# Subnuclear organization and mislocalization of plasmids reduce transgene expression

## TL;DR

This study shows that plasmid localization in the nucleus affects gene expression, and combining different promoter types on one plasmid disrupts this localization and lowers expression.

## Contribution

The study reveals that plasmids with mixed promoter types mislocalize and reduce transgene expression, linking nuclear organization to transcriptional outcomes.

## Key findings

- Plasmids with Pol III promoters localize to distinct nuclear foci different from Pol II domains.
- Dual-promoter plasmids mislocalize and show reduced expression of both cassettes.
- Nuclear organization and promoter structure jointly influence transcriptional outcomes.

## Abstract

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.

Gu and colleagues demonstrate that plasmids expressing transcripts from various Pol III promoters localize to discrete foci in the nucleus that are distinct from Pol II transcription and processing domains. However, when both Pol II and Pol III promoter-transcript cassettes are on the same plasmid, this localization is disrupted, accompanied by a profound reduction in expression of both cassettes.