Mechanistic dissection of dynamics of transcriptional regulation by chromatin looping

哺乳动物转录调控的一个主要驱动力是基因启动子与 增强子，可能与它们的同源启动子有数百个碱基的距离。增强剂已经被 建议通过环联系启动子来增强转录。更广泛的3D组织 已提出将基因组分成拓扑相关区域(TADS)以限制E-P循环主要 发生在相同的TAD内，而不是在两个TAD之间，从而有助于转录的调节。 TAD结构和增强子活性都会随着发育和许多因子的突变而改变 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在以下方面得出了相互矛盾的结果 基因组结构是否能影响转录。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。 3D基因组组织和染色质环的大多数研究都利用了基因组学或 显微镜方法依赖于固定，因此不允许测量动力学 这些过程中。此外，最近的研究主要集中在单个发育调节基因上。 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TADS和E-P接触对转录调控的贡献的问题，拟议的研究将 使用自下而上的方法构建合成的TADS和E-P对，并测量染色质环和 用新发展的活细胞超分辨显微镜研究活细胞中的转录动力学 接近了。然后将使用聚合物来开发这些过程的功能的机械假说 通过与实验数据的对比和针对性的评估，对环流挤出过程进行模拟 没有内源基因固有的冗余性和复杂性的扰动。总而言之，建议的 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 TAD形成和E-P相互作用的机理理解。这将为今后的工作提供基础 疾病中纠正基因组错误折叠的方法的发展。 拟议的研究将与培训相结合，以发展申请者作为研究科学家的技能 让他在拟议的项目中取得成功，并在他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 圆周率。除了发展申请人的实验和计算专业知识外，来自 赞助商和联合赞助商将接受培训，交流申请者的研究、学生指导、 实验室管理，负责任地进行研究，为未来的职业目标做准备。这次培训将是 在麻省理工学院生物工程系一流的学术环境中进行，在那里 应聘者将在各种领域和丰富的专业知识和设施方面与世界领导人保持密切联系。

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.