Transcription factor mobility

许多重要基因的表达是周期性的，并可能在不同的时间尺度上包括多个周期，如小时尺度上的昼夜循环或超传统周期，以及分钟尺度上的快速和短时间转录爆发。这种循环的机制尚不清楚，但对于药物的正确应用非常重要。在转录水平上，基因表达受DNA启动子和增强子内调控元件的可及性控制。在酵母和哺乳动物细胞中的研究表明，染色质可及性的调节是通过转录因子(TF)和染色质重构体的相互作用来实现的。某些转铁蛋白与其目标DNA序列的结合是高度动态的，在几秒钟的尺度上。此外，一些转铁蛋白在几分钟内经历“缓慢”循环，由交替的“开”和“关”基因状态组成。目前尚不清楚这种转铁蛋白循环与转录循环之间的关系。我们感兴趣的是转录因子在启动子上循环的分子机制及其与转录循环的关系。我们使用了参与重金属胁迫的酵母基因CUP1和参与热休克胁迫的Hsp104作为模型。CUP1被铜结合的Tf Ace1p激活。HSP104被HSF1激活。我们正在利用单分子跟踪技术(SMT)研究这些因子在特定启动子上的动态变化。此前，我们已经为SMT构建了定制仪器，并优化了SMT数据解释。我们已经建立了酵母细胞核SMT的方法和在特定启动子上进行SMT的方法。我们构建了带有CUP1或Hsp104启动子的报告基因。转录可以通过smFISH(荧光原位杂交)或通过荧光标记的mRNA的实时成像来可视化。我们将转录因子在启动子上的动态与转录输出联系起来。我们证明了转录因子的瞬时招募受到快速周期性染色质重塑事件的调节，以确保最佳的动态转录反应。在我们最近的出版物中，我们证明了转录的慢周期由单个基因上表达的快周期(爆发)组成。这是第一个基因模型，其中叠加的慢周期和快周期的转录与叠加的慢周期和快周期的转录因子。最终，这些研究将为分析转录机制组件的体内相互作用奠定基础。我们正在开发的将转录因子生物物理学与转录建模相关联的技术可能会应用于许多其他细胞生物学问题，其中需要转录分子调控的信息。

Expression of many important genes is cyclical, and may incorporate multiple cycles at the different time scale, such as circadian or ultradian cycles on the scale of hours, and fast and short bursts of transcription on the scale of minutes. The mechanisms of this cycling are poorly understood, but very important for the correct application of the drugs. At the transcription level, gene expression is controlled by the accessibility of the regulatory elements within DNA - promoters and enhancers. Research in yeast and mammalian cells indicates that modulation of chromatin accessibility occurs through interaction of transcription factors (TF) and chromatin remodelers. Binding of certain TF to their target DNA sequences is highly dynamic, on the scale of seconds. Also, some TF undergo the "slow" cycling on the scale of minutes, which consist of alternating "ON" and "OFF" gene states. It is unclear how this TF cycling is related to the transcriptional cycling. We are interested in molecular mechanisms of the TF cycling on promoters and its correlation with transcription cycles. We use as a model yeast gene CUP1 involved in heavy metal stress, and HSP104, involved in heat shock stress. CUP1 is activated by copper-bound TF Ace1p. HSP104 is activated by HSF1. We are studying dynamics of these TF factors on specific promoters by Single Molecule Tracking (SMT). Previously, we have built a custom instrument for SMT and optimized SMT data interpretation. We have developed methods for SMT in yeast cell nuclei and methods of performing SMT on specific promoters.We constructed reporter genes with promoters of either CUP1 or HSP104. Transcription may be visualized by smFISH (Fluorescence In Situ Hybridization) or by live imaging of the fluorescently tagged mRNA. We correlate the dynamics of the TF at the promoter with transcriptional output. We demonstrated that the transient recruitment of TFs is regulated by fast cyclical chromatin remodeling events to ensure the best dynamic transcriptional response. In our recent publication we demonstrate that the slow cycle of transcription consists of the fast cycles (bursts) of expression on individual genes. This is the first genetic model where the superimposed slow and fast cycles of transcription correlate with superimposed slow and fast cycling of the transcriptional factor. Ultimately, these studies will lay the groundwork for the analysis of in vivo interactions of the components of the transcriptional machinery. The technique of correlation of the TF biophysics with transcription modeling that we are in process of developing may be applied to a number of other problems of cellular biology where the information for molecular regulation of transcription is desired.