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Abstract
Nuclear spatial positioning plays an important role in the epigenetic regulation of eukaryotic gene expression. Here we show a role for nuclear spatial positioning in regulating episomal transgenes that are delivered by virus-like particles (VLPs). VLPs mediate the delivery of plasmid DNA (pDNA) to cell nuclei but lack viral factors involved in initiating and regulating transcription. By tracking single fluorescently labeled VLPs, coupled with luciferase reporter gene assays, we found that VLPs transported pDNA to cell nuclei efficiently but transgenes were immediately silenced by the cell. An investigation of the nuclear location of fluorescent VLPs revealed that the pDNAs were positioned next to centromeric heterochromatin. The activation of transcription by providing viral factors or inhibiting histone deacetylase activity resulted in the localization to euchromatin regions. Further, the activation of transcription induced the recruitment of PML nuclear bodies (PML-NBs) to the VLPs. This association did not play a role in regulating transgene expression, but PML protein was necessary for the inhibition of transgene expression with alpha interferon (IFN-α). These results support a model whereby cells can prevent foreign gene expression at two levels: by positioning transgenes next to centromeric heterochromatin or, if that is overcome, via the type I IFN response facilitated by PML-NB recruitment.
We thank A. Pombo, M. Merkenschlager, K. Linton, P. Hobson, and K. Brown for helpful discussions; W. Earnshaw and A. Pombo for gifts of autoimmune sera against centromeric proteins or Sm; P. Pandolfi for pml − / − and pml +/+ mouse fibroblasts; K. Leppard, P. Freemont, and A. Zelent for E4ORF3, PML-RARα, and PLZF-RARα expressing pDNAs; and A. Porter for the Wellferon. We also thank M. Elliott and N. Bennett for performing the AFM and F. Ramirez for performing chi-squared analysis.
We are grateful to the Medical Research Council United Kingdom and the Hammersmith Hospitals Trust Research Committee (grant no. 20223) for financial support.