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

生物钟(~24小时)是存在于几乎所有哺乳动物细胞中的分子振荡器，驱动生理、新陈代谢和行为的日常变化。时钟错位或紊乱在现代社会中很常见，并与许多广泛传播的疾病有关，如代谢综合征、癌症、精神障碍和心血管疾病。因此，探索在哺乳动物细胞中建立一致的、大幅度和正确的相位的昼夜节律的机制对于理解与功能失调的昼夜节律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调节。以前的研究已经发现时钟蛋白复合体与转录机制相互作用，驱动转录的昼夜节律。主要由于技术的限制，这些研究集中在几个调控因子上，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。在提出的初步工作中，我们成功地采用并验证了一种新的基因组基因座蛋白质组学方法来研究与昼夜节律转录的关键染色质区域(即E-box)结合的蛋白质的时间依赖性变化。简单地说，催化失活的RNA引导核酸酶Cas9(DCAS9)与抗坏血酸过氧化物酶APEX2融合。单引导RNA靶向这种复合体，靶向基因组中的一个特定位置，在那里APEX2被激活，使其附近的蛋白质生物素化。生物素化的蛋白质被提纯，并通过质谱仪进行分析。利用这项新技术，我们的目标是(I)首次全面和公正地表征调控昼夜节律基因表达的昼夜节律蛋白；以及(Ii)识别对昼夜节律动态至关重要的新的转录调控因子。特别令人感兴趣的是那些将昼夜节律机制与其他重要的细胞机制(如细胞周期、细胞间通讯等)联系起来的调节器。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，从而为确定新的靶点，为开发更好的昼夜节律相关疾病的治疗策略奠定基础。

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.