Investigating the genomic mechanisms mediating daily timekeeping in the suprachiasmatic nucleus (SCN) in mammals

通过我的BBSRC发现奖学金，我提议解开主起搏器中管理日常计时的基因调控机制。在这里，我假设环境诱导的昼夜节律变化的3-D染色体构象驱动时空基因调控的中央时钟。地球24小时（hr）绕着自己的轴旋转，导致光和温度的每日循环，并指导几乎所有生物体内的生物钟。这种内在的昼夜节律（大约一天）时钟使分子、行为和生理过程（如睡眠-觉醒周期）与不断变化的日常环境条件保持一致。通常，在多细胞生物体如人类中，环境光从视网膜传播到大脑的特定区域;视交叉上核（SCN）也被称为中央起搏器，并触发一系列有节奏的分子和生化事件。然后，信号被传递到大脑和组织（心脏，肝脏，肾脏等）的不同区域。以同步本地外围时钟并产生相干的生理响应。精确和及时地调节SCN中的基因表达对于昼夜节律计时和整体健身至关重要。然而，使SCN成为一个强大的主振荡器的基因调控机制仍然是未知的，直到最近，由于技术上的限制，外周组织和细胞系被用作真实的SCN的“代理”来研究支撑日常计时的基因调控过程。然而，这显然是不令人满意的，因为它缺乏SCN作为我们的中枢脑起搏器的作用所固有的关键神经元细胞尺寸。我的研究重点是调查基因调控元件和过程，是至关重要的日常计时机制，并最近发现循环组织特异性基因增强子元件在SCN的流行。在我的奖学金项目，我将致力于获得深入的机制洞察基因组调控在中央时钟运作，以了解日常节奏的系统维护。增强子是可以调节近端和远端基因表达的短DNA片段。在发现近40年后，增强子被认为在基因表达的时空控制中起着关键作用。因此，我将首先专注于使用大规模平行报告分析（MPRA）的puppelinemapped SCN增强子的功能表征。接下来，我建议研究关键的DNA环挤出蛋白CTCF（CCCTC结合因子）和cohesin在昼夜节律计时的背景下的全基因组结合。此外，我还想采用先进的Capture-C技术，制作有史以来第一个SCN的染色体接触图，探索增强子和下游靶基因表达之间缺失的环节。最后，我计划进行高通量空间转录组学可视化和定量靶向转录增强子和亚细胞水平的基因表达，总的来说，我的研究结果将清楚地表明如何日常环境刺激调节表观基因组景观，以实现时空基因调控在哺乳动物大脑。拟议的研究将突出组织特异性增强子元素和促进日常计时的相关过程。它将构成理解异常基因调控特征（增强子病）如何导致昼夜节律失调并导致各种疾病和病症发展的起点。此外，这项研究具有很大的潜力，以促进我们目前的理解系统调控染色体构象和DNA拓扑结构和生物学基础的时间保持。

With my BBSRC Discovery Fellowship, I propose to unravel the gene regulatory mechanisms that governs daily timekeeping in the master pacemaker. Here, I hypothesise environmentally induced circadian change in 3-D chromosomal conformation drives the spatiotemporal gene regulation in the central clock. The 24-hour (hr) rotation of the earth around its own axis results in daily cycles of light and temperature and directs the internal body clock present in almost all living creatures. This intrinsic circadian (approximately one day) clocks align the molecular, behavioural and physiological processes such as sleep-wake cycles, to changing daily environmental conditions. Typically, in multicellular organisms such as humans, environmental light travels from the retina to specific region of the brain; suprachiasmatic nuclei (SCN) also known as central pacemaker and triggers a series of rhythmic molecular and biochemical events. The signal is then passed to different regions of the brain and tissues (heart, liver, kidney etc.) to synchronize the local peripheral clocks and generate a coherent physiological response. Precise and timely regulation of the gene expression in the SCN is crucial for circadian timekeeping and overall fitness. However, the gene-regulatory mechanisms that renders SCN as a powerful master oscillator is still unknown.Until recently, and because of technical limitations, peripheral tissues and cell lines were used as a "proxy" for the real SCN to study the gene-regulatory processes that underpin daily timekeeping. However, this is patently unsatisfactory as it lacks the critical neuronal cellular dimension intrinsic to the role of the SCN as our central brain pacemaker. My research is focussed on investigating the gene- regulatory elements and processes that are vital for daily timekeeping mechanisms, and have recently discovered the prevalence of cycling tissue-specific gene enhancer elements in the SCN.In my fellowship project, I will aim to gain in-depth mechanistic insights into the genomic regulation operative in the central clock to understand the systematic maintenance of daily rhythms. Enhancers are short stretches of DNA that can modulate both proximal and distal gene expression. Almost forty years after their discovery, enhancers are recognised as playing a critical role in the spatiotemporal control of gene expression. Therefore, I will initially focus on the functional characterization of the putatively mapped SCN enhancers by using massively parallel reporter assay (MPRA). Next, I propose to study the genome-wide binding of the key DNA loop extrusion proteins CTCF (CCCTC-Binding Factor) and cohesin in the context of circadian timekeeping. In addition, I would also like to adopt advanced Capture-C technology to produce the first ever chromosomal contact map of the SCN and explore the missing link between enhancer and downstream target gene expression. Finally, I plan to carry out high-throughput spatial transcriptomics to visualize and quantify targeted transcribing enhancer and gene expression at sub-cellular level.Overall, my findings will clearly demonstrate how daily environmental stimuli modulate the epigenomic landscape in order to achieve spatiotemporal gene regulation in the mammalian brain. The proposed research will highlight the tissue-specific enhancer elements and involved processes that facilitates daily timekeeping. It will constitute the starting points to understand how aberrant gene -regulatory features (enhanceropathies) could result in circadian misalignment and lead to the development of various diseases and disorders. Moreover, this study holds great potential to advance our current understanding on systemic regulation of chromosomal conformation and DNA topology and the biological basis of time keeping.