Chromosome dynamics

How do chromosomes, the storage centers for our blueprints, achieve their remarkable stability, plasticity and their movements? The secret lies in their structure and their dynamic interactions with nuclear factors, which open or close pockets for accessibility, form flexible scaffolds or tether functional units. Furthermore, chromosomal functions are confined to specific domains, such as kinetochores, loops and core regions and hotspots of recombination. A detailed biochemical and cell biological analysis of these chromosomal domains and their interplay is necessary to understand how genomes segregate. We propose to apply recently developed technologies to overcome current limitations in the field. Limitations are the incomplete knowledge of the identity of important proteins, the poor characterization of their interaction with chromosomes, incomplete understanding of chromosome conformation in vivo and the availability of only few in vitro systems to characterize complex processes involving chromosomes. To overcome these limitations we will 1) comprehensively analyze protein-protein and protein-DNA interactions by using tandem affinity purification, followed by mass spectrometry of protein complexes to identify novel interactions. For the purification we will chose meiotic proteins based on their functional importance, as well as candidates resulting from our ongoing screens; 2) we will analyze Protein-DNA interactions by chromatin immunoprecipitation followed by hybridization to high-resolution tiling arrays; 3) we will perform chromosome conformation capture techniques and live cell imaging using fluorescently marked chromosome sections to establish DNA-DNA proximities and to reconstruct in vivo chromosome structures; and 4) above all, we will conduct our efforts within a wide range of model organisms, covering fungi, animals, plants and a ciliate to put the discovered mechanisms into an evolutionary perspective. The individual groups aim to study various aspects of chromosome dynamics. The regulation of kinetochore - microtubule attachment and the biochemistry of cohesins, both key aspects of segregation, will be studied in meiosis and in mitosis. Chromosome pairing and recombination ensure correct chromosome segregation via chiasma formation specifically in meiosis I. DSB independent pairing will be studied in C. elegans, where we have discovered a mutant exhibiting age dependent segregation defects. Recombination hotspots will be mapped in C. elegans and A. thaliana. The ciliate T. thermophila allows asking whether DSB formation is an evolutionary conserved trait at all, while yeast mini-chromosomes will allow to define the structural requirements for DSB formation. We emphasize that meiotic chromosome missegregation is a leading cause of miscarriages (Down`s syndrome) and most cancer cells are associated with aberrant chromosome number suggesting that basic knowledge on segregation mechanisms is required to understand the etiology of these problems.