Nuclear organization of centromeres
Final Report Abstract
Centromeres are specialised genomic regions that are essential for chromosome segregation. Over the past 40 years, experiments have shown that in many organisms and different cell types, the centromeres are clustered in the nucleus, often in proximity to the nucleolus. However, how they are positioned in the nucleus is still little understood. Using Drosophila Schneider S2 cells and larval hemocytes, we identified a member of the Nucleophosmin/Nucleoplasmin protein family in Drosophila melanogaster, the Nucleoplasmin like protein (NLP), as an important regulator of subnuclear centromere organization during interphase. NLP binds specifically to the centromere region of the chromosomes and depletion of NLP leads to declustering and repositioning of centromeres. During the duration of our DFG research grant, we could show that NLP promotes clustering of centromeres by interacting with Cenp-ACID, the insulator protein CTCF and the nucleolin homologue Modulo. We observed that centromere clusters in interphase co-localize with NLP, CTCF and the periphery of nucleolar Modulo staining. Co-IP experiments confirmed the interaction between the three proteins and Cenp-ACID. Knockdown experiments and fly mutants showed that loss of any of the three proteins leads to a disruption of centromere clustering and detachment of the centromeres from the periphery of the nucleolus. We found clustering to be an intrinsic property of centromeric chromatin and not dependent on a specific DNA sequence, as plasmids that do not contain any centromeric DNA cluster upon induction of neocentromeres. While intact heterochromatin is dispensable for centromere clustering, the declustering of centromeres resulted in a disruption of the spatial organization of heterochromatin. Concomitantly this also led to a partial loss of silencing, suggesting that cells tether centromeres to the nucleolus to help heterochromatin mediated transcriptional repression. Moreover, declustering of centromeres further leads to increase in DNA repair foci and defects during chromosome segregation, thus affecting general genome stability (Padeken et al., 2013). We are now investigating the molecular details of NLP's role at the centromere. To this end we have created mutated versions of NLP that cannot oligomerise anymore into a reported pentameric complex (Namboodiri et al., 2003). Interestingly, these NLP variants also lost the ability to localize to the centromere. We are currently investigating their ability to bind reconstituted canonical and centromeric nucleosomes. In summary our work identifies NLP as a new nuclear organizer, which mediates chromatin interactions in trans. We would like to propose that clustering and tethering of specialized chromatin domains (e.g. centromeres, telomeres) serves as a mechanism to spatially organize associated heterochromatin domains, allowing proper silencing of transposons and protection against recombination of repetitive DNA elements.
Publications
- (2013), The Nucleoplasmin homologue NLP mediates centromere clustering and anchoring to the nucleolus, Molecular Cell, 50, 236-249
Padeken, J., Mendiburo, M.J., Chlamydas, S., Schwarz, H.J., Kremmer, E., Heun, P.
(See online at https://doi.org/10.1016/j.molcel.2013.03.002) - Centromeres in Nuclear organization, Cell Cycle 2013, 12:3455–3456
Padeken, J. and Heun, P.
- Nucleolus and Nuclear Periphery: Velcro for heterochromatin, Curr Opin Cell Biol. 2014, 28, 54-60
Padeken, J. and Heun, P.
(See online at https://doi.org/10.1016/j.ceb.2014.03.001)
