Circadian clocks (∼24 h) are responsible for daily physiological, metabolic, and behavioral changes. Central to these oscillations is the regulation of gene transcription. Previous research has identified clock protein complexes that interact with the transcriptional machinery to orchestrate circadian transcription, but technological constraints have limited the identification of de novo proteins. Here we use a novel genomic locus-specific quantitative proteomics approach to provide a new perspective on time of day-dependent protein binding at a critical chromatin locus involved in circadian transcription: the E-box. Using proximity labeling proteomics at the E-box of the clock-controlled Dbp gene in mouse fibroblasts, we identified 69 proteins at this locus at the time of BMAL1 binding. This method successfully enriched BMAL1 as well as HDAC3 and HISTONE H2A.V/Z, known circadian regulators. New E-box proteins include the MINK1 kinase and the transporters XPO7 and APPL1, whose depletion in human U-2 OS cells results in disrupted circadian rhythms, suggesting a role in the circadian transcriptional machinery. Overall, our approach uncovers novel circadian modulators and provides a new strategy to obtain a complete temporal picture of circadian transcriptional regulation.

中文翻译：

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蛋白质组学对昼夜节律转录调控的见解：通过邻近标记揭示的新型 E-box 交互子


生物钟 （∼24 h） 负责日常生理、代谢和行为变化。这些振荡的核心是基因转录的调节。以前的研究已经确定了与转录机制相互作用以协调昼夜节律转录的时钟蛋白复合物，但技术限制限制了从头蛋白的鉴定。在这里，我们使用一种新的基因组位点特异性定量蛋白质组学方法，为参与昼夜节律转录的关键染色质位点 E-box 的日依赖性蛋白质结合时间提供了新的视角。在小鼠成纤维细胞中时钟控制的 Dbp 基因的 E-box 处使用邻近标记蛋白质组学，我们在 BMAL1 结合时在该位点鉴定了 69 个蛋白质。该方法成功富集了 BMAL1 以及 HDAC3 和 HISTONE H2A。V/Z，已知的昼夜节律调节因子。新的 E-box 蛋白包括 MINK1 激酶和转运蛋白 XPO7 和 APPL1，它们在人 U-2 OS 细胞中的耗竭导致昼夜节律紊乱，表明在昼夜节律中发挥作用。总体而言，我们的方法发现了新的昼夜节律调节剂，并提供了一种新策略来获得昼夜节律转录调控的完整时间图景。