Cells orchestrate many essential biological processes around their genome, such as retrieval of genetic information for protein synthesis, DNA duplication for propagation, and DNA repair for damage remediation. However, it remains an open question what roles the genome itself plays in such fundamental DNA-directed processes. One critical barrier to answering this question is the lack of quantitative knowledge of how chromatin, the material form of the genome that consists of a complex assembly of nucleic acid and protein, is organized in three dimensional space and how the chromatin organization changes in response to various biological, chemical and physical factors in the cellular environment. This project will develop and use state-of-the-art experimental techniques combined with extensive data-driven computational modeling, with the goal to reconstruct three dimensional structures of chromatin. Through systematic studies of model chromatin systems in wide-ranging solution conditions, this project further aims to elucidate the governing principles of chromatin organization. Along with the research effort, this project will enrich educational experiences for broadly represented student groups, particularly in response to the current challenge of engaging and retaining students in STEM education. Specific emphasis will be placed on incorporating underrepresented groups in research and educational activities.
The objective of this project is to gain systematic, mechanistic understandings of the conformation and dynamics of chromatin modulated by solution conditions, genetic and epigenetic variations, and chromatin-architectural proteins. Folding of chromatin from its open structure of 'beads-on-a-string' is an intricate process that has challenged researchers for decades, in part due to the complex set of governing physical and biochemical factors, and in part due to the lack of experimental approaches at the pertinent length scales above the mono-nucleosome level (i.e., one to tens of nanometers). This project addresses both challenges by applying solution small angle x-ray and neutron scattering (SAXS/SANS) to the biochemically defined system of recombinant nucleosome arrays. The use of strong positioning DNA sequences yields regularly spaced and stable arrays of nucleosomes, and SAXS/SANS measures the distribution of inter-nucleosome mesoscale distances as the array folds. SAXS/SANS further has unique advantages of probing array native structures directly in solution and decomposing protein and DNA components via contrast variation. Significant efforts will be directed towards applying and developing data-driven computational modeling methods to reconstruct the three dimensional structures of nucleosome arrays. To dissect the multifactorial determinants of folding, arrays of increasing lengths (2, 3, 4, 6, and 12 nucleosomes) will be analyzed under relevant conditions of ions, molecular crowding, linker DNAs, histone variations, and chromatin architectural proteins. Completion of this project is expected to elucidate detailed mesoscale structures of nucleosome arrays in solution and the molecular mechanisms of chromatin folding, and to shed light on the genetics/epigenetics-structure-function relationship of chromatin in vivo. This project is supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences.
