Mechanistic dissection of dynamics of transcriptional regulation by chromatin looping

哺乳动物中转录调控的主要驱动力是基因启动子与转录因子的关联。 增强子，其可远离其同源启动子数百个碱基。增强剂已经 提出通过环化以接触启动子来增强转录。更广泛的3D组织 已提出将基因组转化为拓扑相关结构域（TAD）来限制E-P循环，主要用于 发生在同一个转录因子内，而不是在两个TAD之间，从而有助于转录的调节。 基因结构和增强子活性都在发育过程中发生变化，许多因子的突变 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获得了相互矛盾的结果： 基因组结构是否会影响转录。此外，目前还不清楚E-P如何 相互作用与转录有关，甚至与是否需要直接接触有关。 大多数3D基因组组织和染色质循环的研究都采用了基因组学或 显微镜方法依赖于固定，因此不允许测量动力学 这些过程。此外，最近的研究主要集中在单个发育调控基因上， 其可能具有高度的冗余性和复杂性。因此，为了解决这些主要问题， TADs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的TADs和E-P对，并测量染色质环， 通过新开发的活细胞超分辨率显微镜在活细胞中一起转录动力学 接近。这些过程的功能机制假说，然后将开发使用聚合物 模拟环挤压，并通过与实验数据的比较进行评估，并有针对性地 干扰而没有内源基因固有的冗余和复杂性。在一起，拟议的 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 机械的理解，无论是形成和E-P的相互作用。这将为今后的 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配对，以发展申请人作为研究科学家的技能 让他在拟议的项目和他未来的职业生涯中取得成功，因为他的目标是成为一个独立的 Pi.除了发展申请人的实验和计算专业知识，导师从 赞助商和共同赞助商将涉及培训，在沟通申请人的研究，指导学生， 实验室管理，负责任的研究和未来职业目标的准备。将培训以文件形式进行 在麻省理工学院生物工程系领先的学术环境中进行， 申请人将在接近世界领导人在各种领域和丰富的专业知识和设施。

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.