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2011 | 58 | 1 | 67-73
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Sp100 interacts with phage ΦC31 integrase to inhibit its recombination activity

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Phage ΦC31 integrase is a potential vector for the insertion of therapeutic genes into specific sites in the human genome. To understand the mechanism involved in ΦC31 integrase-mediated recombination, it is important to understand the interaction between the integrase and cellular proteins. Using a yeast two-hybrid system with pLexA-ΦC31 integrase as bait, we screened a pB42AD human fetal brain cDNA library for potential interacting cellular proteins. From the 106 independent clones that were screened, 11 potential interacting clones were isolated, of which one encoded C-terminal fragment of Sp100. The interaction between Sp100 and ΦC31 integrase was further confirmed by yeast mating and co-immunoprecipitation assays. The hybridization between a ΦC31 integrase peptide array and an HEK293 cell extract revealed that residues 81RILN84 in the N-terminus of ΦC31 integrase are responsible for the interaction with Sp100. Knocking down endogenous Sp100 with Sp100-specific siRNA increased ΦC31 integrase-mediated recombination but did not impact reporter gene expression. Therefore, endogenous Sp100 may interact with ΦC31 integrase and inhibit the efficiency of ΦC31 integrase-mediated recombination.
Physical description
  • Calos MP (2006) The phiC31 integrase system for gene therapy. Curr Gene Ther 6: 633-645.
  • Chalberg TW, Portlock JL, Olivares EC, Thyagarajan B, Kirby PJ, Hillman RT, Hoelters J, Calos MP (2006) Integration specificity of phage phiC31 integrase in the human genome. J Mol Biol 357: 28-48.
  • Chen JZ, Ji CN, Xu GL, Pang RY, Yao JH, Zhu HZ, Xue JL, Jia W (2006) DAXX interacts with phage ΦC31 integrase and inhibits recombination. Nucleic Acids Res 34: 6298-6304.
  • Ehrhardt A, Engler JA, Xu H, Cherry AM, Kay MA (2006) Molecular analysis of chromosomal rearrangements in mammalian cells after phiC31-mediated integration. Hum Gene Ther 17: 1077-1094.
  • Groth AC, Olivares EC, Thyagarajan B, Calos MP (2000) A phage integrase directs efficient site-specific integration in human cells. Proc Natl Acad Sci USA 97: 5995-6000.
  • Hacein-Bey-Abina S, von Kalle C, Schmidt M, Le Deist F, Wulffraat N, McIntyre E, Radford I, Villeval JL, Fraser CC, Cavazzana-Calvo M, Fischer A (2003) A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 348: 255-256.
  • Held PK, Olivares EC, Aguilar CP, Finegold M, Calos MP, Grompe M (2005) In vivo correction of murine hereditary tyrosinemia type I by ΦC31 integrase-mediated gene delivery. Mol Ther 11: 399-408.
  • Keravala A, Ormerod BK, Palmer TD, Calos MP (2008) Long-term transgene expression in mouse neural progenitor cells modified with phiC31 integrase. J Neurosci Methods 173: 299-305.
  • Kuhstoss S, Rao RN (1991) Analysis of the integration function of the streptomycete bacteriophage ΦC31. J Mol Biol 222: 897-908.
  • Liu Y, Thyagarajan B, Lakshmipathy U, Xue H, Lieu P, Fontes A, MacArthur CC, Scheyhing K, Rao MS, Chesnut JD (2009) Generation of platform human embryonic stem cell lines that allow efficient targeting at a predetermined genomic location. Stem Cells Dev 18: 1459-1472.
  • Maucksch C, Aneja MK, Hennen E, Bohla A, Hoffmann F, Elfinger M, Rosenecker J, Rudolph C (2008) Cell type differences in activity of the Streptomyces bacteriophage phiC31 integrase. Nucleic Acids Res 36: 5462-5471.
  • McEwan AR, Rowley PA, Smith MC (2009) DNA binding and synapsis by the large C-terminal domain of phiC31 integrase. Nucleic Acids Res 37: 4764-4773.
  • Milovic-Holm K, Krieghoff E, Jensen K, Will H, Hofmann TG (2007) FLASH links the CD95 signaling pathway to the cell nucleus and nuclear bodies. EMBO J 26: 391-401.
  • Möller A, Sirma H, Hofmann TG, Staege H, Gresko E, Lüdi KS, Klimczak E, Dröge W, Will H, Schmitz ML (2003) Sp100 is important for the stimulatory effect of homeodomain-interacting protein kinase-2 on p53-dependent gene expression. Oncogene 22: 8731-8737.
  • Naka K, Ikeda K, Motoyama N (2002) Recruitment of NBS1 into PML oncogenic domains via interaction with SP100 protein. Biochem Biophys Res Commun 299: 863-871.
  • Negorev D, Maul GG (2001) Cellular proteins localized at and interacting within ND10/PML nuclear bodies/PODs suggest functions of a nuclear depot. Oncogene 20: 7234-7242.
  • Olivares EC, Hollis RP, Chalberg TW, Meuse L, Kay MA, Calos MP (2002) Site-specific genomic integration produces therapeutic factor IX levels in mice. Nat Biotechnol 20: 1124-1128.
  • Pfafl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29: 2002-2007.
  • Quenneville SP, Chapdelaine P, Rousseau J, Tremblay JP (2007) Dystrophin expression in host muscle following transplantation of muscle precursor cells modified with the phiC31 integrase. Gene Ther 14: 514-22.
  • Rowley PA, Smith MC (2008) Role of the N-terminal domain of phiC31 integrase in attB-attP synapsis. J Bacteriol 190: 6918-6921.
  • Rowley PA, Smith MC, Younger E, Smith MC (2008) A motif in the C-terminal domain of phiC31 integrase controls the directionality of recombination. Nucleic Acids Res 36: 3879-3891.
  • Seeler JS, Marchio A, Losson R, Desterro JM, Hay RT, Chambon P, Dejean A (2001) Common properties of nuclear body protein SP100 and TIF1alpha chromatin factor: role of SUMO modification. Mol Cell Biol 21: 3314-3324.
  • Seeler JS, Marchio A, Sitterlin D, Transy C, Dejean A (1998) Interaction of SP100 with HP1 proteins: a link between the promyelocytic leukemia-associated nuclear bodies and the chromatin compartment. Proc Natl Acad Sci USA 95: 7316-7321.
  • Szostecki C, Guldner HH, Netter HJ, Will H (1990) Isolation and characterization of cDNA encoding a human nuclear antigen predominantly recognized by autoantibodies from patients with primary biliary cirrhosis. J Immunol 145: 4338-4347.
  • Tavalai N, Stamminger T (2008) New insights into the role of the subnuclear structure ND10 for viral infection. Biochim Biophys Acta 1783: 2207-2221.
  • Thorpe HM, Smith MC (1998) In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of resolvase/invertase family. Proc Natl Acad Sci USA 95: 5505-5510.
  • Wang BY, Xu GL, Zhou CH, Tian L, Xue JL, Chen JZ, Jia W (2010) PhiC31 integrase interacts with TTRAP and inhibits NFkappaB activation. Mol Biol Rep 37: 2809-2816
  • Xie K, Lambie EJ, Snyder M (1993) Nuclear dot antigens may specify transcriptional domains in the nucleus. Mol Cell Biol 13: 6170-6179.
  • Yordy JS, Li R, Sementchenko VI, Pei H, Muise-Helmericks RC, Watson DK (2004) SP100 expression modulates ETS1 transcriptional activity and inhibits cell invasion. Oncogene 23: 6654-6665.
  • Zhang MX, Li ZH, Fang YX, Zhu HZ, Xue JL, Chen JZ, Jia W (2009) TAT-phiC31 integrase mediates DNA recombination in mammalian cells. J Biotechnol 142: 107-113.
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