Subnuclear organization and mislocalization of plasmids reduce transgene expression.

The expression of DNA within the nucleus is controlled by both promoter structure and sequence and higher-order organization of the nucleus, but how these two affect each other has not been greatly studied. We have previously shown that plasmids carrying Pol II expression cassettes localize to areas of Pol II transcription and processing while those carrying Pol I expression cassettes localize to the nucleolus. Using a microinjection approach in individual cells and imaging both DNA location and gene expression, we find that plasmids carrying Pol III expression cassettes localize in a distinct manner from the other two promoter types in a transcription-dependent manner, similar to all three promoter types. However, when both a Pol II expression cassette and a Pol III expression cassette are carried on the same plasmid, not only does the dual-promoter-class plasmid fail to distribute in a manner similar to plasmids carrying either promoter alone but also expressions of both cassettes are reduced compared to plasmids carrying single-expression cassettes. Taken together, these results confirm that both nuclear organization and promoter structure affect transcriptional outcome.