Transcription Imaging Consortium, Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, USA.
Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, USA.
Cell. 2014 Mar 13;156(6):1274-1285. doi: 10.1016/j.cell.2014.01.062.
Enhancer-binding pluripotency regulators (Sox2 and Oct4) play a seminal role in embryonic stem (ES) cell-specific gene regulation. Here, we combine in vivo and in vitro single-molecule imaging, transcription factor (TF) mutagenesis, and ChIP-exo mapping to determine how TFs dynamically search for and assemble on their cognate DNA target sites. We find that enhanceosome assembly is hierarchically ordered with kinetically favored Sox2 engaging the target DNA first, followed by assisted binding of Oct4. Sox2/Oct4 follow a trial-and-error sampling mechanism involving 84-97 events of 3D diffusion (3.3-3.7 s) interspersed with brief nonspecific collisions (0.75-0.9 s) before acquiring and dwelling at specific target DNA (12.0-14.6 s). Sox2 employs a 3D diffusion-dominated search mode facilitated by 1D sliding along open DNA to efficiently locate targets. Our findings also reveal fundamental aspects of gene and developmental regulation by fine-tuning TF dynamics and influence of the epigenome on target search parameters.
增强子结合多能性调节因子（Sox2 和 Oct4）在胚胎干细胞（ES）细胞特异性基因调控中发挥着重要作用。在这里，我们将体内和体外单分子成像、转录因子（TF）突变和 ChIP-exo 作图相结合，以确定 TF 如何动态地搜索并组装到其同源 DNA 靶位上。我们发现，增强子组装是按层次顺序进行的，动力学上有利的 Sox2 首先与靶 DNA 结合，随后 Oct4 辅助结合。Sox2/Oct4 遵循一种试错采样机制，涉及 3D 扩散（3.3-3.7 s）的 84-97 次事件，其间穿插短暂的非特异性碰撞（0.75-0.9 s），然后在特定的靶 DNA 上获取和停留（12.0-14.6 s）。Sox2 通过沿开放 DNA 的 1D 滑动来促进 3D 扩散主导的搜索模式，从而有效地定位靶标。我们的研究结果还揭示了通过精细调整 TF 动力学和表观基因组对目标搜索参数的影响，基因和发育调控的基本方面。
