In eukaryotic nuclei, chromatin loops mediated through cohesin are critical structures that regulate gene expression and DNA replication. Here, we demonstrate a new method to see endogenous genomic loci using synthetic zinc-finger proteins harboring repeat epitope tags (ZF probes) for signal amplification via binding of tag-specific intracellular antibodies, or frankenbodies, fused with fluorescent proteins. We achieve this in two steps: First, we develop an anti-FLAG frankenbody that can bind FLAG-tagged proteins in diverse live-cell environments. The anti-FLAG frankenbody complements the anti-HA frankenbody, enabling two-color signal amplification from FLAG- and HA-tagged proteins. Second, we develop a pair of cell-permeable ZF probes that specifically bind two endogenous chromatin loci predicted to be involved in chromatin looping. By coupling our anti-FLAG and anti-HA frankenbodies with FLAG- and HA-tagged ZF probes, we simultaneously see the dynamics of the two loci in single living cells. This shows a close association between the two loci in the majority of cells, but the loci markedly separate from the triggered degradation of the cohesin subunit RAD21. Our ability to image two endogenous genomic loci simultaneously in single living cells provides a proof of principle that ZF probes coupled with frankenbodies are useful new tools for exploring genome dynamics in multiple colors.

中文翻译：

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使用合成锌指蛋白与活细胞中的抗 FLAG 和抗 HA frankenbody 可视化两个内源基因组基因座的循环

在真核细胞核中，通过黏连蛋白介导的染色质环是调节基因表达和 DNA 复制的关键结构。在这里，我们展示了一种查看内源基因组位点的新方法，该方法使用含有重复表位标签（ZF 探针）的合成锌指蛋白通过与荧光蛋白融合的标签特异性细胞内抗体或 frankenbody 的结合进行信号放大。我们通过两个步骤实现这一目标：首先，我们开发了一种抗 FLAG frankenbody，它可以在不同的活细胞环境中结合 FLAG 标记的蛋白质。anti-FLAG frankenbody 与 anti-HA frankenbody 互补，能够从 FLAG 和 HA 标记的蛋白质中放大双色信号。其次，我们开发了一对细胞可渗透的 ZF 探针，它们特异性结合两个预计参与染色质环的内源性染色质基因座。通过将我们的 anti-FLAG 和 anti-HA frankenbody 与带有 FLAG 和 HA 标记的 ZF 探针相结合，我们同时看到了单个活细胞中两个基因座的动态。这表明大多数细胞中两个基因座之间存在密切关联，但该基因座与引发的黏连蛋白亚基 RAD21 的降解明显分开。我们在单个活细胞中同时成像两个内源基因组位点的能力提供了原理证明，即 ZF 探针与 frankenbody 结合是探索多种颜色的基因组动力学的有用新工具。但该基因座与黏连蛋白亚基 RAD21 的触发降解明显分开。我们在单个活细胞中同时成像两个内源基因组位点的能力提供了原理证明，即 ZF 探针与 frankenbody 结合是探索多种颜色的基因组动力学的有用新工具。但该基因座与黏连蛋白亚基 RAD21 的触发降解明显分开。我们在单个活细胞中同时成像两个内源基因组位点的能力提供了原理证明，即 ZF 探针与 frankenbody 结合是探索多种颜色的基因组动力学的有用新工具。