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

昼夜节律染色质景观和生物钟新型转录调节因子的鉴定

• 批准号: 532624261
• 负责人: Professor Dr. Achim Kramer
• 金额: --
• 依托单位: Fachbereich Chronobiologie (CVK)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/532624261?language=en
• 关键词: Circadian; chromatin; landscape; identification; novel

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.

昼夜节律（约24小时）是存在于所有哺乳动物细胞中的分子振荡器，并驱动生理学，代谢和行为的每日变化。现代社会中的未对准或中断的时钟很常见，并且与许多广泛的疾病有关，例如代谢综合征，癌症，精神病和心血管病理。因此，探索建立哺乳动物细胞中建立连贯，较大幅度和正确分阶段的昼夜节律的机制对于理解与功能失调的昼夜节律时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。先前的研究已经确定了与转录机械相互作用的时钟蛋白复合物，以驱动转录的昼夜节律。这些研究主要是由于技术局限性，重点是一些调节剂，并在我们对昼夜节律动态的理解中留下了许多差距。随着定量基因组特异性蛋白质组学的最新进展，现在有机会克服这些局限性。在提出的初步工作中，我们成功地改编并验证了一种新型的基因组蛋白质组学方法，以研究蛋白质结合与昼夜节律转录的关键染色质区域（即E-box）的依赖性变化。简而言之，将催化无效的RNA引导的核酸酶Cas9（DCAS9）融合到抗坏血酸酯过氧化物酶Apex2。单引导RNA靶向该复合物的靶向基因组中的一个特定基因座，在该基因组中，APEX2在其接近度中被激活至生物素蛋白。纯化生物素化蛋白并通过质谱法分析。使用这项新技术，我们的目标是（i）提供调节昼夜节律基因表达的昼夜节律蛋白的首个全面和公正的特征； （ii）确定对昼夜动力至关重要的新型转录调节剂。特别感兴趣的是那些将昼夜节律与其他重要的细胞机制联系起来的调节器（例如细胞周期，细胞 - 细胞通信等）。总体而言，该项目将提供有关昼夜节振荡器功能机理的新信息，从而为确定新目标奠定了基础，以制定与昼夜节律相关疾病的更好治疗策略。