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Development of an in Vitro Assay for the Proteolytic Processing of the CDP/Cux Transcription Factor

  • Hebert, Sherry (Molecular Oncology Group, McGill University Health Center) ;
  • Berube, Ginette (Molecular Oncology Group, McGill University Health Center) ;
  • Nepveu, Alain (Molecular Oncology Group, McGill University Health Center)
  • Received : 2003.01.07
  • Accepted : 2003.02.27
  • Published : 2003.07.31
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Abstract

The CDP/Cux transcription factor was previously shown to be proteolytically processed at the G1/S transition. In view of characterizing and eventually identifying the protease responsible for CDP/Cux processing, we have established an in vitro proteolytic processing assay. CDP/Cux recombinant proteins expressed in mammalian or bacterial cells were efficiently processed in vitro using as a source of protease either whole cell extracts, the nuclear or the cytoplasmic fraction. Processing was found to take place optimally at a lower pH, to be insensitive to variations in salt concentration, and to be inhibited by the protease inhibitors MG132 and E64D. Interestingly, the bacterially-produced substrate was more efficiently processed than the substrate purified from mammalian cells. Moreover, processing in vitro was more efficient when CDP/Cux substrates were purified from populations of cells enriched in the S phase than in the G1 phase of the cell cycle. Altogether, these results suggest that post-translational modifications of CDP/Cux in mammalian cells inhibits processing and contributes to the cell cycle-dependent regulation of processing. The in vitro processing assay described in this study will provide a useful tool for the purification and identification of the protease responsible for the processing of CDP/Cux.

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References

  1. Andres, V., Chiara, M. D. and Mahdavi, V. (1994) A new bipartite DNA-binding domain: cooperative interaction between the cut repeat and homeo domain of the cut homeo proteins. Genes Dev. 8, 245-257. https://doi.org/10.1101/gad.8.2.245
  2. Aufiero, B., Neufeld, E. J.,and Orkin, S. H. (1994a) Sequencespecific DNA binding of individual Cut repeats of the human CCAAT displacement/Cut homeodomain protein. Proc. Natl. Acad. Sci. USA 91, 7757-7761. https://doi.org/10.1073/pnas.91.16.7757
  3. Aufiero, B., Neufeld, E. J. and Orkin, S. H. (1994b) Sequencespecific DNA binding of individual Cut repeats of the human CCAAT displacement/Cut homeodomain protein. Proc. Natl. Acad. Sci. USA 91, 7757-7761. https://doi.org/10.1073/pnas.91.16.7757
  4. Blochlinger, K., Bodmer, R., Jack, J., Jan, L. Y. and Jan, Y. N.(1988) Primary structure and expression of a product from cut, a locus involved in specifying sensory organ identity in Drosophila. Nature 333, 629-635. https://doi.org/10.1038/333629a0
  5. Benov, L. and Al-Ibraheem, J. (2002) Disrupting Escherichia coli: a comparison of methods. J. Biochem. Mol. Biol. 35, 428-431. https://doi.org/10.5483/BMBRep.2002.35.4.428
  6. Blochlinger, K., Bodmer, R., Jan, L. Y. and Jan, Y. N. (1990) Patterns of expression of cut, a protein required for external sensory organ development in wild-type and cut mutant Drosophila embryos. Genes Dev. 4, 1322-1331. https://doi.org/10.1101/gad.4.8.1322
  7. Blochlinger, K., Jan, L. Y. and Jan, Y. N. (1991) Transformation of sensory organ identity by ectopic expression of Cut in Drosophila. Genes Dev. 5, 1124-1135. https://doi.org/10.1101/gad.5.7.1124
  8. Bodmer, R., Barbel, S., Shepherd, S., Jack, J. W., Jan, L. Y. andJan, Y. N. (1987) Transformation of sensory organs by mutations of the cut locus of D. melanogaster. Cell 51, 293-307. https://doi.org/10.1016/0092-8674(87)90156-5
  9. Cai, H. N. and Levine, M. (1997) The gypsy insulator can function as a promoter-specific silencer in the Drosophila embryo. EMBO J. 16, 1732-1741. https://doi.org/10.1093/emboj/16.7.1732
  10. Catt, D., Luo, W. and Skalnik, D. G. (1999) DNA-binding properties of CCAAT displacement protein cut repeats [In Process Citation]. Cell. Mol. Biol. (Noisy-Le-Grand) 45, 1149-1160.
  11. Coqueret, O., Berube, G. and Nepveu, A. (1996) DNA Binding By Cut Homeodomain Proteins Is Down-Modulated By Protein Kinase C. J. Biol. Chem. 271, 24862-24868. https://doi.org/10.1074/jbc.271.40.24862
  12. Coqueret, O., Berube, G. and Nepveu, A. (1998a) The mammalian Cut homeodomain protein functions as a cell-cycle dependent transcriptional repressor which downmodulates p21WAF1/CIP1/ SDI1 in S phase. EMBO J. 17, 4680-4694. https://doi.org/10.1093/emboj/17.16.4680
  13. Coqueret, O., Martin, N., Berube, G., Rabbat, M., Litchfield, D.W. and Nepveu, A. (1998b) DNA binding by cut homeodomain proteins is down-modulated by casein kinase II. J. Biol. Chem. 273, 2561-2566. https://doi.org/10.1074/jbc.273.5.2561
  14. Dorsett, D. (1993) Distance-independent inactivation of an enhancer by the suppressor of Hairy-wing DNA-binding protein of Drosophila. Genetics 134, 1135-1144.
  15. Ellis, T., Gambardella, L., Horcher, M., Tschanz, S., Capol, J.,Bertram, P., Jochum, W., Barrandon, Y. and Busslinger, M.(2001) The transcriptional repressor CDP (Cutl1) is essential for epithelial cell differentiation of the lung and the hair follicle. Genes Dev. 15, 2307-2319. https://doi.org/10.1101/gad.200101
  16. Harada, R., Berube, G., Tamplin, O. J., Denis-Larose, C. andNepveu, A. (1995) DNA-binding specificity of the cut repeats from the human cut-like protein. Mol. Cell. Biol. 15, 129-140.
  17. Harada, R., Dufort, D., Denis-Larose, C. and Nepveu, A. (1994) Conserved cut repeats in the human cut homeodomain protein function as DNA binding domains. J. Biol. Chem. 269, 2062-2067.
  18. Hoffmann, I., Draetta, G. and Karsenti, E. (1994) Activation of the phosphatase activity of human cdc25A by a cdk2-cyclin E dependent phosphorylation at the G1/S transition. EMBO J. 13, 4302-4310.
  19. Holthuis, J., Owen, T. A., van Wijnen, A. J., Wright, K. L.,Ramsey-Ewing, A., Kennedy, M. B., Carter, R., Cosenza, S. C.,Soprano, K. J., Lian, J. B., Stein, J. L. and Stein, G. S. (1990) Tumor cells exhibit deregulation of the cell cycle histone gene promoter factor HiNF-D. Science 247, 1454-1457. https://doi.org/10.1126/science.2321007
  20. Jack, J. and DeLotto, Y. (1995) Structure and regulation of a complex locus: the cut gene of Drosophila. Genetics 139, 1689-1700.
  21. Jack, J., Dorsett, D., Delotto, Y. and Liu, S. (1991) Expression of the cut locus in the Drosophila wing margin is required for cell type specification and is regulated by a distant enhancer. Development 113, 735-747.
  22. Jinno, S., Suto, K., Nagata, A., Igarashi, M., Kanaoka, Y., Nojima,H. and Okayama, H. (1994) Cdc25A is a novel phosphatase functioning early in the cell cycle. EMBO J. 13, 1549-1556.
  23. Kim, S. K., Park, P. J., Kim, J. B. and Shahidi, F. (2002) Purification and characterization of a collagenolytic protease from the filefish, Novoden modestrus. J. Biochem. Mol. Biol. 35, 165-171. https://doi.org/10.5483/BMBRep.2002.35.2.165
  24. Ledford, A. W., Brantley, J. G., Kemeny, G., Foreman, T. L.,Quaggin, S. E., Igarashi, P., Oberhaus, S. M., Rodova, M.,Calvet, J. P. and Vanden Heuvel, G. B. (2002) Deregulated Expression of the Homeobox Gene Cux-1 in Transgenic Mice Results in Downregulation of p27(kip1) Expression during Nephrogenesis, Glomerular Abnormalities, and Multiorgan Hyperplasia. Dev. Biol. 245, 157-171. https://doi.org/10.1006/dbio.2002.0636
  25. Lee, S. J. and Park, M. T. (2003) Cell cycle and cancer. J. Biochem. Mol. Biol. 36, 60-65. https://doi.org/10.5483/BMBRep.2003.36.1.060
  26. Li, S., Aufiero, B., Schiltz, R. L. and Walsh, M. J. (2000)Regulation of the homeodomain CCAAT displacement/cut protein function by histone acetyltransferases p300/CREBbinding protein (CBP)- associated factor and CBP. Proc. Natl. Acad. Sci. USA 97, 7166-7171. https://doi.org/10.1073/pnas.130028697
  27. Liu, S. and Jack, J. (1992) Regulatory interactions and role in cell type specification of the Malpighian tubules by the cut, kruppel, and caudal genes of Drosophila. Dev. Biol. 150, 133-143. https://doi.org/10.1016/0012-1606(92)90013-7
  28. Liu, S., McLeod, E. and Jack, J. (1991) Four distinct regulatory regions of the cut locus and their effect on cell type specification in Drosophila. Genetics 127, 151-159.
  29. Luong, M. X., van der Meijden, C. M., Xing, D., Hesselton, R.,Monuki, E. S., Jones, S. N., Lian, J. B., Stein, J. L., Stein, G.S., Neufeld, E. J. and van Wijnen, A. J. (2002) Genetic ablation of the CDP/Cux protein C terminus results in hair cycle defects and reduced male fertility. Mol. Cell. Biol. 22, 1424-1437. https://doi.org/10.1128/MCB.22.5.1424-1437.2002
  30. Modolell, J., Bender, W. and Meselson, M. (1983) Drosophila melanogaster mutations suppressible by the suppressor of Hairy-wing are insertions of a 7.3-kilobase mobile element. Proc. Natl. Acad. Sci. USA 80, 1678-1682. https://doi.org/10.1073/pnas.80.6.1678
  31. Moon, N. S., Berube, G. and Nepveu, A. (2000) CCAAT displacement activity involves Cut repeats 1 and 2, not the Cut homeodomain. J. Biol. Chem. 275, 31325-31334. https://doi.org/10.1074/jbc.M002912200
  32. Moon, N. S., Premdas, P., Truscott, M., Leduy, L., Berube, G. andNepveu, A. (2001) S phase-specific proteolytic cleavage is required to activate stable DNA binding by the CDP/Cut homeodomain protein. Mol. Cell. Biol. 21, 6332-6345. https://doi.org/10.1128/MCB.21.18.6332-6345.2001
  33. Moon, N. S., Rong Zeng, W., Premdas, P., Santaguida, M.,Berube, G. and Nepveu, A. (2002) Expression of N-terminally truncated isoforms of CDP/CUX is increased in human uterine leiomyomas. Int. J. Cancer 100, 429-432. https://doi.org/10.1002/ijc.10510
  34. Nepveu, A. (2001) Role of the multifunctional CDP/Cut/Cux homeodomain transcription factor in regulating differentiation, cell growth and development. Gene 270, 1-15. https://doi.org/10.1016/S0378-1119(01)00485-1
  35. Neufeld, E. J., Skalnik, D. G., Lievens, P. M. and Orkin, S. H.(1992) Human CCAAT displacement protein is homologous to the Drosophila homeoprotein, cut. Nat. Genet. 1, 50-55. https://doi.org/10.1038/ng0492-50
  36. Quaggin, S. E., Vandenheuvel, G. B., Golden, K., Bodmer, R. andIgarashi, P. (1996) Primary structure, neural-specific expression, and chromosomal localization of Cux-2, a second murine homeobox gene related to Drosophila Cut. J. Biol. Chem. 271, 22624-22634. https://doi.org/10.1074/jbc.271.37.22624
  37. Santaguida, M., Ding, Q., Berube, G., Truscott, M., Whyte, P. andNepveu, A. (2001) Phosphorylation of the CCAAT displacement protein (CDP)/Cux transcription factor by cyclin A-Cdk1 modulates Its DNA binding activity in G2. J. Biol. Chem. 276, 45780-45790. https://doi.org/10.1074/jbc.M107978200
  38. Sinclair, A. M., Lee, J. A., Goldstein, A., Xing, D., Liu, S., Ju, R.,Tucker, P. W., Neufeld, E. J. and Scheuermann, R. H. (2001) Lymphoid apoptosis and myeloid hyperplasia in CCAAT displacement protein mutant mice. Blood 98, 3658-3667. https://doi.org/10.1182/blood.V98.13.3658
  39. Tufarelli, C., Fujiwara, Y., Zappulla, D. C. and Neufeld, E. J. (1998) Hair defects and pup loss in mice with targeted deletion of the first cut repeat domain of the Cux/CDP homeoprotein gene. Dev. Biol. 200, 69-81. https://doi.org/10.1006/dbio.1998.8950
  40. Valarche, I., Tissier-Seta, J. P., Hirsch, M. R., Martinez, S.,Goridis, C. and Brunet, J. F. (1993) The mouse homeodomain protein Phox2 regulates Ncam promoter activity in concert with Cux/CDP and is a putative determinant of neurotransmitter phenotype. Development 119, 881-896.
  41. van Wijnen, A. J., Choi, T. K., Owen, T. A., Wright, K. L., Lian,J. B., Jaenisch, R., Stein, J. L. and Stein, G. S. (1991) Involvement of the cell cycle-regulated nuclear factor HiNF-D in cell growth control of a human H4 histone gene during hepatic development in transgenic mice. Proc. Natl. Acad. Sci. USA 88, 2573-2577. https://doi.org/10.1073/pnas.88.6.2573
  42. van Wijnen, A. J., van Gurp, M. F., de Ridder, M. C., Tufarelli,C., Last, T. J., Birnbaum, M., Vaughan, P. S., Giordano, A.,Krek, W., Neufeld, E. J., Stein, J. L. and Stein, G. S. (1996) CDP/cut is the DNA-binding subunit of histone gene transcription factor HiNF-D: a mechanism for gene regulation at the G1/S phase cell cycle transition point independent of transcription factor E2F. Proc. Natl. Acad. Sci. USA 93, 11516-11521. https://doi.org/10.1073/pnas.93.21.11516
  43. van Wijnen, A. J., Wright, K. L., Lian, J. B., Stein, J. L. andStein, G. S. (1989) Human H4 histone gene transcription requires the proliferation-specific nuclear factor HiNF-D. Auxiliary roles for HiNF-C (Sp1-like) and HiNF-A (high mobility group-like). J. Biol. Chem. 264, 15034-15042.
  44. Wright, K. L., Dell’Orco, R. T., van Wijnen, A. J., Stein, J. L. andStein, G. S. (1992) Multiple mechanisms regulate the proliferation-specific histone gene transcription factor HiNF-D in normal human diploid fibroblasts. Biochemistry 31, 2812-2818. https://doi.org/10.1021/bi00125a023

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