Transcriptional regulation is affected by subnuclear targeting of reporter plasmids to PML nuclear bodies.
Whereas the PML protein has been reported to have both transcriptional coactivator and corepressor potential, the contribution of the PML nuclear body (PML NB) itself to transcriptional regulation is not well understood. Here we demonstrate that plasmid DNA artificially tethered to PML or the PML NB-targeting domain of Sp100 is preferentially localized to PML NBs. Using the tethering technique, we targeted a simian virus 40 promoter-driven luciferase reporter plasmid to PML NBs, resulting in the repression of the transgene transcriptional activity. Conversely, the tethering of a cytomegalovirus promoter-containing reporter plasmid resulted in activation. Targeting a minimal eukaryotic promoter did not affect its activity. The expression of targeted promoters could be modulated by altering the cellular concentration of PML NB components, including Sp100 and isoforms of the PML protein. Finally, we demonstrate that ICP0, the promiscuous herpes simplex virus transactivator, increases the level of transcriptional activation of plasmid DNA tethered to the PML NB. We conclude that when PML NB components are artificially tethered to reporter plasmids, the PML NB contributes to the regulation of the tethered DNA in a promoter-dependent manner. Our findings demonstrate that transient transcription assays are sensitive to the subnuclear localization of the transgene plasmid.
Block
Gregory J
GJ
The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada.
Eskiw
Christopher H
CH
Dellaire
Graham
G
Bazett-Jones
David P
DP
D022003:Cell Nucleus Structures / Q000502:physiology*; D005456:Fluorescent Dyes; D005786:Gene Expression Regulation / Q000502:physiology*; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D007211:Indoles; D008156:Luciferases / Q000378:metabolism; D009363:Neoplasm Proteins / Q000502:physiology*; D009687:Nuclear Proteins / Q000502:physiology*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D000071156:Promyelocytic Leukemia Protein; D020033:Protein Isoforms / Q000235:genetics / Q000502:physiology; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection; D019076:Transgenes; D025521:Tumor Suppressor Proteins / Q000502:physiology*
D005456:Fluorescent Dyes; D007211:Indoles; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D000071156:Promyelocytic Leukemia Protein; D020033:Protein Isoforms; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C070576:PML protein, human; C007293:DAPI; D008156:Luciferases