Hagedorn Claudia, Gogol-Döring Andreas, Schreiber Sabrina, Epplen Jörg T, Lipps Hans J
University of Witten/Herdecke, ZBAF, Institute of Cell Biology, Stockumer Strasse 10, 58453 Witten, Germany.
Technische Hochschule Mittelhessen (University of Applied Sciences), Department of Bioinformatics, Wiesenstrasse 14, 35390 Gießen, Germany.
Nucleic Acids Res. 2017 Jul 27;45(13):7841-7854. doi: 10.1093/nar/gkx522.
Autonomously replicating vectors represent a simple and versatile model system for genetic modifications, but their localization in the nucleus and effect on endogenous gene expression is largely unknown. Using circular chromosome conformation capture we mapped genomic contact sites of S/MAR-based replicons in HeLa cells. The influence of cis-active sequences on genomic localization was assessed using replicons containing either an insulator sequence or an intron. While the original and the insulator-containing replicons displayed distinct contact sites, the intron-containing replicon showed a rather broad genomic contact pattern. Our results indicate a preference for certain chromatin structures and a rather non-dynamic behaviour during mitosis. Independent of inserted cis-active elements established vector molecules reside preferentially within actively transcribed regions, especially within promoter sequences and transcription start sites. However, transcriptome analyses revealed that established S/MAR-based replicons do not alter gene expression profiles of host genome. Knowledge of preferred contact sites of exogenous DNA, e.g. viral or non-viral episomes, contribute to our understanding of episome behaviour in the nucleus and can be used for vector improvement and guiding of DNA sequences to specific subnuclear sites.
自主复制载体是一种用于基因改造的简单且通用的模型系统，但其在细胞核中的定位以及对内源基因表达的影响在很大程度上尚不清楚。我们利用环状染色体构象捕获技术绘制了HeLa细胞中基于基质附着区域（S/MAR）的复制子的基因组接触位点。使用含有绝缘子序列或内含子的复制子评估顺式作用序列对基因组定位的影响。虽然原始的和含绝缘子的复制子显示出不同的接触位点，但含内含子的复制子呈现出相当广泛的基因组接触模式。我们的结果表明，在有丝分裂期间，其偏好特定的染色质结构且行为相当不活跃。与插入的顺式作用元件无关，已建立的载体分子优先位于活跃转录区域内，特别是在启动子序列和转录起始位点内。然而，转录组分析表明，已建立的基于S/MAR的复制子不会改变宿主基因组的基因表达谱。了解外源DNA（如病毒或非病毒附加体）的偏好接触位点，有助于我们理解附加体在细胞核中的行为，并可用于载体改进以及将DNA序列引导至特定的核内亚位点。
