In the chromosomes within the cell nuclei of eukaryotic organisms, DNA is periodically coiled around histone protein cores to form nucleosome arrays. These arrays are further folded in a zig-zag manner into higher order chromatin structures. For proper cell functioning, the higher order chromatin structures must unfold to make DNA accessible to transcription, replication, recombination, and repair. Alternatively, it can adopt a condensed and repressed state known as heterochromatin. The objective of this project is to deduce the basic principles of nucleosome array architecture that support the condensed heterochromatin state and may prevent chromatin unfolding. This project is aimed to test the hypothesis that nucleosome array compaction in condensed heterochromatin is determined by a) nucleosome orientation in zig-zag chromatin arrays and b) protein factors that promote inter-array nucleosome bridging (nucleosome array self association caused by a protein factor connecting two or more separate arrays) over intra-array nucleosome folding (compaction within a single nucleosome array). The experimental design includes two specific aims. One specific aim is to determine the internucleosome orientations and the architectural protein factors that promote either folding or bridging of the nucleosome arrays. The second specific aim is to determine the topography of nucleosome interactions in compact chromatin. This work combines the utilization of established biochemical and electron microscopic experimental techniques with the development of novel approaches to assess chromatin structure, such as electron microscopy and polymerase chain reaction-assisted nucleosome interaction capture techniques.
Broader Impacts: More than 50 years since the discovery of DNA double helix, its higher order packing in condensed chromatin and chromosomes is still unknown. This work should help to solve the long-standing problem of spatial organization of higher-order chromatin and uncover the nature of chromatin structural transitions involved in heterochromatin formation and chromatin condensation. The new information and methodology that is being developed under this project is crucial for understanding spatial organization of DNA in chromatin and its relationship to fundamental mechanisms of heterochromatin formation, gene silencing, and cell differentiation and thus should significantly contribute to basic science education in molecular biology and genetics. In addition to the general scientific knowledge, this project is aimed to provide new research training and education opportunities for undergraduate students from the Penn State Summer Undergraduate Research program and other colleges in central Pennsylvania. Many of these students represent minority groups and come from environments that have little previous exposure to experimental science. This research will be also essential for developing educational infrastructure to facilitate graduate student training in molecular imaging and electron microscopy. This project includes a number of tasks (such as chromatin template "construction kit", histone isolation, and nucleosome reconstitution) especially suitable for undergraduates and entry-level graduate students, that will allow them to relate biochemical experiments to visual changes in chromatin structure as observed by electron microscopy and to develop the initial confidence necessary for them to promote their interest and motivation for scientific research.
