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Mechanisms of CENP-A Assembly and Propagation at Fission Yeast Centromeres

Final Report Summary - S.P CENP-A (Mechanisms of CENP-A Assembly and Propagation at Fission Yeast Centromeres)

Stable maintenance of genome integrity is prerequisite for the unperturbed propagation of genetic information. Centromeres serve as the specialised sites on chromosomes where kinetochores that govern spindle microtubule attachment assemble, and ensure accurate chromosome segregation during cell division. CENP-A is an essential kinetochore protein that maintains centromere identity. Although it has been long known that CENP-A is an integral component of centromeric chromatin and is required for centromere function, the mechanisms that promote incorporation and propagation of CENP-A at centromeres have remained elusive. Through the research proposed, the researcher aimed to define the complex mechanisms involved in the assembly of CENP-A chromatin in fission yeast. In this endeavour, multidisciplinary approaches combining state-of-the-art proteomics and genomics techniques with biochemistry, cell and molecular biology methods were to be employed. Specifically, the researcher sought to use proteomic and genetic screens to identify novel factors that influence chromatin integrity at the centromere (objective 1). Next, the researcher proposed to characterise the novel factors found to affect CENP-A chromatin integrity through careful molecular analyses (objective 2). Finally, the researcher proposed a highly innovative approach that for the first time, would uncover the molecular basis for the ability of CENP-A to serve as an epigenetic mark that can determine centromere identity (objective 3).

During the course of the project, the researcher was able to successfully perform large-scale proteomic and genomic screens as proposed. Through the proteomic screens (collaboration with Juri Rappsilber), she was able to identify two previously uncharacterised proteins Eic1 and Eic2, both of which localise to centromeres. While Eic1 appears to directly influence CENP-A assembly in concert with other kinetochore proteins, Eic2 might serve independent functions. During the project duration, the researcher also attempted to perform biochemical analyses of Eic1 interactions (collaboration with Jeyaprakash Arulanandam), yielding some interesting insights. The researcher is currently preparing a manuscript describing her efforts directed at pinpointing the exact mechanisms that Eic1 and Eic2 employ to influence CENP-A assembly and function.

Genome-wide genetic screens led to the identification of a whole range of factors that affect CENP-A chromatin integrity to varying extents. The researcher decided to pursue the top 15 factors that affect CENP-A chromatin the most. With assistance from a Masters student (from LMU, Munich), she characterized these factors further through careful molecular genetic analyses, and narrowed down on the four most promising factors that influence CENP-A levels the most. Current experiments are focused on dissecting the mechanisms that these factors utilise to influence CENP-A chromatin assembly and maintenance.

Although the researcher’s Marie Curie IIF funding has run out, the researcher will continue to work on the project through independent funding that she was able to secure from EMBO (2013-2014). Her current and future experiments are expected to provide a detailed understanding of how the factors that she has identified regulate CENP-A chromatin, and thereby influence kinetochore integrity in concert with known kinetochore protein complexes.

Centromeres are essential for chromosome segregation in all eukaryotes. Defective chromosome segregation causes aneuploidy, which can contribute to cancer progression, and in meiosis can result in trisomic individuals with reduced quality of life. A complete understanding of the processes that lead to full centromere-kinetochore assembly are required in order to dissect, and ultimately intervene in, the mechanism of chromosome segregation.