Nature | 发文多维组学手段揭示小鼠肝脏昼夜节律调控机制

所谓昼夜节律，是指生物体为了适应外界环境变化而进化出的一套适应性生理系统。它广泛影响着生物体的生理状态与行为活动【1】。前期针对哺乳动物多个组织器官的转录组研究也指出，有超过半数的基因转录受到昼夜节律的调控【2】。昼夜节律影响着几乎所有组织器官的生理功能变化，节律紊乱则与众多疾病的发生密切相关。分子机制上，目前普遍认为昼夜节律的调控核心是基于转录因子控制的以转录-翻译为基础的多层次、多维度的复杂反馈调节网络【3】。作为网络核心的转录因子一直以来受到昼夜节律研究者的广泛关注。2017年的诺贝尔生理医学奖的颁给了首次克隆到节律调控中关键转录因子的三位科学家，这也再次印证了转录因子在节律研究中的重要性。

昼夜节律如此普遍的存在看似易于研究，实则不然。由于技术的局限性，尤其在蛋白水平上对于低丰度转录因子的鉴定及DNA结合活性的定量上存在困难，对于昼夜节律中节律性转录因子的研究多集中在基因组和转录组水平上。但由于转录后调控、翻译后修饰等复杂过程的存在，基因转录水平与最终蛋白表达水平存在一定差异；仅从基因组水平和转录组水平无法直接、全面地揭示转录因子节律性。不仅如此，鉴于昼夜节律是一个多维度的调控网络，如何能全方位、多维度的阐明昼夜节律对组织器官生理功能的调控，也是阻碍昼夜节律研究的瓶颈之一。

4月19日，复旦大学丁琛课题组、国家蛋白质科学中心（北京）秦钧课题组与军事科学院贺福初课题组合作在Nature Communications杂志上发表了题为 The proteomics landscape of Circadian Rhythm in Mouse liver 的研究论文，该研究揭示了转录因子在小鼠肝脏昼夜节律中的核心调控作用，构建了以转录因子为核心的多维度昼夜节律调控网络，填补了昼夜节律研究在蛋白质组层面的空白，为了解肝脏昼夜节律功能转换机制，节律相关疾病的防诊治的研究提供依据。

研究人员利用前期开发的catTFRE技术【4】，结合多种组学研究方法，以小鼠肝脏为模型在48小时的周期中共获得了了包括：转录因子DNA结合活性谱；磷酸化蛋白谱；泛素化蛋白谱；肝脏的全蛋白谱；肝脏的转录组与核蛋白谱在内的六组不同维度的组学数据。通过对多维组学数据的整合，该研究建立了以转录因子为核心的昼夜节律调控网络，并发现在该网络的调控之下小鼠肝脏会在昼夜间完成免疫调控、糖代谢、脂代谢以及细胞周期调控在内的四大功能转换。

该项研究同时鉴定了一系列的肝脏主效节律转录因子（dominant regulatory transcription factors，DR-TFs），并证明DR-TFs通过调控基因的节律性转录和节律性蛋白表达，最终达到驱动肝脏生理功能的昼夜转化的作用。

同时，值得一提的是，研究人员发现小鼠肝脏昼夜间的免疫反应存在差异。针对这一现象，研究人员利用小鼠肝脏巨噬细胞（Kupffer Cells）在细胞分辨率水平对昼夜节律进行了蛋白质组研究，发现相对于肝脏，Kupffer Cells中的免疫反应相关蛋白表现出更加显著的昼夜节律性。

该研究不仅在蛋白组水平上首次对昼夜节律中的调控核心-转录因子进行了解析，并结合多维组学的方法系统性的描绘了节律调控网络对肝脏生理功能昼夜变化的影响。同时，肝脏和Kupffer Cells在昼夜间的蛋白质组学研究，体现出蛋白在细胞水平和组织水平中昼夜节律的差异，为深入解析器官昼夜生理功能差异的分子机制奠定了良好的基础，也为后续肝脏昼夜生理功能和节律相关疾病的研究提供理论指导和新的研究靶点。

复旦大学丁琛、国家蛋白质科学中心（北京）的秦钧和军事科学院贺福初院士为本文的共同通讯作者；复旦大学博士生王云之，清华大学博士生宋雷为本文的共同第一作者。

专家点评

Jake Chen（德州大学休斯敦健康科学中心）

Comments：Constructing a panoramic view of biological timing

As the adage goes, timing is everything. Doing the right thing but at the wrong time would wreak havoc in your social life. Likewise, in our body, all essential physiological and metabolic processes are temporally ordered to achieve optimal performance or segregated to avoid futile cycles and deleterious consequences. Such biological timing is governed by the so-called circadian clock, with the word circadian coined from “circa” and “diem” in Latin, meaning approximately one day. In the past 40 years, active circadian research has uncovered profound mechanisms of how the circadian clock works, which culminated in the 2017 Nobel Prize in Physiology/Medicine for the cloning of the first animal circadian gene. We now know a great deal about our biological timer. It consists of molecular oscillators present in every cell of our body, which are synchronized by a master pacemaker in the brain to perform tissue-specific functions. Liver, as the central metabolic organ, has been a favorite subject for circadian studies. Indeed, numerous metabolic pathways and their rate-limiting components are controlled by the liver clock. However, much remains unknown. The current study (Wang et al. ) now provides us a far-reaching framework toward a full understanding of biological timing in this essential organ. 

This technological tour de force, a collaboration of Fuchu He, Jun Qin, Chen Ding and their colleagues, is a multi-pronged effort to construct a panoramic view of hepatic timing, with a particular emphasis on proteomic fluctuation. The centerpiece of this study is an innovative tenique called concatenated tandem-array of consensus transcription factor (TF) response elements (catTFRE). In a high temporal resolution design, they queried liver samples collected every three hours, over two consecutive full days, from mice under normal light:dark cycles. On average, slightly over 200 transcription factors were identified to show cyclic DNA-binding activity (DBA) at each time point, with remarkably robust reproducibility between the two circadian cycles. Importantly, whereas previous circadian proteomic studies failed to reveal the core circadian TFs, including CLOCK and BMAL1, the current study clearly demonstrates the oscillatory pattern of these factors, along with two other clock components. This is an initial clue that this catTFRE platform affords a superior technical sensitivity and precision, and the 200 or so TFs uncovered in this proteomic screen represent a gold mine for the circadian research community. 

Based on this initial success, the study went on to illustrate how the cyclic DBA survey can synergize with previous and new profiling studies to teach us important insight on global hepatic timing. While somewhat surprisingly, the rhythmic TF DBA did not seem to correlate with nuclear abundance of TFs (see also below), there is a strong concordance between DBA and phosphorylation of TFs, as evaluated by unbiased phosphorylation proteomic profiling. Of note, the core circadian TFs CLOCK and BMAL1 were found to demonstrate a phosphorylation pattern mirroring their DBA. Another protein modification they surveyed is ubiquitylation. Interestingly, both modification profiling revealed several functional clusters, including immune response and lipid metabolism during the day and cell cycle and glucose metabolism at night. 

In another major breakthrough finding, they identified a group of rhythmic TFs that appear to play a predominant role in driving circadian gene expression in the liver. These TFs, which they dubbed “dominant rhythmic TFs” or DR-TFs, were first identified via an integrative bioinformatic analysis of both DBA and transcriptomic databases. Furthermore, by comparing DBA and liver whole proteome to derive a second set of DR-TFs, the team observed an astoundingly high level of overlap between these two sets, thus validating appropriately 40 TFs that are mainly responsible for the clock-driven gene regulation program in the liver. This finding represents a major step to reconcile a discrepancy between gene expression and regulator dynamics (TF in this case) commonly observed in previous studies, and unveil a core TF network for decoding tissue-specific circadian regulation. As an extension of this key insight on hepatic TFs, their data also revealed a central importance of the Mediator complex as a transcriptional coregulator. Approximately two thirds of Mediator components were recovered in their TFRE dataset, and many exhibited oscillatory retention at cognate DNA binding sequences. 

In the aforementioned whole tissue proteomics, they observed cyclic abundance of proteins involved in toll-like receptor and NF-kB signaling pathways. When they performed the same proteomic analysis using isolated liver-resident macrophage cells, Kupffer cells (KCs), they made another startling discovery: whereas 690 proteins were found to oscillate in KCs, only 61 of them met the same oscillatory criteria in the whole tissue dataset. This is a vivid example of tissue- or cell type-specific circadian regulation at the protein level. Finally, they conducted mouse based assays to evaluate innate immunity.  At two diagonally spaced circadian times, mouse survival and tissue response showed distinct patterns in response to acute insults. 

Like any groundbreaking stories, this work raises several other important questions. First, since the mice were placed in light:dark cycles, it will be interesting to conduct future studies in a constant condition, namely constant darkness. This will eliminate the masking effects by light, the dominant external cue for the circadian system. Second, circadian transcription factors, such as BMAL1, are known to undergo a “Kamikaze” destruction by ubiquitylation when they bind to promoters and activate transcription. As a result, paradoxically, their highest activity has been found to correlate with their lowest abundance. The dynamic nature of this process may constitute a confounding factor for the apparent lack of correlation between DBA and nuclear TF steady-state amount. Third, this study reveals several core networks driving circadian timing in either liver or liver-associated functions/cell types. These highly prioritized networks are now experimentally amenable to functional and mechanistic characterization. Such follow-up studies will provide a refined time phase and elucidate biological logic for key TFs in the liver. Last but not least, metabolomic studies in liver have also been published. Given a predominant role of liver in systemic metabolism, future integrative studies should be performed to complete the pipeline, from gene regulation in the nucleus to metabolite flux throughout the cell. 

Even after the Nobel Prize, it is clear that we still have much to learn about biological timing. This landmark study, with unmatched breadth and rigor, lays a comprehensive foundation for many exciting discoveries in the future.    

参考文献

1. Saini, C., Suter, D. M., Liani, A., Gos, P. & Schibler, U. The mammalian circadian timing system: synchronization of peripheral clocks. Cold Spring Harb. Symp. Quant. Biol. 76, 39–47 (2011).

2. Zhang, R., et al., A circadian gene expression atlas in mammals: implications for biology and medicine. Proc Natl Acad Sci U S A, 2014. 111(45): p. 16219-24.

3. Takahashi, J.S., et al., The genetics of mammalian circadian order and disorder: implications for physiology and disease. Nat Rev Genet, 2008. 9(10): p. 764-75.

4. Ding, C. et al. Proteome-wide profiling of activated transcription factors with a concatenated tandem array of transcription factor response elements. Proc.Natl Acad. Sci. USA 110, 6771–6776 (2013).