Epigenetic inheritance and organisation of centromeres
Our lab is interested in the epigenetic inheritance and organization of centromeres. Epigenetic transmission of centromere identity through many cell generations is required 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 propagated?
Centromeres are found at the primary constriction of chromosomes in mitosis where they remain connected before cell division. This structure is essential for an equal distribution of chromosomes 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); Using the LacO/LacI tethering system, we are interested in dissecting the function of centromere factors in Drosophila and human cells for propagation of CENP-AcenH3. This approach has been successfully introduced into a heterologous system comprised of human centromere factors expressed in Drosophila Schneider S2 cells (Logsdon et al., 2015). We are currently trying to reconstitute the loading and self-propagation of either human or Drosophila CENP-A at the ectopic LacO locus (Figure 1c).
What is the role of transcription at the centromere?
Loading of CENP-A at the centromere occurs outside of S-phase and requires the removal of H3 placeholder" nucleosomes. Transcription at centromeres has been linked to the deposition of new CENP-A, although the molecular mechanism is not understood. Interestingly, transcription is able to evict nucleosomes, which can be recycled by the histone chaperone Spt6. We find that centromeric localization of Spt6, RNAPII and centromere-associated transcripts temporally coincides with dCENP-A loading from mitosis to G1. Using fast acting transcriptional inhibitors in combination with a newly developed CENP-A loading system, we demonstrate that centromeric transcription is required for dCENP-A loading by evicting placeholder nucleosomes and promoting dCENP-A transition from chromatin association to nucleosome incorporation. In contrast, loss of parental CENP-A in Spt6 depleted cells underlines the importance of CENP-A maintenance during transcription. Thus, co-operated actions of transcription and Spt6-mediated nucleosome recycling are essential for the stability of the epigenetic centromere mark dCENP-A.
Barth, T.K., Schade, G.O., Schmidt, A., Vetter, I., Wirth, M., Heun, P.*, Thomae, A.W.*, and Imhof, A*. (2014). Identification of novel Drosophila centromere-associated proteins. Proteomics 14, 2167-2178 (*corresponding authors).
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.
Barrey, EJ and Heun, P. (2017). Artificial Chromosomes and Strategies to Initiate Epigenetic Centromere Establishment. Prog Mol Subcell Biol. 56, 193-212