Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA.
Davidson School of Chemical Engineering, Purdue University, West Lafayette, IN 47907, USA.
Genes (Basel). 2024 Sep 21;15(9):1232. doi: 10.3390/genes15091232.
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.
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.
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.
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.
背景/目的：转基因的应用范围从基因治疗到稳定细胞系和生物体的开发，都依赖于维持转基因的表达。迄今为止，基于质粒的转基因的应用受到其在进入靶细胞后表达迅速丧失的限制。基于质粒的转基因的短暂表达主要归因于宿主细胞介导的转基因降解和/或沉默。开发基于染色质的基因传递策略有可能有助于确定建立表观遗传状态的要求，并增强许多应用的转基因表达。
为了评估“启动”基于质粒的转基因以采用可及染色质状态来促进基因表达的影响，在转基因启动子处引入核小体定位元件，并在将载体引入细胞之前，预先将其组装成含有未经修饰组蛋白的核小体或模拟组蛋白 H3 残基 9 和 14 或组蛋白 H4 残基 16 处组成型乙酰化状态的突变体，然后监测转基因的表达随时间的变化。
能够定位核小体的 DNA 序列可以在没有预先组装成染色质的情况下，以依赖距离的方式对相邻转基因的表达产生积极影响。有趣的是，与未预先包装成染色质的质粒相比，将质粒预先组装成染色质有助于转基因的延长表达。还评估了预先组装的染色质状态与核小体定位对表达的相互作用，一般来说，核小体定位在影响基因表达方面相对于用超乙酰化染色质状态启动发挥主导作用。
在核内传递之前结合核小体定位元件和将质粒预先组装成染色质的策略可以调节基于质粒的转基因的表达。
