Investigating the epigenetic inheritance of human centromere identity in a heterologous system

Interested individuals must follow Steps 1, 2 and 3 at this link on how to apply
http://www.ed.ac.uk/biology/prospective-students/postgraduate/pgr/how-to-apply

Centromeres are specialized genomic regions required for proper chromosome segregation and critical to maintain genome stability. In most eukaryotes centromeres are identified epigenetically rather than through DNA sequence. The centromere-specific histone H3 variant CENP-A (also known a cenH3) plays a key role in centromere determination and is responsible for the targeting of many downstream centromere factors.
To systematically dissect the process of human CENP-A deposition and maintenance, we developed a novel in vivo reconstitution method by introducing human centromere genes into the evolutionary distant Drosophila Schneider S2 cell line. Using the LacI/LacO tethering system, we have targeted various LacI fusions of human centromere proteins to an array of LacO repeats either on an extra-chromosomal plasmid or integrated into the arm of a Drosophila chromosome and investigated the recruitment of specific human factors.
We have been able to reproduce the contacts between CENP-A and its known direct binding partners CENP-C and CENP-N and identified the minimal regions of CENP-A involved in each of these interactions. Furthermore, we demonstrated that every step in the current model of the CENP-A propagation pathway; namely HJURP-mediated deposition of CENP-A, association of CENP-C with CENP-A, M18BP1 recruitment to CENP-C, Mis18 complex assembly, and HJURP association with the Mis18 complex; can be reconstituted in the absence of any other human factor. We are now interested in further investigating the factors involved in inheritance of CENP-A, including centromeric DNA sequences, DNA binding factors, and other centromere components. We will particularly focus on the abilitiy of CENP-A to spread along chromatin fibers during its replenishment every cell cycle in late M-phase/early G1. However, it is largely unclear what factors promote or impede propagation of this epigenetic mark to achieve the correct amount of CENP-A at the centromere. Using human tissue culture as a model, we will use targeted loading of CENP-A on synthetic arrays of centromeric alpha-satellite DNA containing short stretches of unique DNA sequences to investigate CENP-A self-propagation.
To dissect the molecular mechanism of this process, we plan to manipulate the centromeric DNA sequence, the chromatin context and the CENP-A loading machinery. Eventually, the goal is to create a system that fully recapitulates the maintenance of human centromere identity in a heterologous system. Techniques relevant for this study include general molecular biology, microscopy, chromatin immunoprecipitation, quantitative PCR, genome editing and synthetic biology. The knowledge generated in this project will further our understanding of how epigenetic information is inherited using centromeres as a paradigm.

The student will be based in the Heun lab with a team of molecular biologists. We have weekly group meetings alternating journal clubs and progress report. Students are given the opportunity to visit at least on international conference a year.