High-resolution mapping of centromere factors on stretched chromatin fibers
The centromere is a unique chromatin domain important for proper segregation of chromosomes during mitosis. In most organisms, the position of the centromere is determined epigenetically by the centromere-specific incorporation of the H3-variant CENP-A. However, a thorough understanding of centromere organisation is still missing. We have developed a novel microscopy-based approach using stretched chromatin fibers that allows the mapping not only of centromeric nucleosomes but also of the associated centromere factors in high resolution. For this project we take advantage of protein fusions to biotinylation enzyme APEX and TurboID to mark the underlying chromatin fibers with biotin and map their “footprint” relative position to the CENP-A nucleosome. We are planning to map a variety of different endogenous centromere factors whose precise location on the centromere chromatin is currently unknown. This involves CrispR-Cas9 genome editing for tagging of endogenous proteins such as the chromosomal passenger complex CPC, members of the transcription machinery and CENP-A chaperones and licensing factors. In parallel we also plan to use super-resolution microscopy to validate the 2D mapping results in 3D. Overall, the aim of this Ph.D. project is to gain a better understanding of the 2D and 3D organisation of the centromere. http://www.wcb.ed.ac.uk/research/patrick-heun
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1. McKinley and Cheeseman IM, The molecular basis for centromere identity and function. Nat Rev Mol Cell Biol. 2016, 17:16-29 Review
2. Bobkov G, Gilbert N and Heun P, Centromere transcription allows CENP-A to transit from chromatin association to stable incorporation. J Cell Biol 2018, 217: 1957-1972
3. Kyriacou E and Heun P: High-resolution mapping of centromeric protein binding using APEX-chromatin fibers, in revision (available upon request)
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