Department of Cell Biology, Yale University School of Medicine, New Haven, CT, 06510, USA.
Sci Rep. 2017 May 18;7(1):2065. doi: 10.1038/s41598-017-02240-y.
Promoter activation drives gene transcriptional output. Here we report generating site-specifically integrated single-copy promoter transgenes and measuring their expression to indicate promoter activities at single-mRNA level. mRNA counts, Pol II density and Pol II firing rates of the Ccnb1 promoter transgene resembled those of the native Ccnb1 gene both among asynchronous cells and during the cell cycle. We observed distinct activation states of the Ccnb1 promoter among G1 and G2/M cells, suggesting cell cycle-independent origin of cell-to-cell variation in Ccnb1 promoter activation. Expressing a dominant-negative mutant of NF-YA, a key transcriptional activator of the Ccnb1 promoter, increased its "OFF"/"ON" time ratios but did not alter Pol II firing rates during the "ON" period. Furthermore, comparing H3K4me2 and H3K79me2 levels at the Ccnb1 promoter transgene and the native Ccnb1 gene indicated that the enrichment of these two active histone marks did not predispose higher transcriptional activities. In summary, this experimental system enables bridging transcription imaging with molecular analysis to provide novel insights into eukaryotic transcriptional regulation.
启动子激活驱动基因转录输出。在这里，我们报告了生成特异性整合的单拷贝启动子转基因，并测量了它们的表达，以指示单个 mRNA 水平的启动子活性。Ccnb1 启动子转基因的 mRNA 计数、Pol II 密度和 Pol II 引发率在细胞周期中以及在异步细胞中与天然 Ccnb1 基因相似。我们观察到 G1 和 G2/M 细胞中 Ccnb1 启动子的激活状态不同，这表明 Ccnb1 启动子激活的细胞间变异具有细胞周期独立性。表达 Ccnb1 启动子的关键转录激活因子 NF-YA 的显性负突变体增加了其“OFF”/“ON”时间比，但在“ON”期间并未改变 Pol II 引发率。此外，比较 Ccnb1 启动子转基因和天然 Ccnb1 基因的 H3K4me2 和 H3K79me2 水平表明，这两种活性组蛋白标记的富集并不会导致更高的转录活性。总之，该实验系统使转录成像与分子分析相结合，为真核转录调控提供了新的见解。
