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。我们的工作集中在使用FRAP来研究转录因子在特定启动子位点和整个细胞核的其他通用位点的流动性。在这项工作中，我们使用了含有GFP标记的糖皮质激素受体(GFP-GR)的小鼠细胞系，我们能够在与MMTV启动子靶点串联阵列结合的活细胞中观察到GFP-GR。GFP-GR的FRAP显示，即使转录持续数小时，GFP-GR的快速交换也在不到一分钟的时间内完全恢复荧光。这一令人惊讶的结果引发了人们对快速交换的机制和目的的质疑。我们最近的工作已经开始解决这些问题，表明伴侣和蛋白酶体参与调节交换，并且交换率似乎与转录结合在一起。在酵母的相关研究中，我们发现转录因子在细胞核中的移动反映了非特异性DNA结合，这些动态相互作用被染色质重构体加速。为了更好地了解FRAP恢复数据并从这些数据中提取有关结合的定量信息，我们正在开发数学模型，以解释存在扩散和结合相互作用时的FRAP恢复。

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 FRAP to investigate the mobility of transcription factors both at specific promoter sites and also at other generic sites throughout the nucleus. In much of this work, we have used a mouse cell line containing a GFP-tagged glucocorticoid receptor (GFP-GR), which we were able to visualize in live cells binding to a tandem array of MMTV promoter target sites. FRAP of GFP-GR at this site revealed rapid exchange of GFP-GR with full fluorescence recovery in less than a minute, even though transcription persists for several hours. This surprising result raised questions about both the mechanism and purpose of rapid exchange. Our recent work has begun to address these questions by showing that chaperones and proteasomes are involved in regulating exchange, and that exchange rate appears coupled with transcription. In related studies in yeast, we have found that transcription factor mobility throughout the nucleus reflects non-specific DNA binding and these dynamic interactions are accelerated by a chromatin remodeler. To better understand FRAP recovery data and extract quantitative information about binding from these data, we are developing mathematical models to account for FRAP recoveries in the presence of diffusion and binding interactions.