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; 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 Discovery奖学金，我建议揭开控制大师Pacemaker日常计时的基因调节机制。在这里，我假设3-D染色体构象中环境诱导的昼夜节律变化驱动了中间时钟中的时空基因调节。地球围绕其自身轴的24小时（HR）旋转会导致光和温度的每日周期，并指导几乎所有生物中存在的内部时钟。这种内在的昼夜节律（大约一天）时钟将分子，行为和生理过程（例如睡眠效果周期）对齐，以改变日常环境条件。通常，在人类等多细胞生物中，环境光从视网膜到大脑的特定区域传播。外丘脑核（SCN）也称为中央起搏器，并触发了一系列节奏的分子和生化事件。然后，该信号传递给大脑和组织的不同区域（心脏，肝脏，肾脏等），以同步局部外围时钟并产生连贯的生理反应。 SCN中基因表达的精确和及时调节对于昼夜节律和整体适应性至关重要。然而，将SCN作为强大的主振荡器的基因调节机制尚不清楚。直到最近，由于技术局限性，外围组织和细胞系被用作实际SCN的“代理”来研究基因调控过程，以支撑日常工作。但是，这显然是不令人满意的，因为它缺乏SCN作为我们中央脑的起搏器作用固有的关键神经元细胞维度。我的研究重点是研究对每日计时机制至关重要的基因调节元素和过程，最近发现了我的奖学金项目中循环组织特异性基因增强子元素的流行率，我将旨在使每日consectation Contrantic conterative in Comeltative of Synectation in Synectation in Synectation in Synectation in Synectation in Synectation in Synectity in Synectity in Synectity in notectation in Synditation in Synectity in Synditation in Synectic in note in Systone conterical in the Mondy consection。增强子是可以调节近端和远端基因表达的简短DNA。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至关重要的作用。因此，我最初将通过使用大量并行的报告基准测定法（MPRA）来关注推定映射的SCN增强子的功能表征。接下来，我建议在昼夜节时间内研究中研究关键DNA环挤出蛋白CTCF（CCCTC结合因子）和粘着蛋白的全基因组结合。此外，我还想采用先进的Capture-C技术来生成SCN的第一个染色体接触图，并探索增强子和下游靶基因表达之间缺失的联系。最后，我计划进行高通量的空间转录组学，以可视化和量化靶向转录的攻击增强子和基因表达在亚细胞水平上。除此之外，我的发现将清楚地证明，每天的环境刺激如何调节表观基因组景观，以实现时空基因调节哺乳动物脑中的时空基因调节。拟议的研究将突出组织特定的增强子元素，并涉及促进日常计时的过程。它将构成了解异常基因调节特征（增强性疾病）如何导致昼夜节律错位的起点，并导致各种疾病和疾病的发展。此外，这项研究具有巨大的潜力，可以促进我们目前对染色体构象和DNA拓扑的系统性调节以及时间保留的生物学基础的理解。