Centromere identity, strength, and regulation

Project Summary/Abstract Chromosomes are the functional unit of inheritance and must segregate with high fidelity every time a cell divides. For eukaryotes, a common mechanism is employed, where sister chromatids are physically attached to each other and bidirectionally oriented towards poles of the microtubule-based spindle that physically move complete sets of chromosomes to the daughter cells. This bidirectionally-oriented attachment is mediated by a proteinaceous structure called the kinetochore, which forms at the microtubule/chromosome interface during mitosis. The site of kinetochore formation is defined by the centromere. Without functional centromeres, chromosomes are mis-segregated at cell division, leading to a genetic catastrophe—aneuploidy—in the daughter cells. Over the last 15+ years, the centromere has been the major focus of our attention, and altogether we have contributed to elucidating the molecular basis for centromere identity, the epigenetic pathway that propagates centromeric chromatin in perpetuity, the relationship between epigenetic and genetic information in driving centromere evolution in eukaryotes, and key steps in the quality control pathway that ensures proper chromosome segregation at cell division. Over the last ~five years, some of our most important contributions emerging from MIRA-supported projects have come through harnessing this knowledge to develop new and powerful types of human artificial chromosomes, and using standard structural biology approaches as well as solution biophysical approaches to advance our understanding of the proteins that drive chromosome inheritance at cell division. A major focus for our proposed studies will be to use cryo-electron tomography to visualize the centromere in its natural chromosomal context. In the first research area, we propose to visualize the centromere during mitosis. This work will fill a major gap in our understanding of how centromeric chromatin demarcates the site of kinetochore formation. In the second research area, we will advance our current understanding of how centromeric chromatin propagates centromere identity by visualizing this part of the chromosome at key steps through the cell cycle. Altogether, the two research areas will provide answers to vital questions related to how chromosomes are faithfully inherited through cell division.