Circadian chromatin landscape and identification of novel transcriptional regulators of the circadian clock

昼夜节律（~24小时）时钟是基本上存在于所有哺乳动物细胞中的分子振荡器，并驱动生理、代谢和行为的日常变化。失调或紊乱的生物钟在现代社会中很常见，并且与许多广泛的疾病有关，例如代谢综合征、癌症、精神疾病和心血管疾病。因此，探索在哺乳动物细胞中建立连贯的、大幅度的和正确定相的昼夜节律的机制对于理解与功能失调的生物钟相关的病理生理机制至关重要。分子振荡器的中心是基因转录的昼夜节律调节。以前的研究已经确定了与转录机制相互作用的时钟蛋白复合物，以驱动转录的昼夜节律。由于技术限制，这些研究主要集中在少数调控因子上，在我们对昼夜节律转录动力学的理解中留下了许多空白。随着定量基因组位点特异性蛋白质组学的最新进展，现在有机会克服这些限制。在所提出的初步工作中，我们成功地调整和验证了一种新的基因组位点蛋白质组学方法，以研究蛋白质结合昼夜节律转录的关键染色质区域（即E盒）的时间依赖性变化。简而言之，将无催化活性的RNA引导的核酸酶CAS9（dCAS 9）与抗坏血酸过氧化物酶APEX 2融合。单向导RNA靶向该复合物靶向基因组中的特定位点，其中APEX 2被激活以使其附近的蛋白质生物素化。生物素化的蛋白质通过质谱法纯化和分析。使用这项新技术，我们的目标是（i）提供第一个全面和公正的昼夜节律蛋白质调控昼夜节律基因表达的表征;（ii）确定新的转录调控昼夜节律动力学的关键。特别感兴趣的是那些将昼夜节律机制与其他重要细胞机制（例如细胞周期，细胞-细胞通信等）联系起来的调节剂。总体而言，该项目将提供有关昼夜节律振荡器功能机制的新信息，从而为确定新靶点奠定基础，以便为昼夜节律时钟相关疾病制定更好的治疗策略。

Circadian (~24 hr) clocks are molecular oscillators present in essentially all mammalian cells and drive daily changes in physiology, metabolism and behavior. Misaligned or disrupted clocks are common in modern society and are associated with many widespread diseases, such as metabolic syndromes, cancer, psychiatric disorders and cardiovascular pathologies. Thus, exploring the mechanisms that establish coherent, large amplitude and correctly phased circadian rhythms in mammalian cells is crucial for understanding the pathophysiological mechanisms associated with dysfunctional circadian clocks. At the center of the molecular oscillator is the circadian regulation of gene transcription. Previous studies have identified clock protein complexes interacting with the transcriptional machinery to drive circadian rhythms of transcription. Primarily due to technological limitations, these studies focused on a few regulators and left many gaps in our understanding of circadian transcription dynamics. With recent advances in quantitative genomic locus-specific proteomics, there is now an opportunity to overcome these limitations. In the preliminary work presented, we successfully adapted and validated a novel genomic locus proteomics method to study the time-of-day dependent change in protein binding to a key chromatin region of circadian transcription (i.e. the E-box). Briefly, catalytically inactive RNA-guided nuclease CAS9 (dCAS9) is fused to ascorbate peroxidase APEX2. Single guide RNAs target this complex targets a specific locus in the genome where APEX2 is activated to biotinylate proteins in its proximity. Biotinylated proteins are purified and analyzed via mass spectrometry. Using this new technology, our goal is (i) to provide the first comprehensive and unbiased characterization of circadian proteins regulating circadian gene expression; and (ii) to identify novel transcriptional regulators critical to circadian dynamics. Of particular interest are those regulators that link the circadian machinery to other important cellular mechanisms (e.g. cell cycle, cell-cell communication, etc.). Overall, this project will provide new information on the mechanism of circadian oscillator function, thus laying the groundwork for the identification of new targets for the development of better treatment strategies for circadian clock-related disorders.