3D structural and mechanistic underpinnings of the eu/heterochromatin dichotomy

DNA in chromosomes of human and other eukaryotic cells is packed into open and active euchromatin or repressed and condensed heterochromatin. Packing into these alternative chromatin states governs DNA accessibility and expression of the genetic code without altering the sequence, akin to obtaining information from open but not closed pages of a book. Resolving the structures and mechanisms underpinning euchromatin and heterochromatin formation is crucial for understanding fundamental biological processes underlying gene expression, cell differentiation, chromosomal stability, and genetic abnormalities. This project will investigate 3-dimensional (3D) structures and mechanisms that direct native chromosome folding into euchromatin and heterochromatin and thus find new ways of regulating chromosome accessibility and gene expression without altering DNA sequences. The research will be integrated with postdoctoral training and graduate and undergraduate education and provide new opportunities to inspire student enthusiasm for scientific discoveries and careers in STEM. The goal of this project is to reveal the 3D structural motifs and molecular mechanism by which the natural nucleosome spacing distribution directs the differential chromatin folding underlying genomic euchromatin and heterochromatin states and associated genetic functions. A recent breakthrough in understanding native chromatin folding was achieved by applying cryo-electron tomography (Cryo-ET) that challenged established models of regular chromatin higher-order folding. Three specific aims will employ Cryo-ET to accomplish the overall goal: Aim 1 is to determine 3D chromatin structures and associated linker DNA length distributions in isolated human eu- and heterochromatin fractions; Aim 2 is to determine nucleosome array folding paths and compaction associated with eu- and heterochromatin compartments in mouse cells cryo-vitrified in situ; Aim 3 is to generate reconstituted nucleosome arrays reproducing native nucleosome spacing diversity and the eu/heterochromatin structural dichotomy for Cryo-ET studies. This work is expected to uncover fundamental mechanism(s) governing higher-order folding of eukaryotic chromatin, and provide new native-like chromatin models, imaging tools, and datasets, with which specific structural motifs and nucleosome interactions mediating native chromatin accessibility for transcription and other DNA-dependent functions can be readily visualized in 3D and reproduced in chromatin computational models. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.