Forschungsgruppe Diekmann(Zur Zeit nur in Englisch verfügbar)
The centromere/kinetochore complex
Mechanisms of cellular senescence
We pulled-down centromeric nucleosomes containing tagged CENP-A and analysed their structure by EM and AFM. In collaboration with the Dalal lab (NIH, USA) we found that human CENP-A containing nucleosomes can adopt a hemisome structure consisting of only one copy of histones CENP-A, H4, H2A and H2B each (Dimitriadis et al., 2010). In a following study we found that CENP-A containing nucleosomal structures form octamers only during S-phase, but aggregate to tetramers during the rest of the cell cycle (Bui et al., 2012).
We detected that CENP-N assembly into the CCAN constantly changes over the cell cycle (Hellwig et al., 2011). Next we studied the properties of CENP-T and CENP-W (collaboration with K. Sullivan, Galway, Ireland). We analysed the cell cycle regulation, timing of assembly, generational persistence, and requirement for function of CENP-T and CENP-W in the cell cycle in human cells. The CENP-T/W complex assembles through a dynamic exchange mechanism in late S-phase and G2, is required for mitosis in each cell cycle and does not persist across cell generations (Prendergast et al., 2011), properties reciprocal to those of CENP-A. We thus proposed that the CENP-A and H3-CENP-T/W nucleosome components of the centromere are specialized for centromeric and kinetochore activities, respectively. We speculate that these two separate functions allow the cell to switch between chromatin configurations that reciprocally support (i) the replication of the centromere and (ii) its conversion to a mitotic state on post-replicative chromatin. We continued this work by analyzing the behavior of CENP-S and CENP-X which associate with CENP-T and CENP-W at the kinetochore (Dornblut et al., in revision). Additionally, we studied the CENP-Q/U/P/O/R sub-complex of the inner kinetochore (Eskat et al., 2012). We found that the proteins of this sub-complex bind to the kinetochore as single units mostly in S-phase. Once bound to the kinetochore, two of these proteins (CENP-Q and -U) multimerise suggesting a maturatioin step of centromeric chromatin before mitosis.
Within the BMBF-funded JenAge (Jena Centre for Systems Biology of Ageing) research project we analysed the transcriptome(s) of human primary fibroblasts during their ageing in vitro. Massive parallel mRNA sequencing was performed for two human primary fibroblast cell types at 5 different time points of their lifespan. Ageing-associated differentially expressed genes (aaDEGs) are currently being compared to aaDEGs from other model organisms of the JenAge consortium (H. sapiens, M. musculus, D. rerio, N. furzeri, C. elegans). We hope to identify commonly regulated gene expression networks which might reveal new pathways of ageing.
In parallel we finished analyses of anti-ageing effects of anti-oxidative compounds on primary human blood cells (Marthandan et al., 2011).
We developed a systems-biology cellular senescence model (collaboration with S. Schuster, FSU Jena) establishing a new mathematical model which quantitatively describes the ageing process of human fibroblast during replicative senescence (Schäuble et al., 2012). Applying this model, we quantitatively described the growth curve of several cell lines and their transition into senescence due to different origins of stress. The model makes specific predictions which we are testing experimentally.
Eskat A, Deng W, Hofmeister A, Rudolphi S, Emmerth S, Hellwig D, Ulbricht T, Döring V, Bancroft JM, McAinsh AD, Cardoso MC, Meraldi P, Hoischen C, Leonhardt H, Diekmann S (2012) Step-wise assembly, maturation and dynamic behavior of the human CENP-P/O/R/Q/U kinetochore sub-complex. PLoS One, 7, e44717 [PLos One]
Bui M, Dimitriadis E K, Hoischen C, An E, Quénet D, Giebe S, Nita-Lazar A, Diekmann S, Dalal Y (2012) Cell-cycle-dependent structural transitions in the human CENP-A nucleosome in vivo. Cell, 150, 317-326 [PubMed]
Hellwig D, Emmerth S, Ulbricht T, Döring V, Hoischen C, Martin R, Samora CP, McAinsh AD, Carroll CW, Straight AF, Meraldi P, Diekmann S (2011) Dynamics of CENP-N kinetochore binding during the cell cycle. J Cell Sci, 124, 3871-3883 [PubMed]
- Prendergast L, van Vuuren C, Kaczmarczyk A, Doering V, Hellwig D, Quinn N, Hoischen C, Diekmann S, Sullivan KF (2011) Premitotic assembly of human CENPs -T and -W switches centromeric chromatin to a mitotic state. PLoS Biol, 9, e1001082 [PubMed]
Dimitriadis EK, Weber C, Gill RK, Diekmann S, Dalal Y (2010) Tetrameric organization of vertebrate centromeric nucleosomes. Proc Natl Acad Sci U S A, 107, 20317-20322 [PubMed]