Ross Nikki L, Sullivan Millicent O
Department of Chemical and Biomolecular Engineering, University of Delaware , 150 Academy Street, Newark, Delaware 19716, United States.
Mol Pharm. 2015 Dec 7;12(12):4488-97. doi: 10.1021/acs.molpharmaceut.5b00645. Epub 2015 Oct 23.
For successful gene delivery, plasmid DNA must be able to access the nucleus in order to be transcribed. Numerous studies have shown that gene delivery occurs more readily in dividing cells, which is attributed to increased nuclear access when the nuclear envelope disassembles during mitosis; however, nonviral carriers continue to have low transfection efficiencies and require large quantities of DNA per cell to achieve reasonable gene transfer, even in dividing cells. Therefore, we hypothesized that using histone-derived nuclear localization sequences (NLS)s to target polyplexes might enhance nuclear delivery by facilitating interactions with histone effectors that mediate nuclear partitioning and retention during mitosis. We discovered a novel interaction between polyplexes linked to histone 3 (H3) N-terminal tail peptides and the histone nuclear import protein importin-4, as evidenced by strong spatial colocalization as well as significantly decreased transfection when importin-4 expression was reduced. A fraction of the histone-targeted polyplexes was also found to colocalize with the retrotranslocon of the endoplasmic reticulum, Sec61. Super resolution microscopy demonstrated a high level of polyplex binding to chromatin postmitosis, and there also was a significant decrease in the amount of chromatin binding following importin-4 knockdown. These results provide evidence that natural histone effectors mediate both nuclear entry and deposition on chromatin by histone-targeted polyplexes, and a translocation event from the endoplasmic reticulum into the cytosol may occur before mitosis to enable the polyplexes to interact with these essential cytoplasmic proteins.
为了成功实现基因传递，质粒DNA必须能够进入细胞核才能进行转录。大量研究表明，基因传递在分裂细胞中更容易发生，这归因于在有丝分裂期间核膜解体时核进入增加；然而，即使在分裂细胞中，非病毒载体的转染效率仍然很低，并且每个细胞需要大量的DNA才能实现合理的基因转移。因此，我们假设使用组蛋白衍生的核定位序列（NLS）来靶向多聚体可能通过促进与在有丝分裂期间介导核分配和保留的组蛋白效应物的相互作用来增强核传递。我们发现与组蛋白3（H3）N端尾肽连接的多聚体与组蛋白核输入蛋白输入蛋白-4之间存在一种新相互作用，这通过强烈的空间共定位以及当输入蛋白-4表达降低时转染显著减少得到证明。还发现一部分组蛋白靶向的多聚体与内质网的反转录转运体Sec61共定位。超分辨率显微镜显示有丝分裂后多聚体与染色质的结合水平很高，并且在输入蛋白-4敲低后染色质结合量也显著减少。这些结果证明天然组蛋白效应物介导组蛋白靶向的多聚体进入细胞核并沉积在染色质上，并且在有丝分裂之前可能发生从内质网到细胞质的转运事件，以使多聚体能够与这些必需的细胞质蛋白相互作用。
