Laboratory for Biomolecular Dynamics, University of Leuven, Leuven, Flanders, B-3000, Belgium.
Nucleic Acids Res. 2011 Mar;39(4):1310-25. doi: 10.1093/nar/gkq933. Epub 2010 Oct 25.
Nearly all cellular and disease related functions of the transcriptional co-activator lens epithelium-derived growth factor (LEDGF/p75) involve tethering of interaction partners to chromatin via its conserved integrase binding domain (IBD), but little is known about the mechanism of in vivo chromatin binding and tethering. In this work we studied LEDGF/p75 in real-time in living HeLa cells combining different quantitative fluorescence techniques: spot fluorescence recovery after photobleaching (sFRAP) and half-nucleus fluorescence recovery after photobleaching (hnFRAP), continuous photobleaching, fluorescence correlation spectroscopy (FCS) and an improved FCS method to study diffusion dependence of chromatin binding, tunable focus FCS. LEDGF/p75 moves about in nuclei of living cells in a chromatin hopping/scanning mode typical for transcription factors. The PWWP domain of LEDGF/p75 is necessary, but not sufficient for in vivo chromatin binding. After interaction with HIV-1 integrase via its IBD, a general protein-protein interaction motif, kinetics of LEDGF/p75 shift to 75-fold larger affinity for chromatin. The PWWP is crucial for locking the complex on chromatin. We propose a scan-and-lock model for LEDGF/p75, unifying paradoxical notions of transcriptional co-activation and lentiviral integration targeting.
转录共激活因子晶状体上皮衍生生长因子（LEDGF/p75）的几乎所有细胞和疾病相关功能都涉及通过其保守的整合酶结合域（IBD）将相互作用伙伴锚定到染色质上，但关于体内染色质结合和锚定的机制知之甚少。在这项工作中，我们结合不同的定量荧光技术，实时研究了活 HeLa 细胞中的 LEDGF/p75：点荧光恢复后光漂白（sFRAP）和半核荧光恢复后光漂白（hnFRAP）、连续光漂白、荧光相关光谱（FCS）和改进的 FCS 方法研究染色质结合的扩散依赖性，可调焦 FCS。LEDGF/p75 以转录因子典型的染色质跳跃/扫描模式在活细胞的核内运动。LEDGF/p75 的 PWWP 结构域对于体内染色质结合是必要的，但不是充分的。与 HIV-1 整合酶通过其 IBD（一种通用的蛋白质-蛋白质相互作用基序）相互作用后，LEDGF/p75 的动力学转变为对染色质的亲和力增加 75 倍。PWWP 对于将复合物锁定在染色质上至关重要。我们提出了一个用于 LEDGF/p75 的扫描和锁定模型，统一了转录共激活和慢病毒整合靶向的矛盾概念。
