Syed Sahla, Duan Yifei, Lim Bomyi
Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA 19104.
Master of Biotechnology Program, University of Pennsylvania, Philadelphia, PA 19104.
bioRxiv. 2023 Jan 5:2023.01.05.522923. doi: 10.1101/2023.01.05.522923.
It is well known that enhancers regulate the spatiotemporal expression of their target genes by recruiting transcription factors (TFs) to the cognate binding sites in the region. However, the role of multiple binding sites for the same TFs and their specific spatial arrangement in determining the overall competency of the enhancer has yet to be fully understood. In this study, we utilized the MS2-MCP live imaging technique to quantitatively analyze the regulatory logic of the distal enhancer in early embryos. Through systematic modulation of Dorsal and Twist binding motifs in this enhancer, we found that a mutation in any one of these binding sites causes a drastic reduction in transcriptional amplitude, resulting in a reduction in total mRNA production of the target gene. We provide evidence of synergy, such that multiple binding sites with moderate affinities cooperatively recruit more TFs to drive stronger transcriptional activity than a single site. Moreover, a Hidden Markov-based stochastic model of transcription reveals that embryos with mutated binding sites have a higher probability of returning to the inactive promoter state. We propose that TF-DNA binding regulates spatial and temporal gene expression and drives robust pattern formation by modulating transcriptional kinetics and tuning bursting rates.
众所周知，增强子通过将转录因子（TFs）招募到该区域的同源结合位点来调节其靶基因的时空表达。然而，同一转录因子的多个结合位点及其特定空间排列在决定增强子整体活性方面的作用尚未完全了解。在本研究中，我们利用MS2-MCP活细胞成像技术定量分析早期胚胎中远端增强子的调控逻辑。通过系统地调节该增强子中背侧蛋白（Dorsal）和扭曲蛋白（Twist）的结合基序，我们发现这些结合位点中任何一个发生突变都会导致转录幅度急剧降低，从而导致靶基因的总mRNA产量减少。我们提供了协同作用的证据，即多个具有中等亲和力的结合位点比单个位点协同招募更多的转录因子以驱动更强的转录活性。此外，基于隐马尔可夫模型的转录随机模型表明，结合位点发生突变的胚胎恢复到无活性启动子状态的概率更高。我们提出，转录因子与DNA的结合通过调节转录动力学和调整爆发频率来调控基因表达的时空模式并驱动稳健的模式形成。
