Ligand Binding Shifts Highly Mobile Retinoid X Receptor to the Chromatin-Bound State in a Coactivator-Dependent Manner, as Revealed by Single-Cell Imaging
Ligand Binding Shifts Highly Mobile Retinoid X Receptor to the Chromatin-Bound State in a Coactivator-Dependent Manner, as Revealed by Single-Cell Imaging
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DOI：
10.1128/mcb.01097-13
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发表时间：
2014-04-01
影响因子：
5.3
通讯作者：
Vamosi, Gyoergy
中科院分区：
文献类型：
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作者：
Brazda, Peter;Krieger, Jan;Vamosi, Gyoergy
维甲酸X受体(RXR)是一种杂交性核受体，与其他几种受体形成异二聚体，激活不同的基因。经激动剂处理后，其基因组结合区的占有率增加，但染色质免疫沉淀和测序显示其结合部位的数量仅有轻微变化，表明其行为相当静态。然而，这种全基因组和生化方法没有考虑转录因子的动态行为。因此，我们使用活细胞成像在亚秒尺度上表征了RXR在单细胞激活过程中的核动态。通过应用光漂白后的荧光恢复和荧光相关光谱(FCS)这两种不同时间和空间分辨率的技术，可以揭示出一种高度动态的行为，这一行为最好地被受体迁移率的两态模型(慢和快)所描述。在非连接状态下，大多数RXR属于快速群体，慢速染色质结合部分的RXR约为15%。在激动剂治疗后，由于立即和可逆的重新分配，这一比例增加到类似的43%。共激活子结合对于再分布似乎是不可或缺的，并对染色质的结合有重要贡献。用光片显微镜-FCS记录的核迁移率图表明，配体诱导的从快态到慢态的跃迁发生在整个细胞核中。我们的结果支持一个模型，在该模型中，RXR具有独特的、高度动态的核行为，并在激活时遵循打了就跑的动力学。
Retinoid X receptor (RXR) is a promiscuous nuclear receptor forming heterodimers with several other receptors, which activate different sets of genes. Upon agonist treatment, the occupancy of its genomic binding regions increased, but only a modest change in the number of sites was revealed by chromatin immunoprecipitation followed by sequencing, suggesting a rather static behavior. However, such genome-wide and biochemical approaches do not take into account the dynamic behavior of a transcription factor. Therefore, we characterized the nuclear dynamics of RXR during activation in single cells on the subsecond scale using live-cell imaging. By applying fluorescence recovery after photobleaching and fluorescence correlation spectroscopy (FCS), techniques with different temporal and spatial resolutions, a highly dynamic behavior could be uncovered which is best described by a two-state model (slow and fast) of receptor mobility. In the unliganded state, most RXRs belonged to the fast population, leaving similar to 15% for the slow, chromatin-bound fraction. Upon agonist treatment, this ratio increased to similar to 43% as a result of an immediate and reversible redistribution. Coactivator binding appears to be indispensable for redistribution and has a major contribution to chromatin association. A nuclear mobility map recorded by light sheet microscopy-FCS shows that the ligand-induced transition from the fast to the slow population occurs throughout the nucleus. Our results support a model in which RXR has a distinct, highly dynamic nuclear behavior and follows hit-and-run kinetics upon activation.