Higher-Order Structure Of Chromatin

This project addresses the molecular mechanisms that enable several meters of DNA (the genome) to be packaged into a cell nucleus which has a diameter that is approximately 100,000 times smaller than this. This amazing degree of compaction of the DNA is achieved by repeatedly coiling of the DNA double helix around multiple spools, each of which is composed of protein (histone) cores. The DNA is thus formed into a 'beads-on-a-string' array of DNA/histone particles called nucleosomes. The nucleosome arrays are then further folded into structures that include multiple loops where the DNA is physically constrained. Beyond a basic understanding of this structure, the architectural organization of the DNA-protein complex in the cell nucleus (known as chromatin), remains enigmatic. This research will use biochemical experiments and computational 3-dimensionsal modeling to describe the structural interactions of DNA in living cells and to predict how the structure impacts function. The project will promote education and training for graduate students and for undergraduates, including those from underrepresented groups and underserved areas of rural Pennsylvania who typically have little opportunity to participate in fundamental research.

In most current textbooks, the higher-order structure of DNA is modeled based on zigzag or solenoidal folding of nucleosomes forming linear chromatin fibers. However, recent in vivo studies suggested a dynamic looping of flexible and disordered nucleosome arrays rather than linear fibers in most cell types. This project is centered on a hypothesis that different lengths and conformations of the linker DNA connecting nucleosome beads impose distinct topological states and orders of nucleosome packing that segregate the chromatin domains into either flexible nucleosome chains facilitating functional interactions between distant DNA elements or, alternatively, tightly packed nucleosomes that inhibit interactions between distant chromosomal sites. This hypothesis will be tested using a unique electron microscopy nucleosome interaction capture method, DNA topology assays, and computational modeling to determine patterns of nucleosome interactions and DNA topology of both circular arrays of precisely positioned nucleosomes and circular minichromosomes isolated from yeast cells. The results will provide a more detailed picture of chromatin organization as it is likely to exist in the cell, and this knowledge will serve as a framework for better understanding of how chromatin features regulate gene expression.

This award is co-funded by programs in Genetics Mechanism (Division of Molecular and Cellular Biosciences) and the Physics of Living Systems (Division of Physics).