Dissecting enhancer-promoter looping and gene induction dynamics in differentiation

剖析分化过程中的增强子-启动子循环和基因诱导动态

• 批准号: 10642028
• 负责人: Michele Gabriele
• 金额: $ 12.5万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10642028
• 关键词: 3-Dimensional; Acute; Address; Advisory Committees; Bioinformatics; Biological; Biological Models; Biomedical Engineering; Cancer Etiology; Cell Differentiation process; Cells; Chromatin; Chromatin Loop; Computer Models; Consensus; Dedications; Defect; Development; Disease; Distal; Educational process of instructing; Enhancers; Experimental Designs; FOXG1B gene; Gene Activation; Gene Expression; Genes; Genetic Transcription; Genomic approach; Genomics; Goals; Histones; Knowledge; Label; Lead; Maintenance; Malignant Neoplasms; Measures; Mentors; Methodology; Methods; Modeling; Molecular Conformation; Mus; Neuronal Differentiation; Neurons; Outcome; Pathologic; Pathology; Phase; Physical condensation; Physiological; Physiology; Play; Polymers; Population; Principal Investigator; Proteins; Regulation; Regulatory Element; Reporting; Research; Research Personnel; Research Proposals; Resolution; Role; Structure; Techniques; Testing; Time; Tissues; Training; Transcription Initiation Site; Transcriptional Activation; Transcriptional Regulation; Visualization; Work; Writing; cell fixing; chromosome conformation capture; developmental disease; embryonic stem cell; equity, diversity, and inclusion; gene induction; genome editing; genome-wide; histone modification; image processing; imaging system; improved; insight; live cell imaging; outreach; pluripotency; promoter; prototype; simulation; single cell analysis; skills; spatiotemporal; success; temporal measurement; ultra high resolution

Project Summary/Abstract The precise activation of transcription in specific tissues and developmental stages is regulated by enhancer and promoter interactions (EPIs), whose alterations cause developmental defects and cancer. Indeed, it is critical to understand the mechanism by which enhancers are responsible for tissue- and time-specific gene activation. Despite the importance of EPIs, the mechanisms by which EPIs are formed are still largely unknown, and available models are still debated. In particular, the central open questions can be listed as follows: 1) Is contact between enhancer and promoter necessary, or is proximity enough? 2) Are the EPIs stable or dynamic? To address these knowledge gaps, in his K99 Aim1, Dr. Gabriele will set up a super-resolution 3D live-cell imaging (SRLCI) system to visualize the role of EPIs in transcription activation in a prototype gene. He will focus Aim1 on Foxg1, which is expressed after differentiating pluripotent cells to cortical neurons. Also, in neuronal lineages, the Foxg1 promoter displays a long-range interaction with an enhancer region, absent in pluripotency, thus making Foxg1 the ideal candidate to answer the study EPIs during differentiation. Notably, Dr. Gabriele has previously established an SRLCI methodology to study chromatin looping and found that these structures are rare and transitory. Here, he will dedicate the previous methodology to studying EPIs. In Aim2 (R00 phase), he will build on his SRLCI work of Aim1 to investigate the role of chromatin regulators and histone modification in regulating EPIs and establishing and maintaining transcription after cell differentiation. Then, to measure to what extent the identified EPIs mechanism generalizes to all the EPIs, in Aim3 (between K99 and R00) he will employ sequencing methods to identify all the newly formed EPIs that result in transcription activation while differentiating mouse embryonic stem cells to cortical neurons. Dr. Gabriele’s long-term goal is to achieve independence as a principal investigator to dedicate his study to understanding the physiological and pathological mechanisms involved in the regulation of cell identity. For this purpose, he will be supported by his mentor and Scientific Advisory Committee. During the K99 phase, he will be trained in the experimental processing and analysis of single-cell genomics techniques such as SHARE- seq and Micro-C, and 3D polymer simulations. Moreover, he will improve his imaging processing analysis and deepen his bioinformatics knowledge. In addition, during the K99 phase, Dr. Gabriele will improve his scientific writing, outreach, mentoring and teaching, and management skills with a focus on diversity, equity, and inclusion values. Completing the K99 research and training will significantly facilitate Dr. Gabriele’s transition to independence and success as an independent investigator and mentor.

项目总结/摘要 在特定组织和发育阶段，转录的精确激活受增强子和 启动子相互作用（EPI），其改变导致发育缺陷和癌症。事实上， 了解增强子负责组织和时间特异性基因激活的机制。 尽管EPI很重要，但EPI形成的机制在很大程度上仍然未知， 现有的模式仍在争论中。特别是，中心开放式问题可以列举如下：1）是联系 增强子和启动子之间的距离是必要的，还是接近就足够了？2)环境绩效指标是稳定的还是动态的？到 为了弥补这些知识空白，Gabriele博士将在他的K99 Aim 1中建立一个超分辨率3D活细胞成像系统， （SRLCI）系统来可视化EPI在原型基因中的转录激活中的作用。他将专注于目标1 在Foxg 1上，Foxg 1在多能细胞分化为皮质神经元后表达。同样，在神经元谱系中， Foxg 1启动子显示与增强子区域的长程相互作用，在多能性中不存在，因此 这使得Foxg 1成为在分化过程中回答研究EPI的理想候选者。值得注意的是，加布里埃尔博士 先前建立了SRLCI方法来研究染色质循环，并发现这些结构是 罕见且短暂在这里，他将致力于以前的方法来研究环境绩效指标。 在Aim 2（R 00阶段），他将在Aim 1的SRLCI工作的基础上研究染色质的作用 调节因子和组蛋白修饰在调节EPI和建立和维持细胞后转录中的作用 分化 然后，为了衡量所确定的环境绩效指标机制在多大程度上适用于所有环境绩效指标，目标3 （在K99和R 00之间）他将采用测序方法来鉴定所有新形成的EPI， 转录激活，同时将小鼠胚胎干细胞分化为皮质神经元。 Gabriele博士的长期目标是作为一名主要研究者实现独立， 了解参与细胞身份调节的生理和病理机制。为 为此，他将得到他的导师和科学咨询委员会的支持。在K99阶段，他 将接受单细胞基因组学技术（例如SHARE-）的实验处理和分析培训 seq和Micro-C以及3D聚合物模拟。此外，他将提高他的成像处理分析， 加深他的生物信息学知识。 此外，在K99阶段，Gabriele博士将提高他的科学写作，推广，指导和 教学和管理技能，重点是多样性，公平和包容性的价值观。完成K99 研究和培训将大大促进加布里埃尔博士的过渡到独立和成功， 独立调查员和导师