The preceding project components will produce topological reference maps (TRMs) for cells at different stages of differentiation of hESCs, and for human fibroblasts at different positions of the cell cycle, for natural and engineered TADs, and as a function of chromatin modifications. A piece of the overall spatial organization picture missing from the above analysis is direct measurement of actual 3D/4D organization of chromosome organization, i.e., visualization and analysis of dynamics of the absolute spatial layout of chromatin loci, rather than the relative position of pairs of loci. This component will attack this part of the problem, as well as developing approaches that directly address other current limitations of 3C-based methods, by development of entirely new high-risk approaches to map and perturb the 4D nucleome. Specific Aim 3. Use micromanipulation and microelasticity assays, combined with CRISPR/Cas9-fusion-protein-based labeling methods, to analyze the internal organization of metaphase chromosomes, and to carry out similar experiments on prophase chromosomes to determine the dynamics of mitotic chromosome folding. Using microdissection and micromanipulation, Marko’s lab will analyze metaphase chromosome mechanics and carry out high-resolution fluorescence imaging of their internal structure, with an objective of directly observing the loop-domain structure thought to underlie metaphase chromosome organization. We will also use chromosome stretching to boost imaging resolution. Labeling will be carried out using Cas9-gfp fusions targeted to specific genomic loci, and will be done in coincidence with condensin SMC complex antibody labeling. Experiments will be carried out to develop parallel techniques to analyze prophase chromosomes, in order to study the dynamics of chromosome folding during early mitosis, so as to test both 4D TRM data and the theoretical modeling results.
|Effective start/end date||9/30/15 → 7/31/16|
- University of Massachusetts Medical School (WA00363875/OSP2016103//1-U54-DK1079)
- National Institute of Diabetes and Digestive and Kidney Diseases (WA00363875/OSP2016103//1-U54-DK1079)
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