Key Technology Engineering Center for New Veterinary Vaccine and Industry of Yunnan Provincial Education Department, Kunming University, Kunming, Yunnan, China.
Pharmaceutical Department, Second Affiliated Hospital of Xinxiang Medical University, Xinxiang, Henan, China.
Epigenetics. 2024 Dec;19(1):2337085. doi: 10.1080/15592294.2024.2337085. Epub 2024 Apr 9.
The PhiC31 integration system allows for targeted and efficient transgene integration and expression by recognizing pseudo attP sites in mammalian cells and integrating the exogenous genes into the open chromatin regions of active chromatin. In order to investigate the regulatory patterns of efficient gene expression in the open chromatin region of PhiC31 integration, this study utilized Ubiquitous Chromatin Opening Element (UCOE) and activating RNA (saRNA) to modulate the chromatin structure in the promoter region of the PhiC31 integration vector. The study analysed the effects of DNA methylation and nucleosome occupancy changes in the integrated promoter on gene expression levels. The results showed that for the OCT4 promoter with moderate CG density, DNA methylation had a smaller impact on expression compared to changes in nucleosome positioning near the transcription start site, which was crucial for enhancing downstream gene expression. On the other hand, for the SOX2 promoter with high CG density, increased methylation in the CpG island upstream of the transcription start site played a key role in affecting high expression, but the positioning and clustering of nucleosomes also had an important influence. In conclusion, analysing the DNA methylation patterns, nucleosome positioning, and quantity distribution of different promoters can determine whether the PhiC31 integration site possesses the potential to further enhance expression or overcome transgene silencing effects by utilizing chromatin regulatory elements.
PhiC31 整合系统通过识别哺乳动物细胞中的假 attP 位点，并将外源基因整合到活性染色质的开放染色质区域，实现了靶向和高效的转基因整合和表达。为了研究 PhiC31 整合开放染色质区域中高效基因表达的调控模式，本研究利用普遍染色质开放元件（UCOE）和激活 RNA（saRNA）来调节 PhiC31 整合载体启动子区域的染色质结构。该研究分析了整合启动子中 DNA 甲基化和核小体占有率变化对基因表达水平的影响。结果表明，对于 CG 密度适中的 OCT4 启动子，DNA 甲基化对表达的影响小于转录起始位点附近核小体定位的变化，这对于增强下游基因表达至关重要。另一方面，对于 CG 密度较高的 SOX2 启动子，转录起始位点上游 CpG 岛中增加的甲基化在影响高表达方面起着关键作用，但核小体的定位和聚集也有重要影响。总之，分析不同启动子的 DNA 甲基化模式、核小体定位和数量分布，可以确定 PhiC31 整合位点是否具有通过利用染色质调节元件进一步增强表达或克服转基因沉默效应的潜力。
