The physical properties of the genome play a central role in controlling essential processes, including gene expression, DNA repair, and DNA replication. All eukaryotic organisms, from yeast to humans, organize their genomic DNA by repeatedly wrapping it around DNA-associated proteins, called histones, into small spools known as nucleosomes. These nucleosomes, which are about a millionth of a centimeter (10 nanometers) in size, are strung like beads on a string to form long chromatin fibers that ultimately make up chromosomes. The spatial arrangement of these nucleosomes within the chromatin fibers continually changes, often through coordinated movements. However, current technologies are limited in quantifying these structural rearrangements, and this limitation has in turn limited our understanding of how chromatin controls gene expression. This project will monitor structural changes in chromatin as gene expression is switched between the 'on' and 'off' states by developing DNA-based nanometer-sized calipers to detect structural changes in chromatin at the 10 nanometer to 100 nanometer scale, which covers a critical range for events during the regulation of gene expression. Graduate and undergraduate students will receive training in molecular biology, biochemistry, DNA nanotechnology, and single molecule detection, which will position them to become significant contributors as the next generation of biotechnology scientists. The PIs will also work with outreach programs, including the OSU Minority Engineering Program, the Women in Engineering Program, and the Masters to Ph.D. Bridge Program, to improve diversity and interest in the fields of biotechnology and biophysics.
The conversion of chromatin between open euchromatin and compact heterochromatin is essential for the regulation of transcription and cellular differentiation. This conversion is regulated by histone post-translational modifications (PTMs) and chromatin architectural proteins that recognize these PTMs to dynamically control chromatin structure. However, the dynamic chromatin structural properties that regulate transcription are currently not well understood. These properties includes changes in the distance between nucleosomes, changes in nucleosome orientation, and the time for these structural changes to occur. To directly investigate chromatin structural dynamics, this project is using DNA origami nanotechnology to develop DNA-based nano-calipers that quantify 10 nm to 100 nm distance changes within large macromolecular complexes. The project will use this DNA nano-caliper design to: (i) determine the long range structural dynamics of heterochromatin that is formed by Heterochromatin Protein 1 as it interacts with the histone H3 PTM, lysine 9 trimethylation, and (ii) determine the long range structural and dynamic response of compacted heterochromatin to the binding of transcription activating complexes. This approach involves integrating PTM-containing chromatin molecules into DNA nano-calipers where each end is attached to opposite nano-caliper arms. The DNA nano-caliper conformational changes will then be detected separately with transmission electron microscopy to determine the distribution of chromatin conformations and single molecule fluorescence to determine the transition rates between distinct chromatin structural states. These studies will provide key distance and time measurements of the structural dynamics that occur during heterochromatin formation and heterochromatin decompaction as transcription activators bind to their target sites. More broadly, the development of this DNA origami nanotechnology will be widely applicable to the study of numerous biological complexes that undergo structural changes on the 10 nm to 100 nm length scale.
This project is jointly supported by the Genetic Mechanisms and the Molecular Biophysics Programs of the Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences, and by the Biomaterials Program of the Division of Materials Research in the Directorate for Physical and Mathematical Sciences.
