Physical organizing principles and technologies for genome organization

Abstract

Chromatin, a nucleic acid and protein-based polymer, is dynamically organized into spatially andfunctionally distinct compartments. Given the densely crowded, polymeric nature of chromatin, the 3D architecture of the genome may be organized by phase transitions, including compartmentalization by liquid-liquid phase separation (LLPS). Telomeres are a particularly interesting genomic compartment comprised of kilobases of repetitive DNA and a six-protein complex named shelterin that work together as a unit to protect the single-stranded ends of chromosomes from aberrant recognition by DNA damage repair proteins. In this thesis, we tested the hypothesis that telomere organization is driven by LLPS and characterized the biophysical phase behavior of telomeres through quantitative imaging methods and in vitro experiments. However, because telomeres rarely encounter one another in living cells due to their constrained subdiffusive motion, it is difficult to test in cells whether two telomeres can coalesce, a key aspect of the liquid condensate model. To overcome this challenge, we developed a novel optogenetic approach, VECTOR, that brings two telomeres into close proximity, using capillary forces and found that telomeres readily coalesce into and remain as a single liquid-like droplet. These findings are consistent with in vitro experiments, which reveal that shelterin complex proteins readily phase separate together with telomeric DNA. Further studies will investigate the downstream functional consequences of these telomere merger events that lead to signs of genome instability, i.e. the formation of chromatin bridges, micronuclei, nucleolar defects with perturbed shelterin stoichiometry that has major implications in cancer, disease, and accelerated aging. This thesis work also includes efforts towards developing a new emerging model organism to study aging, the Euprymna berryi squid. We present an optimized protocol to isolate primary cells from squids of any life stage. Future work will investigate changes in nuclear organization with increasing age and will link molecular determinants of aging to physical signs of deterioration at the organismal level. This thesis uncovers physical organizing principles of genomic and nuclear compartmentalization and develops new technologies and systems to probe and measure properties and dynamics of the 3D nuclear space in living cells.