Gene Center and Department of Biochemistry, Center for Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377, München, Germany.
Faculty of Biology, Ludwig-Maximilians-Universität München, Großhaderner Str. 2, 82152, Planegg-Martinsried, Germany.
Commun Biol. 2020 Nov 12;3(1):663. doi: 10.1038/s42003-020-01375-5.
The gene regulatory network governing anterior-posterior axis formation in Drosophila is a well-established paradigm to study transcription in developmental biology. The rapid temporal dynamics of gene expression during early stages of development, however, are difficult to track with standard techniques. We optimized the bright and fast-maturing fluorescent protein mNeonGreen as a real-time, quantitative reporter of enhancer expression. We derive enhancer activity from the reporter fluorescence dynamics with high spatial and temporal resolution, using a robust reconstruction algorithm. By comparing our results with data obtained with the established MS2-MCP system, we demonstrate the higher detection sensitivity of our reporter. We used the reporter to quantify the activity of variants of a simple synthetic enhancer, and observe increased activity upon reduction of enhancer-promoter distance or addition of binding sites for the pioneer transcription factor Zelda. Our reporter system constitutes a powerful tool to study spatio-temporal gene expression dynamics in live embryos.
调控果蝇体轴形成的基因调控网络是研究发育生物学中转录的一个成熟范例。然而，在胚胎发育的早期阶段，基因表达的快速时间动态很难用标准技术来跟踪。我们优化了明亮且快速成熟的荧光蛋白 mNeonGreen，将其作为增强子表达的实时定量报告器。我们使用强大的重建算法，从报告荧光动力学中以高空间和时间分辨率得出增强子活性。通过将我们的结果与使用既定的 MS2-MCP 系统获得的数据进行比较，我们证明了我们的报告器具有更高的检测灵敏度。我们使用该报告器来量化简单合成增强子变体的活性，并观察到增强子-启动子距离减小或添加先驱转录因子 Zelda 的结合位点时活性增加。我们的报告器系统构成了研究活体胚胎中时空基因表达动态的有力工具。
