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Ku80 Participates in the Targeting of Retroviral Transgenes to the Chromatin of CHO Cells

Christel Masson,¹ Stéphanie Bury-Moné,^1, Elvire Guiot,¹ Asier Saez-Cirion,² Damien Schoëvaërt-Brossault,^3,4 Corinne Brachet-Ducos,¹ Olivier Delelis,¹ Frédéric Subra,¹ Laurence Jeanson-Leh,^1, and Jean-François Mouscadet^1*

LBPA, CNRS, E.N.S. Cachan, 61 Avenue du Président Wilson, 94235 Cachan, France,¹ Régulation des Infections Rétrovirales, Institut Pasteur, 75015 Paris, France,² Laboratoire d'Analyse d'Images en Pathologie Cellulaire, Institut Universitaire d'Hématologie, Hôpital St. Louis, 1 Avenue Vellefaux, 75010 Paris, France,³ Laboratoire d'Andrologie, CHU Bicêtre, 94270 Kremlin, Bicêtre, France⁴

Received 15 September 2006/ Accepted 8 May 2007

The heterodimer Ku70/80 Ku is the DNA-binding component of the DNA-PK complex required for the nonhomologous end-joining pathway. It participates in numerous nuclear processes, including telomere and chromatin structure maintenance, replication, and transcription. Ku interacts with retroviral preintegration complexes and is thought to interfere with the retroviral replication cycle, in particular the formation of 2-long terminal repeat (LTR) viral DNA circles, viral DNA integration, and transcription. We describe here the effect of Ku80 on both provirus integration and the resulting transgene expression in cells transduced with retroviral vectors. We found that transgene expression was systematically higher in Ku80-deficient xrs6 cells than in Ku80-expressing CHO cells. This higher expression was observed irrespective of the presence of the viral LTR and was also not related to the nature of the promoter. Real-time PCR monitoring of the early viral replicative steps demonstrated that the absence of Ku80 does not affect the efficiency of transduction. We analyzed the transgene distributions localization in nucleus by applying a three-dimensional reconstruction model to two-dimensional fluorescence in situ hybridization images. This indicated that the presence of Ku80 resulted in a bias toward the transgenes being located at the periphery of the nucleus associated with their being repressed; in the absence of this factor the transgenes tend to be randomly distributed and actively expressed. Therefore, although not strictly required for retroviral integration, Ku may be involved in targeting retroviral elements to chromatin domains prone to gene silencing.

Published ahead of print on 16 May 2007.

Present address: IGM, CNRS-UMR8621, Université Paris-Sud, Orsay Cedex 91405, France.

Present address: Genéthon, 1 Bis Rue de l'Internationale, BP 60, 91002 Evry, France.