Centromere structure, assembly and function

The centromere and the kinetochore that assembles upon it are crucial for proper chromosome segregation during mitosis or meiosis. The microtubules forming the mitotic spindle attach to the kinetochore and facilitate the movement of the sister chromatids towards the spindle poles. Defects in this chromosome segregation lead to the presence of extra or missing chromosomes in daughter cells (aneuploidy), which causes birth defects and is related to cancer progression. The origin of this segregation defects is thought to be found in the structure of the centromere-kinetochore complex. In order to understand the function and assembly of this complex we will utilize the fission yeast Schizosaccharomyces pombe as a model system. Those centromeres have many features in common with metazoan ones as they are very large (40-120 kb) and they are composed of repetitive elements, the outer repeat regions (otr) that are packaged in heterochromatin. These heterochromatic regions flank the central core domain (cc/cnt and innermost repeats; imr), where the histone H3 is replaced by its variant CENP-A Cnp1 what is maintained epigenetically. We propose that the 3 dimensional structure and the chromatin organization of the centromere is important for proper chromosome segregation. One possibility is that the symmetrical outer and inner repeats mediated an interaction between the left and right sides; indeed preliminary 3C data support such an interaction. Our specific aims are: 1. Measure the distance and interaction between the outer repeats (otr) of the centromeres and determine the proteins these interactions are dependent on. 2. Determine if these interactions are correlated with distinct cell cycle stages. 3. Analyze the distribution of the histone variant CENP-ACnp1 within the centromere at nucleosomal resolution. These aims will be fulfilled by applying the molecular biology techniques: 1. 3C (Chromosome conformation capture) 2. ChIP (Chromatin immuno precipitation) 3. Deep sequencing using SOLEXA or 454 systems 4. Microscopy (fluorescent-, confocal fluorescent- and structured illumination- microscopy) These techniques will be applied singly and in combinations. The main aim of this project is to find out what factors influence the normal architecture at fission yeast centromeres and how this contributes to proper kinetochore assembly and function.