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Patrick Heun


Eduard Anselm, Irene Andrade-Zapata, Evelyne Barrey, Thomas van Emden,Eftychia Kyriacou, Vasilik Lazou, Virginie Roure, Emilija Ruksenaite, Georg Schade, Judith Zich

Epigenetic regulation and nuclear organization ofchromosomes

Our lab is interested in the epigenetic inheritance and nuclear organization of centromeres. The DNA-sequence independent epigenetic transmission of centromere identity through many cell generations is highly relevant for proper genome regulation and when perturbed can lead to genome instability and cellular malfunction. We use the fruit fly Drosophila melanogaster and human cells as a model organism to address the following questions:

How is the epigenetic identity of centromeres regulated?

Centromeres are found at the primary constriction of chromosomes in mitosis where they remain connected before cell division. This structure is essential for an equivalent chromosomes distribution to the daughter cells.

The centromere specific histone H3-variant CENP-AcenH3 is essential for kinetochore formation and centromere function. We have recently established a biosynthetic approach to target dCENP-AcenH3 to specific non-centromeric sequences such as the Lac Operator and follow the formation of functional neocentromeres. Using this approach we were able to directly demonstrate that a dCENP-AcenH3 -LacI fusion is sufficient to induce centromere formation as well as self-propagation and inheritance of the epigenetic centromere mark (Figure 1a and b)(Mendiburo et al., 2011). Using the LacO/LacI tethering system, we are interested in dissecting the function of dCENP-AcenH3 in Drosophila (also known as CID)
and human cells for centromere targeting, kinetochore formation and self-propagation (Logsdon et al., 2015). This approach has been successfully introduced into a heterologous system comprised of human centromere factors expressed in Drosophila Schneider S2 cells. We are currently trying to reconstitute the loading and self-propagation of human CENP-A at an ectopic locus in the Drosophila genome (Figure 1c).

How are centromeres organized in the nucleus?
The compartmentalization of the eukaryotic cell helps regulating proper genome function and gene expression. In D. melanogaster centromeres are not randomly positioned in the nucleus, but instead cluster together and are tethered to periphery of the nucleolus (Figure 2a). We could recently show that the protein NLP (Nucleoplasmin Like Protein) plays a major role for this centromere-specific positioning (Padeken et al., 2013). Elimination of NLP leads to declustering of centromeres, dissociation from the nucleolus, loss of silencing of repetitive DNA elements, DNA damage and mitotic defects. We are investigating how NLP and its binding factors contribute to higher-order genome organization and maintenance of genome integrity (Figure 2b). To study the interactions within this network of proteins at a molecular level, we are currently using recombinant wildtype and mutated factors involved in this network for an in vitro reconstitution of centromeric chromatin.

Selected publications:

Logsdon, G.A., Barrey, E.J., Bassett, E.A., DeNizio, J.E., Guo, L.Y., Panchenko, T., Dawicki-McKenna, J.M., Heun, P., and Black, B.E. (2015). Both tails and the centromere targeting domain of CENP-A are required for centromere establishment. J Cell Biol 208, 521-531.

Mendiburo, M.J., Padeken, J., Fulop, S., Schepers, A., and Heun, P. (2011). Drosophila CENH3 is sufficient for centromere formation. Science 334, 686-690.

Padeken, J., Mendiburo, M.J., Chlamydas, S., Schwarz, H.J., Kremmer, E., and Heun, P. (2013). The nucleoplasmin homolog NLP mediates centromere
clustering and anchoring to the nucleolus. Molecular cell 50, 236-249.

1. CENP-A-LacI is efficiently targeted to a LacO plasmid, where it induces de novo centromere formation.

These neocentromeres recruit other centromere factors like dCENP-C (a) and associate with spindle microtubules(b). Using this biosynthetic approach we aim to reconstitute a human centromere in evolutionary distant Drosophila cells.
2. Centromere clustering is mediated by a network of proteins around NLP. a) Centromeres form clusters at the periphery of the nucleolus in Drosophila Schneider S2 cells. b) NLP (nucleoplasmin) and dCTCF contribute to centromere clustering, while dNucleolin, serves as a nucleolus anchor. (Padeken et al., Mol. Cell 2013)