Transcription factor mobility

蛋白质在细胞核内移动，并与那里的结合部位短暂地相互作用，但在大多数情况下，我们不知道它们为什么如此可移动或它们与什么结合。我们的工作重点是利用光漂白后的荧光恢复和荧光相关光谱来研究转录因子在特定启动子位置和整个细胞核中其他普通位置的迁移率。我们之前已经在小鼠细胞系中表明，GFP标记的糖皮质激素受体与特定启动子结合的时间最长为60秒，即使转录持续几个小时。为了准确估计糖皮质激素受体与启动子结合的时间，我们建立了数学模型来分析光漂白实验后荧光恢复过程中受体的扩散和结合作用。我们的模型预测，单个糖皮质激素受体与启动子结合的时间不到一秒。这种非常短暂的结合提出了新的问题，即如何在转录因子的停留时间如此短的情况下组装转录复合体。同时，我们已经证明，对光漂白后的荧光恢复的不同分析方法可以产生不同的停留时间估计。这表明活细胞结合参数的估计仍然存在不确定性，这将需要开发替代的测量程序来得出一致的估计。为此，我们现在已经开发了一个数学模型来从荧光相关光谱数据中提取结合估计，并使用这个模型来比较通过光漂白后的荧光恢复和通过荧光相关光谱获得的同一分子的结合估计。我们已经证明，这两种方法是一致的，但前提是对荧光相关光谱的标准方法进行修正。校正必须考虑到测量过程中发生的漂白。因此，我们的交叉验证程序有助于识别其中一个程序中的错误，同时也有助于增加我们对当前活细胞结合估计的信心。我们还扩展了这些活细胞结合程序，以检查活细胞内单个分子的合作相互作用。为此，我们使用了连接子组蛋白H1，并通过使用我们的分析程序来估计结合的H1分子的比例，分析了它与染色质的结合。我们对野生型H1分子以及一系列缺乏各种关键结合域的突变体进行了这一研究。通过比较不同突变体中结合的分子比例，我们可以确定哪些结构域在结合过程中协同作用。简单地说，协同相互作用域是指当两个都存在时，相对于当其中一个单独存在时，结合分数之和要高得多的区域。这一过程对于研究活细胞中分子的协同结合将是一种普遍有用的方法。最后，我们开发了单分子跟踪技术来监控转录因子在活细胞核内的运动。我们正在使用这个程序通过测量转录因子保持不动的时间来估计它们的结合停留时间。这反映了它们与染色质结合的时间。初步数据表明，这些时间比用荧光相关光谱和光漂白后的荧光恢复测得的停留时间快2-5倍。这种差异可能是由于单分子跟踪揭示的复杂扩散过程，这些过程没有被纳入用于分析荧光相关光谱或光漂白后的荧光恢复的模型中。

Proteins move in the nucleus and transiently interact with binding sites there, but in most cases we do not know why they are so mobile or what they are bound to. Our work has focused on using fluorescence recovery after photobleaching and fluorescence correlation spectroscopy to investigate the mobility of transcription factors both at specific promoter sites and also at other generic sites throughout the nucleus. We have previously shown in a mouse cell line that the GFP-tagged glucocorticoid receptor is bound at a specific promoter for at most 60 seconds, even though transcription persists for several hours. To obtain a precise estimate of how long the glucocorticoid receptor remains bound to the promoter, we have developed mathematical models to analyze the diffusion and binding interactions of the receptor that occur during the fluorescent recovery after photobleaching experiment. Our model predicts that individual glucocorticoid receptors are bound at the promoter for less than a second. This very transient binding raises new questions about how the transcription complex can be assembled with such short residence times of the transcription factor. At the same time, we have shown that different analysis procedures for fluorescence recovery after photobleaching can yield different estimates of residence times. This shows that there are still uncertainties in the estimation of live cell binding parameters that will require developing alternate measurement procedures to arrive at consensus estimates. Towards this end, we have now developed a mathematical model to extract binding estimates from fluorescence correlation spectroscopy data, and used this to compare binding estimates obtained for the same molecule by fluorescence recovery after photobleaching and by flourescence correlation spectroscopy. We have shown that the two approaches agree, but only if a correction is made in the standard approach for fluorescence correlation spectroscopy. The correction must account for the bleaching which occurs during the measurement process. Thus, our cross validation procedure has helped to identify an error in one of the procedures, and at the same time has helped increase our confidence in our current live cell binding estimates. We have also extended these live cell binding procedures to examine cooperative interactions of a single molecule inside of a live cell. For this purpose, we used the linker histone H1 and analyzed its binding to chromatin by using our analysis procedures to estimate the fraction of bound H1 molecules. We did this for the wild type H1 molecule as well as for a series of mutants that lack various key binding domains. By comparing the fraction of molecules bound in the different mutants, we could determine which domains interact cooperatively in the binding process. In simple terms, cooperatively interacting domains are those for which a much higher fraction is bound when both are present compared to the sum of the bound fractions when either is present by itself. This procedure will be a generally useful one for investigating cooperative binding of molecules in live cells. Finally, we have developed single molecule tracking techniques to monitor transcription factor movement inside of live cell nuclei. We are using this procedure to estimate the binding residence times of transcription factors by measuring how long they remain immobile. This reflects the time that they are bound to chromatin. Preliminary data indicate that these times are somewhat faster by a factor of 2-5 than the residence times measured by fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. The difference may be due to complex diffusion processes revealed by single molecule tracking which are not incorporated into the models used to analyze fluorescence correlation spectroscopy or fluorescence recovery after photobleaching.