Mechanistic dissection of dynamics of transcriptional regulation by chromatin looping

染色质环转录调控动力学的机制剖析

• 批准号: 10313180
• 负责人: Miles Kocur Huseyin
• 金额: $ 6.6万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2024-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10313180
• 关键词: 3-Dimensional; Address; Behavior; Biological; Biological Models; Biological Process; Cell physiology; Cells; Chromatin; Chromatin Loop; Chromatin Modeling; Communication; Complement; Conflict (Psychology); Data; Development; Developmental Gene; Disease; Dissection; Distal; Engineering; Enhancers; Environment; Excision; Frequencies; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Genomics; Goals; Hi-C; Image; Individual; Investigation; Link; Location; Malignant Neoplasms; Mammals; Measurement; Measures; Mentorship; Messenger RNA; Microscopy; Modeling; Molecular; Monitor; Mutation; Nuclear; Performance; Polymers; Positioning Attribute; Preparation; Process; Proteins; Regulatory Element; Reporter; Reporting; Research; Resolution; Scientist; Structure; Students; System; Systems Biology; Testing; Training; Transcriptional Activation; Transcriptional Regulation; base; career; cell type; cohesin; design; developmental disease; imaging modality; innovation; insight; live cell imaging; promoter; responsible research conduct; sample fixation; simulation; skills; synthetic biology; synthetic construct; whole genome

A major driver of transcriptional regulation in mammals is the association of gene promoters with enhancers, which can be hundreds of kilobases away from their cognate promoters. Enhancers have been proposed to potentiate transcription through looping to contact the promoter. The broader 3D organization of the genome into topologically associated domains (TADs) has been proposed to restrict E-P looping to primarily occur inside the same TAD and not between two TADs, and to thereby contribute to regulation of transcription. Both TAD structure and enhancer activities change through development, and mutations of many of the factors involved in regulating genome organization have been identified as causative features of both developmental disease and cancers. However, recent studies of developmental genes have obtained conflicting results on whether genome organization can influence transcription. Furthermore, it remains unclear as to how E-P interactions are related to transcription, and whether direct contact is even required. The majority of studies of 3D genome organization and chromatin looping have employed genomics or microscopy approaches which rely on fixation, and therefore do not allow for the measurement of the dynamics of these processes. Additionally, most recent studies have focused on single, developmentally regulated genes which may be subject to a high degree of redundancy and complexity. Therefore, in order to address these major questions of the contribution of TADs and E-P contact to transcriptional regulation, the proposed research will employ a bottom-up approach to construct synthetic TADs and E-P pairs, and measure chromatin looping and transcription dynamics together in live cells through newly developed live-cell super resolution microscopy approaches. Mechanistic hypotheses for the function of these processes will then be developed using polymer simulations of loop extrusion and evaluated through comparison with experimental data and targeted perturbations without the redundancy and complexity inherent to endogenous genes. Together, the proposed research will uncover the interplay between genome organization and transcriptional regulation and develop a mechanistic understanding of both TAD formation and E-P interactions. This will provide a basis for future development of approaches to correct misfolding of the genome in disease. The proposed research will be paired with training to develop the applicant's skills as a research scientist to allow him to succeed in the proposed project and in his future career as he aims to become an independent PI. In addition to developing the applicant's experimental and computational expertise, mentorship from the sponsor and co-sponsor will involve training in communicating the applicant's research, mentorship of students, lab management, responsible conduct of research and preparation for future career goals. This training will be conducted in a leading academic environment in MIT's Department of Biological Engineering, where the applicant will be in close proximity to world leaders in a variety fields and a wealth of expertise and facilities.

哺乳动物转录调控的主要驱动力是基因启动子与 增强子，可能与其同源启动子相距数百千碱基。增强剂已 提出通过环接触启动子来增强转录。更广泛的 3D 组织 已提出将基因组划分为拓扑相关域 (TAD) 以将 E-P 循环限制为主要 发生在同一个 TAD 内而不是在两个 TAD 之间，从而有助于转录调节。 TAD 结构和增强子活性都会随着发育和许多因素的突变而变化 参与调节基因组组织已被确定为两种发育的致病特征 疾病和癌症。然而，最近对发育基因的研究获得了相互矛盾的结果 基因组组织是否会影响转录。此外，目前还不清楚E-P如何 相互作用与转录有关，以及是否需要直接接触。 大多数 3D 基因组组织和染色质环研究都采用了基因组学或 依赖于固定的显微镜方法，因此不允许测量动力学 这些过程。此外，最近的研究都集中在单一的发育调控基因上 这可能会受到高度冗余和复杂性的影响。因此，为了解决这些重大问题 关于 TAD 和 E-P 接触对转录调控的贡献的问题，拟议的研究将 采用自下而上的方法构建合成 TAD 和 E-P 对，并测量染色质循环和 通过新开发的活细胞超分辨率显微镜观察活细胞中的转录动态 接近。然后将使用聚合物开发这些过程功能的机制假设 模拟环挤出，并通过与实验数据和目标数据的比较进行评估 没有内源基因固有的冗余和复杂性的扰动。共同提出的 研究将揭示基因组组织和转录调控之间的相互作用，并开发一种 对 TAD 形成和 E-P 相互作用的机制理解。这将为未来打下基础 开发纠正疾病中基因组错误折叠的方法。 拟议的研究将与培训相结合，以培养申请人作为研究科学家的技能 让他在拟议的项目和未来的职业生涯中取得成功，因为他的目标是成为一名独立的人 PI。除了培养申请人的实验和计算专业知识外，来自 赞助商和共同赞助商将涉及沟通申请人的研究、对学生的指导、 实验室管理，负责任地进行研究并为未来的职业目标做好准备。此次培训将 该研究在麻省理工学院生物工程系领先的学术环境中进行， 申请人将近距离接触各个领域的世界领导者以及丰富的专业知识和设施。