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。我们使用编码金属硫蛋白的酵母基因CUP1作为模型，该基因在重金属胁迫下表达。CUP1被铜结合的Tf Ace1p激活。我们之前的研究表明，在CUP1有两种类型的Ace1p自行车：快的--在秒的尺度上，和慢--在分钟的尺度上。快周期发生在慢周期内。结合的慢循环可以通过常规的荧光成像和单分子跟踪的快速循环(SMT)在单个细胞中定量。此前，我们已经为SMT构建了定制仪器，并优化了SMT数据解释。我们已经开发了在酵母细胞核中进行SMT的方法，以及在特定启动子上进行SMT的方法。通过SMT，我们测量了染色质重构体RSC和Tf Ace1p与CUP1启动子中特定位点(金属反应元件，MRE)的结合参数。CUP1的转录可以通过smFISH(荧光原位杂交)或通过荧光标记的mRNA的实时成像来观察。最近，我们通过smFISH将CUP1启动子的转录活性与Tf结合位点和特异性停留时间的变化联系起来。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。我们目前的目标是通过分析活细胞中的转录来证实这些发现。初步观察表明，转录的慢周期由单个基因上表达的快周期(爆发)组成。这种观察不能由smFISH在固定的细胞中进行，并提供了关于转录的新信息。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。我们正在用实时TS对慢转录周期和快转录周期进行建模，并用smFISH建模证实观察到的结果。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。通过茎环方法进行的实时转录定量证实了smFISH的数据。实时TS成像显示转录在两个不同的尺度上爆发-慢的和快的。值得注意的是，转录的慢周期与转铁蛋白结合的慢周期相关。目前，我们正在表征转录的快速尖峰以及Tf在这些短尖峰中的作用。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 a yeast gene CUP1 encoding metallothionein expressed in response to heavy metal stress. CUP1 is activated by copper-bound TF Ace1p. Our previous studies demonstrated two types of Ace1p cycling at CUP1: fast - on the scale of seconds, and slow - on the scale of minutes. The fast cycling occurs within the slow cycle. The slow cycle of binding may be quantified in individual cells by the regular fluorescence imaging, and the fast cycling - 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. By SMT, we have measured binding parameters of a chromatin remodeler RSC and TF Ace1p to specific sites (Metal Response Elements, MRE) within CUP1 promoter. Transcription of CUP1 may be observed either by smFISH (Fluorescence In Situ Hybridization) or by live imaging of the fluorescently tagged mRNA. Recently, we correlated the transcriptional activity of CUP1 promoter by smFISH with changes in the search for the binding sites and the specific residence time of TF. We demonstrated that the transient recruitment of TFs is regulated by fast cyclical chromatin remodeling events to ensure the best dynamic transcriptional response. Our current goal is to corroborate these findings with analysis of the transcription in live cells. Preliminary observations indicate that the slow cycle of transcription consists of the fast cycles (bursts) of expression on individual genes. This kind of observations cannot be made by smFISH in fixed cells and provides new information about transcription. 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. We are working on modeling of the slow and fast transcription cycles by live TS and corroborating the observations by smFISH modeling. Ultimately, these studies will lay the groundwork for the analysis of in vivo interactions of the components of the transcriptional machinery. The smFISH data are corroborated by the live transcription quantification in situ by a stem-loop approach. Live TS imaging revealed transcription bursting on two different scales - slow and fast. Notably, slow cycle of transcription correlates with slow cycle of TF binding. Currently, we are characterizing fast spikes of transcription and the role of TF in those short spikes. 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.