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

研究介导哺乳动物视交叉上核（SCN）日常计时的基因组机制

• 批准号: BB/Z514792/1
• 负责人: Akanksha Bafna
• 金额: $ 53.54万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FZ514792%2F1
• 关键词: Investigating; genomic; mechanisms; mediating; daily

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.

通过我的 BBSRC 发现奖学金，我提议解开控制主起搏器日常计时的基因调控机制。在这里，我假设环境引起的 3D 染色体构象的昼夜节律变化驱动了中央时钟的时空基因调控。地球绕地轴 24 小时 (hr) 自转导致每日光和温度循环，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（例如睡眠-觉醒周期）与不断变化的日常环境条件保持一致。通常，在人类等多细胞生物中，环境光从视网膜传播到大脑的特定区域；视交叉上核（SCN）也称为中枢起搏器，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的不同区域（心脏、肝脏、肾脏等），以同步局部外周时钟并产生连贯的生理反应。精确及时地调节视交叉上核中的基因表达对于昼夜节律计时和整体健康至关重要。然而，使SCN成为强大的主振荡器的基因调控机制仍然未知。直到最近，由于技术限制，外周组织和细胞系被用作真实SCN的“代理”来研究基因调控支持日常计时的流程。然而，这显然不能令人满意，因为它缺乏 SCN 作为我们的中枢大脑起搏器的作用所固有的关键神经元细胞维度。我的研究重点是研究对日常计时机制至关重要的基因调控元件和过程，最近发现了 SCN 中循环组织特异性基因增强元件的普遍性。在我的奖学金项目中，我的目标是获得-深入了解中央时钟中基因组调控的机制，以了解日常节律的系统维护。增强子是一段短的 DNA，可以调节近端和远端基因表达。在发现增强子近四十年后，增强子被认为在基因表达的时空控制中发挥着关键作用。因此，我将首先通过使用大规模并行报告分析 (MPRA) 来重点关注推定映射的 SCN 增强子的功能表征。接下来，我建议在昼夜节律计时的背景下研究关键 DNA 环挤出蛋白 CTCF（CCCTC 结合因子）和粘连蛋白的全基因组结合。此外，我还想采用先进的Capture-C技术来制作第一个SCN染色体接触图，并探索增强子和下游靶基因表达之间缺失的联系。最后，我计划进行高通量空间转录组学，以可视化和量化亚细胞水平上的靶向转录增强子和基因表达。总的来说，我的研究结果将清楚地展示日常环境刺激如何调节表观基因组景观，以实现时空基因调控在哺乳动物的大脑中。拟议的研究将强调组织特异性增强元件和促进日常计时的相关过程。它将构成了解异常基因调控特征（增强病）如何导致昼夜节律失调并导致各种疾病和失调的发展的起点。此外，这项研究对于推进我们目前对染色体构象和DNA拓扑的系统调节以及计时的生物学基础的理解具有巨大的潜力。