Dahal Liza, Graham Thomas G W, Dailey Gina M, Heckert Alec, Tjian Robert, Darzacq Xavier
Department of Molecular and Cell Biology, Berkeley, United States.
Howard Hughes Medical Institute, University of California, Berkeley, United States.
Elife. 2025 Jan 10;12:RP92979. doi: 10.7554/eLife.92979.
Type II nuclear receptors (T2NRs) require heterodimerization with a common partner, the retinoid X receptor (RXR), to bind cognate DNA recognition sites in chromatin. Based on previous biochemical and overexpression studies, binding of T2NRs to chromatin is proposed to be regulated by competition for a limiting pool of the core RXR subunit. However, this mechanism has not yet been tested for endogenous proteins in live cells. Using single-molecule tracking (SMT) and proximity-assisted photoactivation (PAPA), we monitored interactions between endogenously tagged RXR and retinoic acid receptor (RAR) in live cells. Unexpectedly, we find that higher expression of RAR, but not RXR, increases heterodimerization and chromatin binding in U2OS cells. This surprising finding indicates the limiting factor is not RXR but likely its cadre of obligate dimer binding partners. SMT and PAPA thus provide a direct way to probe which components are functionally limiting within a complex TF interaction network providing new insights into mechanisms of gene regulation in vivo with implications for drug development targeting nuclear receptors.
II型核受体（T2NRs）需要与共同伴侣维甲酸X受体（RXR）异源二聚化，才能结合染色质中的同源DNA识别位点。基于先前的生化和过表达研究，有人提出T2NRs与染色质的结合受核心RXR亚基有限池竞争的调节。然而，这种机制尚未在活细胞中针对内源性蛋白质进行测试。我们使用单分子追踪（SMT）和邻近辅助光激活（PAPA）技术，监测了活细胞中内源性标记的RXR和视黄酸受体（RAR）之间的相互作用。出乎意料的是，我们发现在U2OS细胞中，RAR而非RXR的高表达会增加异源二聚化和染色质结合。这一惊人发现表明，限制因素不是RXR，而是其专一性二聚体结合伴侣群体。因此，SMT和PAPA提供了一种直接方法，用于探究在复杂的转录因子相互作用网络中哪些成分在功能上具有限制性，从而为体内基因调控机制提供新见解，这对靶向核受体的药物开发具有启示意义。
