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. 1996 Apr;16(4):1479–1489. doi: 10.1128/mcb.16.4.1479

Stress-induced binding of the transcriptional factor CHOP to a novel DNA control element.

M Ubeda 1, X Z Wang 1, H Zinszner 1, I Wu 1, J F Habener 1, D Ron 1
PMCID: PMC231132  PMID: 8657121

Abstract

CHOP (GADD153) is a mammalian nuclear protein that dimerizes with members of the C/EBP family of transcriptional factors. Absent under normal conditions, CHOP is induced by the stress encountered during nutrient deprivation, the acute-phase response, and treatment of cells with certain toxins. The basic region of CHOP deviates considerably in sequence from that of other C/EBP proteins, and CHOP-C/EBP heterodimers are incapable of binding to a common class of C/EBP sites. With respect to such sites, CHOP serves as an inhibitor of the activity of C/EBP proteins. However, recent studies indicate that certain functions of CHOP, such as the induction of growth arrest by overexpression of the wild-type protein and oncogenic transformation by the TLS-CHOP fusion protein, require an intact basic region, suggesting that DNA binding by CHOP may be implicated in these activities. In this study an in vitro PCR-based selection assay was used to identify sequences bound by CHOP-C/EBP dimers. These sequences were found to contain a unique core element PuPuPuTGCAAT(A/C)CCC. Competition in DNA-binding assays, DNase 1 footprint analysis, and methylation interference demonstrate that the binding is sequence specific. Deletions in the basic region of CHOP lead to a loss of DNA binding, suggesting that CHOP participates in this process. Stress induction in NIH 3T3 cells leads to the appearance of CHOP-containing DNA-binding activity. CHOP is found to contain a transcriptional activation domain which is inducible by cellular stress, lending further support to the notion that the protein can function as a positively acting transcription factor. We conclude that CHOP may serve a dual role both as an inhibitor of the ability of C/EBP proteins to activate some target genes and as a direct activator of others.

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Selected References

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  1. Akira S., Isshiki H., Sugita T., Tanabe O., Kinoshita S., Nishio Y., Nakajima T., Hirano T., Kishimoto T. A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J. 1990 Jun;9(6):1897–1906. doi: 10.1002/j.1460-2075.1990.tb08316.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alam T., An M. R., Papaconstantinou J. Differential expression of three C/EBP isoforms in multiple tissues during the acute phase response. J Biol Chem. 1992 Mar 15;267(8):5021–5024. [PubMed] [Google Scholar]
  3. Barone M. V., Crozat A., Tabaee A., Philipson L., Ron D. CHOP (GADD153) and its oncogenic variant, TLS-CHOP, have opposing effects on the induction of G1/S arrest. Genes Dev. 1994 Feb 15;8(4):453–464. doi: 10.1101/gad.8.4.453. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Brasier A. R., Ron D., Tate J. E., Habener J. F. A family of constitutive C/EBP-like DNA binding proteins attenuate the IL-1 alpha induced, NF kappa B mediated trans-activation of the angiotensinogen gene acute-phase response element. EMBO J. 1990 Dec;9(12):3933–3944. doi: 10.1002/j.1460-2075.1990.tb07614.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Buck M., Turler H., Chojkier M. LAP (NF-IL-6), a tissue-specific transcriptional activator, is an inhibitor of hepatoma cell proliferation. EMBO J. 1994 Feb 15;13(4):851–860. doi: 10.1002/j.1460-2075.1994.tb06328.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cao Z., Umek R. M., McKnight S. L. Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. Genes Dev. 1991 Sep;5(9):1538–1552. doi: 10.1101/gad.5.9.1538. [DOI] [PubMed] [Google Scholar]
  8. Carlson S. G., Fawcett T. W., Bartlett J. D., Bernier M., Holbrook N. J. Regulation of the C/EBP-related gene gadd153 by glucose deprivation. Mol Cell Biol. 1993 Aug;13(8):4736–4744. doi: 10.1128/mcb.13.8.4736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen Q., Yu K., Holbrook N. J., Stevens J. L. Activation of the growth arrest and DNA damage-inducible gene gadd 153 by nephrotoxic cysteine conjugates and dithiothreitol. J Biol Chem. 1992 Apr 25;267(12):8207–8212. [PubMed] [Google Scholar]
  10. Christy R. J., Kaestner K. H., Geiman D. E., Lane M. D. CCAAT/enhancer binding protein gene promoter: binding of nuclear factors during differentiation of 3T3-L1 preadipocytes. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2593–2597. doi: 10.1073/pnas.88.6.2593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Christy R. J., Yang V. W., Ntambi J. M., Geiman D. E., Landschulz W. H., Friedman A. D., Nakabeppu Y., Kelly T. J., Lane M. D. Differentiation-induced gene expression in 3T3-L1 preadipocytes: CCAAT/enhancer binding protein interacts with and activates the promoters of two adipocyte-specific genes. Genes Dev. 1989 Sep;3(9):1323–1335. doi: 10.1101/gad.3.9.1323. [DOI] [PubMed] [Google Scholar]
  12. Crossley M., Brownlee G. G. Disruption of a C/EBP binding site in the factor IX promoter is associated with haemophilia B. Nature. 1990 May 31;345(6274):444–446. doi: 10.1038/345444a0. [DOI] [PubMed] [Google Scholar]
  13. Crozat A., Aman P., Mandahl N., Ron D. Fusion of CHOP to a novel RNA-binding protein in human myxoid liposarcoma. Nature. 1993 Jun 17;363(6430):640–644. doi: 10.1038/363640a0. [DOI] [PubMed] [Google Scholar]
  14. Ellenberger T. E., Brandl C. J., Struhl K., Harrison S. C. The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted alpha helices: crystal structure of the protein-DNA complex. Cell. 1992 Dec 24;71(7):1223–1237. doi: 10.1016/s0092-8674(05)80070-4. [DOI] [PubMed] [Google Scholar]
  15. Fornace A. J., Jr, Nebert D. W., Hollander M. C., Luethy J. D., Papathanasiou M., Fargnoli J., Holbrook N. J. Mammalian genes coordinately regulated by growth arrest signals and DNA-damaging agents. Mol Cell Biol. 1989 Oct;9(10):4196–4203. doi: 10.1128/mcb.9.10.4196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Freytag S. O., Geddes T. J. Reciprocal regulation of adipogenesis by Myc and C/EBP alpha. Science. 1992 Apr 17;256(5055):379–382. doi: 10.1126/science.256.5055.379. [DOI] [PubMed] [Google Scholar]
  17. Freytag S. O., Paielli D. L., Gilbert J. D. Ectopic expression of the CCAAT/enhancer-binding protein alpha promotes the adipogenic program in a variety of mouse fibroblastic cells. Genes Dev. 1994 Jul 15;8(14):1654–1663. doi: 10.1101/gad.8.14.1654. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Johnson P. F. Identification of C/EBP basic region residues involved in DNA sequence recognition and half-site spacing preference. Mol Cell Biol. 1993 Nov;13(11):6919–6930. doi: 10.1128/mcb.13.11.6919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kreider B. L., Benezra R., Rovera G., Kadesch T. Inhibition of myeloid differentiation by the helix-loop-helix protein Id. Science. 1992 Mar 27;255(5052):1700–1702. doi: 10.1126/science.1372755. [DOI] [PubMed] [Google Scholar]
  21. Landschulz W. H., Johnson P. F., Adashi E. Y., Graves B. J., McKnight S. L. Isolation of a recombinant copy of the gene encoding C/EBP. Genes Dev. 1988 Jul;2(7):786–800. doi: 10.1101/gad.2.7.786. [DOI] [PubMed] [Google Scholar]
  22. Landschulz W. H., Johnson P. F., McKnight S. L. The DNA binding domain of the rat liver nuclear protein C/EBP is bipartite. Science. 1989 Mar 31;243(4899):1681–1688. doi: 10.1126/science.2494700. [DOI] [PubMed] [Google Scholar]
  23. Li X. X., Huang J. H., Rienhoff H. Y., Jr, Liao W. S. Two adjacent C/EBP-binding sequences that participate in the cell-specific expression of the mouse serum amyloid A3 gene. Mol Cell Biol. 1990 Dec;10(12):6624–6631. doi: 10.1128/mcb.10.12.6624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Li X. X., Liao W. S. Expression of rat serum amyloid A1 gene involves both C/EBP-like and NF kappa B-like transcription factors. J Biol Chem. 1991 Aug 15;266(23):15192–15201. [PubMed] [Google Scholar]
  25. Lin F. T., Lane M. D. Antisense CCAAT/enhancer-binding protein RNA suppresses coordinate gene expression and triglyceride accumulation during differentiation of 3T3-L1 preadipocytes. Genes Dev. 1992 Apr;6(4):533–544. doi: 10.1101/gad.6.4.533. [DOI] [PubMed] [Google Scholar]
  26. Luethy J. D., Holbrook N. J. Activation of the gadd153 promoter by genotoxic agents: a rapid and specific response to DNA damage. Cancer Res. 1992 Jan 1;52(1):5–10. [PubMed] [Google Scholar]
  27. Maire P., Wuarin J., Schibler U. The role of cis-acting promoter elements in tissue-specific albumin gene expression. Science. 1989 Apr 21;244(4902):343–346. doi: 10.1126/science.2711183. [DOI] [PubMed] [Google Scholar]
  28. McKnight S. L., Lane M. D., Gluecksohn-Waelsch S. Is CCAAT/enhancer-binding protein a central regulator of energy metabolism? Genes Dev. 1989 Dec;3(12B):2021–2024. doi: 10.1101/gad.3.12b.2021. [DOI] [PubMed] [Google Scholar]
  29. O'Neil K. T., Hoess R. H., DeGrado W. F. Design of DNA-binding peptides based on the leucine zipper motif. Science. 1990 Aug 17;249(4970):774–778. doi: 10.1126/science.2389143. [DOI] [PubMed] [Google Scholar]
  30. Park J. S., Luethy J. D., Wang M. G., Fargnoli J., Fornace A. J., Jr, McBride O. W., Holbrook N. J. Isolation, characterization and chromosomal localization of the human GADD153 gene. Gene. 1992 Jul 15;116(2):259–267. doi: 10.1016/0378-1119(92)90523-r. [DOI] [PubMed] [Google Scholar]
  31. Price B. D., Calderwood S. K. Gadd45 and Gadd153 messenger RNA levels are increased during hypoxia and after exposure of cells to agents which elevate the levels of the glucose-regulated proteins. Cancer Res. 1992 Jul 1;52(13):3814–3817. [PubMed] [Google Scholar]
  32. Rabbitts T. H., Forster A., Larson R., Nathan P. Fusion of the dominant negative transcription regulator CHOP with a novel gene FUS by translocation t(12;16) in malignant liposarcoma. Nat Genet. 1993 Jun;4(2):175–180. doi: 10.1038/ng0693-175. [DOI] [PubMed] [Google Scholar]
  33. Ratajczak T., Williams P. M., DiLorenzo D., Ringold G. M. Multiple elements within the glucocorticoid regulatory unit of the rat alpha 1-acid glycoprotein gene are recognition sites for C/EBP. J Biol Chem. 1992 Jun 5;267(16):11111–11119. [PubMed] [Google Scholar]
  34. Ron D., Brasier A. R., McGehee R. E., Jr, Habener J. F. Tumor necrosis factor-induced reversal of adipocytic phenotype of 3T3-L1 cells is preceded by a loss of nuclear CCAAT/enhancer binding protein (C/EBP). J Clin Invest. 1992 Jan;89(1):223–233. doi: 10.1172/JCI115566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ron D., Habener J. F. CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transcription. Genes Dev. 1992 Mar;6(3):439–453. doi: 10.1101/gad.6.3.439. [DOI] [PubMed] [Google Scholar]
  36. Ryden T. A., Beemon K. Avian retroviral long terminal repeats bind CCAAT/enhancer-binding protein. Mol Cell Biol. 1989 Mar;9(3):1155–1164. doi: 10.1128/mcb.9.3.1155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sadowski I., Ma J., Triezenberg S., Ptashne M. GAL4-VP16 is an unusually potent transcriptional activator. Nature. 1988 Oct 6;335(6190):563–564. doi: 10.1038/335563a0. [DOI] [PubMed] [Google Scholar]
  38. Samuelsson L., Strömberg K., Vikman K., Bjursell G., Enerbäck S. The CCAAT/enhancer binding protein and its role in adipocyte differentiation: evidence for direct involvement in terminal adipocyte development. EMBO J. 1991 Dec;10(12):3787–3793. doi: 10.1002/j.1460-2075.1991.tb04948.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schreiber E., Matthias P., Müller M. M., Schaffner W. Rapid detection of octamer binding proteins with 'mini-extracts', prepared from a small number of cells. Nucleic Acids Res. 1989 Aug 11;17(15):6419–6419. doi: 10.1093/nar/17.15.6419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Shuman J. D., Vinson C. R., McKnight S. L. Evidence of changes in protease sensitivity and subunit exchange rate on DNA binding by C/EBP. Science. 1990 Aug 17;249(4970):771–774. doi: 10.1126/science.2202050. [DOI] [PubMed] [Google Scholar]
  41. Suckow M., von Wilcken-Bergmann B., Müller-Hill B. Identification of three residues in the basic regions of the bZIP proteins GCN4, C/EBP and TAF-1 that are involved in specific DNA binding. EMBO J. 1993 Mar;12(3):1193–1200. doi: 10.1002/j.1460-2075.1993.tb05760.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sun X. H., Baltimore D. An inhibitory domain of E12 transcription factor prevents DNA binding in E12 homodimers but not in E12 heterodimers. Cell. 1991 Jan 25;64(2):459–470. doi: 10.1016/0092-8674(91)90653-g. [DOI] [PubMed] [Google Scholar]
  43. Trus M., Benvenisty N., Cohen H., Reshef L. Developmentally regulated interactions of liver nuclear factors with the rat phosphoenolpyruvate carboxykinase promoter. Mol Cell Biol. 1990 May;10(5):2418–2422. doi: 10.1128/mcb.10.5.2418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Umek R. M., Friedman A. D., McKnight S. L. CCAAT-enhancer binding protein: a component of a differentiation switch. Science. 1991 Jan 18;251(4991):288–292. doi: 10.1126/science.1987644. [DOI] [PubMed] [Google Scholar]
  45. Vinson C. R., Sigler P. B., McKnight S. L. Scissors-grip model for DNA recognition by a family of leucine zipper proteins. Science. 1989 Nov 17;246(4932):911–916. doi: 10.1126/science.2683088. [DOI] [PubMed] [Google Scholar]
  46. Wegner M., Cao Z., Rosenfeld M. G. Calcium-regulated phosphorylation within the leucine zipper of C/EBP beta. Science. 1992 Apr 17;256(5055):370–373. doi: 10.1126/science.256.5055.370. [DOI] [PubMed] [Google Scholar]
  47. Wilson D. R., Juan T. S., Wilde M. D., Fey G. H., Darlington G. J. A 58-base-pair region of the human C3 gene confers synergistic inducibility by interleukin-1 and interleukin-6. Mol Cell Biol. 1990 Dec;10(12):6181–6191. doi: 10.1128/mcb.10.12.6181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zhan Q., Lord K. A., Alamo I., Jr, Hollander M. C., Carrier F., Ron D., Kohn K. W., Hoffman B., Liebermann D. A., Fornace A. J., Jr The gadd and MyD genes define a novel set of mammalian genes encoding acidic proteins that synergistically suppress cell growth. Mol Cell Biol. 1994 Apr;14(4):2361–2371. doi: 10.1128/mcb.14.4.2361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zinszner H., Albalat R., Ron D. A novel effector domain from the RNA-binding protein TLS or EWS is required for oncogenic transformation by CHOP. Genes Dev. 1994 Nov 1;8(21):2513–2526. doi: 10.1101/gad.8.21.2513. [DOI] [PubMed] [Google Scholar]

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