 10.1016/0165-6147(92)90078-k. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Hallenbeck P. L., Marks M. S., Lippoldt R. E., Ozato K., Nikodem V. M. Heterodimerization of thyroid hormone (TH) receptor with H-2RIIBP (RXR beta) enhances DNA binding and TH-dependent transcriptional activation. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5572–5576. doi: 10.1073/pnas.89.12.5572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Hapgood J., Cuthill S., Denis M., Poellinger L., Gustafsson J. A. Specific protein-DNA interactions at a xenobiotic-responsive element: copurification of dioxin receptor and DNA-binding activity. Proc Natl Acad Sci U S A. 1989 Jan;86(1):60–64. doi: 10.1073/pnas.86.1.60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Heyman R. A., Mangelsdorf D. J., Dyck J. A., Stein R. B., Eichele G., Evans R. M., Thaller C. 9-cis retinoic acid is a high affinity ligand for the retinoid X receptor. Cell. 1992 Jan 24;68(2):397–406. doi: 10.1016/0092-8674(92)90479-v. [DOI] [PubMed] [Google Scholar]
  27. Hoffman E. C., Reyes H., Chu F. F., Sander F., Conley L. H., Brooks B. A., Hankinson O. Cloning of a factor required for activity of the Ah (dioxin) receptor. Science. 1991 May 17;252(5008):954–958. doi: 10.1126/science.1852076. [DOI] [PubMed] [Google Scholar]
  28. Härd T., Kellenbach E., Boelens R., Maler B. A., Dahlman K., Freedman L. P., Carlstedt-Duke J., Yamamoto K. R., Gustafsson J. A., Kaptein R. Solution structure of the glucocorticoid receptor DNA-binding domain. Science. 1990 Jul 13;249(4965):157–160. doi: 10.1126/science.2115209. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Kliewer S. A., Umesono K., Mangelsdorf D. J., Evans R. M. Retinoid X receptor interacts with nuclear receptors in retinoic acid, thyroid hormone and vitamin D3 signalling. Nature. 1992 Jan 30;355(6359):446–449. doi: 10.1038/355446a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kliewer S. A., Umesono K., Noonan D. J., Heyman R. A., Evans R. M. Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors. Nature. 1992 Aug 27;358(6389):771–774. doi: 10.1038/358771a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Landers J. P., Bunce N. J. The Ah receptor and the mechanism of dioxin toxicity. Biochem J. 1991 Jun 1;276(Pt 2):273–287. doi: 10.1042/bj2760273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lassar A. B., Davis R. L., Wright W. E., Kadesch T., Murre C., Voronova A., Baltimore D., Weintraub H. Functional activity of myogenic HLH proteins requires hetero-oligomerization with E12/E47-like proteins in vivo. Cell. 1991 Jul 26;66(2):305–315. doi: 10.1016/0092-8674(91)90620-e. [DOI] [PubMed] [Google Scholar]
  34. Leid M., Kastner P., Lyons R., Nakshatri H., Saunders M., Zacharewski T., Chen J. Y., Staub A., Garnier J. M., Mader S. Purification, cloning, and RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently. Cell. 1992 Jan 24;68(2):377–395. doi: 10.1016/0092-8674(92)90478-u. [DOI] [PubMed] [Google Scholar]
  35. Levin A. A., Sturzenbecker L. J., Kazmer S., Bosakowski T., Huselton C., Allenby G., Speck J., Kratzeisen C., Rosenberger M., Lovey A. 9-cis retinoic acid stereoisomer binds and activates the nuclear receptor RXR alpha. Nature. 1992 Jan 23;355(6358):359–361. doi: 10.1038/355359a0. [DOI] [PubMed] [Google Scholar]
  36. Luisi B. F., Xu W. X., Otwinowski Z., Freedman L. P., Yamamoto K. R., Sigler P. B. Crystallographic analysis of the interaction of the glucocorticoid receptor with DNA. Nature. 1991 Aug 8;352(6335):497–505. doi: 10.1038/352497a0. [DOI] [PubMed] [Google Scholar]
  37. Marks M. S., Hallenbeck P. L., Nagata T., Segars J. H., Appella E., Nikodem V. M., Ozato K. H-2RIIBP (RXR beta) heterodimerization provides a mechanism for combinatorial diversity in the regulation of retinoic acid and thyroid hormone responsive genes. EMBO J. 1992 Apr;11(4):1419–1435. doi: 10.1002/j.1460-2075.1992.tb05187.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Miner J. N., Yamamoto K. R. Regulatory crosstalk at composite response elements. Trends Biochem Sci. 1991 Nov;16(11):423–426. doi: 10.1016/0968-0004(91)90168-u. [DOI] [PubMed] [Google Scholar]
  39. Moore M. S., Blobel G. The two steps of nuclear import, targeting to the nuclear envelope and translocation through the nuclear pore, require different cytosolic factors. Cell. 1992 Jun 12;69(6):939–950. doi: 10.1016/0092-8674(92)90613-h. [DOI] [PubMed] [Google Scholar]
  40. Murre C., McCaw P. S., Vaessin H., Caudy M., Jan L. Y., Jan Y. N., Cabrera C. V., Buskin J. N., Hauschka S. D., Lassar A. B. Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence. Cell. 1989 Aug 11;58(3):537–544. doi: 10.1016/0092-8674(89)90434-0. [DOI] [PubMed] [Google Scholar]
  41. Nemoto T., Mason G. G., Wilhelmsson A., Cuthill S., Hapgood J., Gustafsson J. A., Poellinger L. Activation of the dioxin and glucocorticoid receptors to a DNA binding state under cell-free conditions. J Biol Chem. 1990 Feb 5;265(4):2269–2277. [PubMed] [Google Scholar]
  42. Neuhold L. A., Shirayoshi Y., Ozato K., Jones J. E., Nebert D. W. Regulation of mouse CYP1A1 gene expression by dioxin: requirement of two cis-acting elements during induction. Mol Cell Biol. 1989 Jun;9(6):2378–2386. doi: 10.1128/mcb.9.6.2378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Okino S. T., Pendurthi U. R., Tukey R. H. Phorbol esters inhibit the dioxin receptor-mediated transcriptional activation of the mouse Cyp1a-1 and Cyp1a-2 genes by 2,3,7,8-tetrachlorodibenzo-p-dioxin. J Biol Chem. 1992 Apr 5;267(10):6991–6998. [PubMed] [Google Scholar]
  44. Paulson K. E., Darnell J. E., Jr, Rushmore T., Pickett C. B. Analysis of the upstream elements of the xenobiotic compound-inducible and positionally regulated glutathione S-transferase Ya gene. Mol Cell Biol. 1990 May;10(5):1841–1852. doi: 10.1128/mcb.10.5.1841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Perdew G. H. Association of the Ah receptor with the 90-kDa heat shock protein. J Biol Chem. 1988 Sep 25;263(27):13802–13805. [PubMed] [Google Scholar]
  46. Poellinger L., Göttlicher M., Gustafsson J. A. The dioxin and peroxisome proliferator-activated receptors: nuclear receptors in search of endogenous ligands. Trends Pharmacol Sci. 1992 Jun;13(6):241–245. doi: 10.1016/0165-6147(92)90076-i. [DOI] [PubMed] [Google Scholar]
  47. Poland A., Glover E., Ebetino F. H., Kende A. S. Photoaffinity labeling of the Ah receptor. J Biol Chem. 1986 May 15;261(14):6352–6365. [PubMed] [Google Scholar]
  48. 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]
  49. Pongratz I., Mason G. G., Poellinger L. Dual roles of the 90-kDa heat shock protein hsp90 in modulating functional activities of the dioxin receptor. Evidence that the dioxin receptor functionally belongs to a subclass of nuclear receptors which require hsp90 both for ligand binding activity and repression of intrinsic DNA binding activity. J Biol Chem. 1992 Jul 5;267(19):13728–13734. [PubMed] [Google Scholar]
  50. Pongratz I., Strömstedt P. E., Mason G. G., Poellinger L. Inhibition of the specific DNA binding activity of the dioxin receptor by phosphatase treatment. J Biol Chem. 1991 Sep 5;266(25):16813–16817. [PubMed] [Google Scholar]
  51. 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]
  52. Rashbass J., Taylor M. V., Gurdon J. B. The DNA-binding protein E12 co-operates with XMyoD in the activation of muscle-specific gene expression in Xenopus embryos. EMBO J. 1992 Aug;11(8):2981–2990. doi: 10.1002/j.1460-2075.1992.tb05368.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Reyes H., Reisz-Porszasz S., Hankinson O. Identification of the Ah receptor nuclear translocator protein (Arnt) as a component of the DNA binding form of the Ah receptor. Science. 1992 May 22;256(5060):1193–1195. doi: 10.1126/science.256.5060.1193. [DOI] [PubMed] [Google Scholar]
  54. Schüle R., Evans R. M. Cross-coupling of signal transduction pathways: zinc finger meets leucine zipper. Trends Genet. 1991 Nov-Dec;7(11-12):377–381. doi: 10.1016/0168-9525(91)90259-s. [DOI] [PubMed] [Google Scholar]
  55. Sutter T. R., Guzman K., Dold K. M., Greenlee W. F. Targets for dioxin: genes for plasminogen activator inhibitor-2 and interleukin-1 beta. Science. 1991 Oct 18;254(5030):415–418. doi: 10.1126/science.1925598. [DOI] [PubMed] [Google Scholar]
  56. Wiech H., Buchner J., Zimmermann R., Jakob U. Hsp90 chaperones protein folding in vitro. Nature. 1992 Jul 9;358(6382):169–170. doi: 10.1038/358169a0. [DOI] [PubMed] [Google Scholar]
  57. Wilhelmsson A., Cuthill S., Denis M., Wikström A. C., Gustafsson J. A., Poellinger L. The specific DNA binding activity of the dioxin receptor is modulated by the 90 kd heat shock protein. EMBO J. 1990 Jan;9(1):69–76. doi: 10.1002/j.1460-2075.1990.tb08081.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wilhelmsson A., Wikström A. C., Poellinger L. Polyanionic-binding properties of the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. A comparison with the glucocorticoid receptor. J Biol Chem. 1986 Oct 15;261(29):13456–13463. [PubMed] [Google Scholar]
  59. Yu V. C., Delsert C., Andersen B., Holloway J. M., Devary O. V., När A. M., Kim S. Y., Boutin J. M., Glass C. K., Rosenfeld M. G. RXR beta: a coregulator that enhances binding of retinoic acid, thyroid hormone, and vitamin D receptors to their cognate response elements. Cell. 1991 Dec 20;67(6):1251–1266. doi: 10.1016/0092-8674(91)90301-e. [DOI] [PubMed] [Google Scholar]
  60. Zhang X. K., Hoffmann B., Tran P. B., Graupner G., Pfahl M. Retinoid X receptor is an auxiliary protein for thyroid hormone and retinoic acid receptors. Nature. 1992 Jan 30;355(6359):441–446. doi: 10.1038/355441a0. [DOI] [PubMed] [Google Scholar]
  61. Zhang X. K., Lehmann J., Hoffmann B., Dawson M. I., Cameron J., Graupner G., Hermann T., Tran P., Pfahl M. Homodimer formation of retinoid X receptor induced by 9-cis retinoic acid. Nature. 1992 Aug 13;358(6387):587–591. doi: 10.1038/358587a0. [DOI] [PubMed] [Google Scholar]

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