Mechanistic dissection of dynamics of transcriptional regulation by chromatin looping

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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.