The 3-D structure of chromosomes is the richest unexplored territory in cell science. Chromosomes are the largest, most dynamic, and most complex of all cellular organelles. They are most fundamental, underlying cell differentiation, cell physiology, and disease. They are also most enigmatic, exhibiting at the same time structural heterogeneity and order, physical flexibility and rigidity, and functional activity and silencing. We will dispel the mysteries by the determination of chromosome structure. Virtually nothing is known about chromosome structure at the present time. And yet chromosome structure is the key to chromosome function; it is inextricably linked to all DNA transactions, to epigenetics, and to aberrations in disease. We propose a comprehensive solution of the chromosome structure problem. We will trace the path of the chromatin fiber, from nucleosome to nucleosome, through TADs and intervening regions. We will walk the 3-D genome at single nucleosome resolution. We will employ super resolution light microscopy with DNA sequence-specific dye molecules (fluorescence emitters) for the purpose. A resolution of 10-20 nm, comparable to the size of a nucleosome (10 nm) is routinely achieved with current microscopes. The localization of an emitter to an individual nucleosome can be accomplished with the use of specially designed pyrrole-imidazole polyamides and zinc finger proteins. These molecules, once designed, will be applicable to chromosome structure determination in all types of cell, at all stages of the cell cycle, in both normal and disease states. They will transform the field, by providing structural paradigms, and by enabling others to address innumerable problems in genetic chemistry and biology in a facile, penetrating manner. !
The proposed research is relevant to both fundamental studies and to human health. The methods and results will contribute to the eventual understanding of all aspects of the human genome. The elucidation of the the three-dimensional structure of the genome will enable insights into cancer and other diseases, and will lead to novel therapeutics. !