Impacts of Nucleosome Positioning Elements and Pre-Assembled Chromatin States on Expression and Retention of Transgenes

TL;DR

This paper explores how modifying transgenes with nucleosome positioning elements and pre-assembled chromatin can improve their expression and stability in cells.

Contribution

The study introduces a novel approach combining nucleosome positioning and pre-assembled chromatin to enhance transgene expression.

Findings

Nucleosome positioning elements can enhance transgene expression in a distance-dependent manner.

Pre-assembling plasmids into chromatin prolongs transgene expression compared to non-chromatin plasmids.

Nucleosome positioning effects generally outweigh those of hyperacetylated chromatin states on gene expression.

Abstract

Background/Objectives: Transgene applications, ranging from gene therapy to the development of stable cell lines and organisms, rely on maintaining the expression of transgenes. To date, the use of plasmid-based transgenes has been limited by the loss of their expression shortly after their delivery into the target cells. The short-lived expression of plasmid-based transgenes has been largely attributed to host-cell-mediated degradation and/or silencing of transgenes. The development of chromatin-based strategies for gene delivery has the potential to facilitate defining the requirements for establishing epigenetic states and to enhance transgene expression for numerous applications. Methods: To assess the impact of “priming” plasmid-based transgenes to adopt accessible chromatin states to promote gene expression, nucleosome positioning elements were introduced at promoters of transgenes, and vectors were pre-assembled into nucleosomes containing unmodified histones or mutants mimicking constitutively acetylated states at residues 9 and 14 of histone H3 or residue 16 of histone H4 prior to their introduction into cells, then the transgene expression was monitored over time. Results: DNA sequences capable of positioning nucleosomes could positively impact the expression of adjacent transgenes in a distance-dependent manner in the absence of their pre-assembly into chromatin. Intriguingly, the pre-assembly of plasmids into chromatin facilitated the prolonged expression of transgenes relative to plasmids that were not pre-packaged into chromatin. Interactions between pre-assembled chromatin states and nucleosome positioning-derived effects on expression were also assessed and, generally, nucleosome positioning played the predominant role in influencing gene expression relative to priming with hyperacetylated chromatin states. Conclusions: Strategies incorporating nucleosome positioning elements and the pre-assembly of plasmids into chromatin prior to nuclear delivery can modulate the expression of plasmid-based transgenes.

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