Impacts of nucleosome positioning elements and pre-assembled chromatin states on expression and retention of transgenes

Abstract

Transgene applications ranging from gene therapy to development of stable cell lines and organisms rely on maintaining expression of transgenes. To-date, the use of plasmid-based transgenes has been limited by loss of their expression shortly after delivery into target cells. This short-lived expression of plasmid-based transgenes has been largely attributed to host cell-mediated degradation and/or silencing of transgenes. 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, or vectors were pre-assembled into nucleosomes containing unmodified histones or histone 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 transgene expression was monitored over time. DNA sequences capable of positioning nucleosomes could positively impact expression of adjacent transgenes in a distance-dependent manner in the absence of pre-assembly into chromatin. Intriguingly, pre-assembly of plasmids into chromatin facilitated 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 of reporter genes were also assessed and, generally, nucleosome positioning played the predominant role in influencing gene expression relative to priming with hyperacetylated chromatin states.