Transcription factor mobility

蛋白质在细胞核中移动，并与那里的结合位点短暂地相互作用，但在大多数情况下，我们不知道它们为什么如此移动的，也不知道它们与什么结合。 我们的工作集中在使用荧光恢复后的光漂白和荧光相关光谱研究转录因子的流动性在特定的启动子位点，也在整个细胞核的其他通用网站。我们先前已经在小鼠细胞系中显示，GFP标记的糖皮质激素受体与特定启动子结合最多60秒，即使转录持续数小时。 为了获得一个精确的估计多久的糖皮质激素受体保持绑定的启动子，我们已经开发了数学模型来分析的扩散和结合的相互作用的受体发生在荧光恢复后的光漂白实验。我们的模型预测，个别糖皮质激素受体结合在启动子不到一秒钟。这种非常短暂的结合提出了新的问题，即转录因子如何在如此短的停留时间内组装转录复合物。在同一时间，我们已经表明，不同的分析程序的荧光恢复后，光漂白可以产生不同的估计停留时间。这表明活细胞结合参数的估计仍存在不确定性，需要开发替代测量程序以达成共识估计。为此，我们现在已经开发了一个数学模型，从荧光相关光谱数据中提取结合估计，并使用此来比较结合估计获得相同的分子的荧光恢复后，光漂白和荧光相关光谱。我们已经表明，这两种方法是一致的，但只有当在荧光相关光谱的标准方法中进行校正。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协同相互作用。为此，我们使用连接体组蛋白H1，并通过使用我们的分析程序来估计结合的H1分子的分数来分析其与染色质的结合。我们对野生型H1分子以及一系列缺乏各种关键结合结构域的突变体进行了研究。通过比较不同突变体中结合的分子的分数，我们可以确定哪些结构域在结合过程中协同相互作用。简而言之，协同相互作用的结构域是当两者都存在时，与当任一者单独存在时结合分数的总和相比，结合分数高得多的那些结构域。这一程序将是一个普遍有用的一个研究合作结合的分子在活细胞。最后，我们开发了单分子跟踪技术来监测活细胞核内的转录因子运动。我们使用这个程序来估计转录因子的结合停留时间，通过测量它们保持不动的时间。这反映了它们与染色质结合的时间。初步数据表明，这些时间是稍微快了2-5倍，比荧光相关光谱和荧光恢复后光漂白测量的停留时间。这种差异可能是由于复杂的扩散过程所揭示的单分子跟踪，没有纳入用于分析荧光相关光谱或荧光恢复后光漂白的模型。

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.