Structure and Function of a Eukaryotic Centromere

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Project Summary Goals: The centromere serves as the binding site for the kinetochore and is essential for the faithful segregation of chromosomes throughout cell division. The point centromere in yeast is encoded by a ~115 bp specific DNA sequence, whereas regional centromeres span 6-10 kbp in fission yeast to 5-10 Mbp in human. Despite the apparent diversity in centromere organization, the distance between sister kinetochores in metaphase ranges from 800 nm to 1,000 nm in yeast, worms, flies, flower moths, plants, horses and human. Understanding the physical structure of centromere chromatin (pericentromere in yeast, defined as the chromatin between sister kinetochores) will provide fundamental insights how centromere DNA is organized into a stiff spring that resists microtubule pulling forces during mitosis. Approach: Our laboratory develops computational tools to interrogate the structure and dynamics of hundreds of kilobase pairs of pericentromeric DNA. Together with experimentally obtained images of fluorescent probes of pericentromeric structure (e.g. pericentromere DNA, cohesin, condensin) we make quantitative comparisons between simulations and experimental results through transformation of in silico models into microscope images (model convolution). We will test the proposal that the mechanism for building tension between sister kinetochores is a chromatin bottlebrush organized by the loop-extruding proteins condensin and cohesin. The bottlebrush provides a biophysical mechanism that transforms pericentromeric chromatin into a spring due to the steric repulsion between radial loops. The bottlebrush as an organizing principle for chromosome organization has emerged from multiple approaches in the field. We will leverage the powerful features of chromosome engineering in yeast to explore the consequences of reducing the number of centromeres, and exploit synthetic bottlebrushes and statistical physics of polymer models to reveal basic principles linking bottlebrush structure to the functional readout of force/tension. Innovation: We will combine our experience in chromosome engineering and advanced bioimaging in yeast with the expertise of collaborators in statistical physics and applied math (Forest UNC-CH) and synthetic bio-inspired materials (Freeman UNC-CH). Testing our hypotheses will elucidate important information about the organization and function of centromeres, potentially providing a paradigm shifting foundation for the remarkable conservation of distance between sister kinetochores throughout phylogeny.