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Journal of Virology, November 2005, p. 13497-13508, Vol. 79, No. 21
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.21.13497-13508.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Dynamic DNA Methylation and Histone Modifications Contribute to Lentiviral Transgene Silencing in Murine Embryonic Carcinoma Cells

Jin He, Qing Yang, and Lung-Ji Chang*

Department of Molecular Genetics and Microbiology, Powell Gene Therapy Center and McKnight Brain Institute, University of Florida, Gainesville, Florida 32610-0266

Received 31 March 2005/ Accepted 26 July 2005

Embryonic stem cells are subjected to a dynamic genome regulation during development. Here we report that the ectopic lentiviral transgenes are quickly silenced in murine embryonic carcinoma P19 cells. The silencing was correlated with CpG hypermethylation in the transgene promoter. Using high-resolution sodium bisulfite genome sequencing, we detected distinct DNA methylation kinetics in different proviral regions. DNase I sensitivity and chromatin immunoprecipitation assays revealed condensed chromatin structure and histone code switch during silencing. Longitudinal analysis of nonsilenced and silenced identical single-cell clones revealed that the silencing was coupled with CpG methylation in the promoter, as well as a global histone H3 deacetylation. Interestingly, the primer binding site and the packaging signal region appeared to serve as a DNA methylation initiation center which was rapidly hypermethylated regardless of transgene silencing and chromatin modifications. Analysis of cellular genes 45 to 50 kbp upstream and downstream of the integration site indicated that transcriptional activities of the flanking host genes were not affected. Genetic modifications of stem cells have great therapeutic potentials and our results picture a dynamic embryonic genome response to ectopic transgene integration that may have important implications in the future safety and efficacy modifications of stem cells.

* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, Powell Gene Therapy Center and McKnight Brain Institute, University of Florida, 1600 SW Archer Rd., ARB, R1-252, Box 100266, Gainesville, FL 32610-0266. Phone: (352) 392-3315. Fax: (352) 392-3133. E-mail: lchang{at}mgm.ufl.edu.

Journal of Virology, November 2005, p. 13497-13508, Vol. 79, No. 21
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.21.13497-13508.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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