Epigenetic inheritance and organisation of centromeres
Our lab is interested in the epigenetic inheritance and organization of centromeres. The DNA sequence independent 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 previously 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 (Mendiburo et al., 2011). Using the LacO/LacI tethering system, we are interested in dissecting the function of CENP-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 recently 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 1).
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 centromereassociated transcripts temporally coincides with dCENP-A loading in mitosis to G1. Using fast acting transcriptional inhibitors in combination with a newly developed dCENP-A loading system, we find that centromeric transcription is required for dCENP-A loading by stabilizing new dCENP-A. In contrast, loss of parental dCENP-A in Spt6 depleted cells underlines its importance in dCENP-A maintenance. Thus, co-operated actions of transcription and Spt6mediated nucleosome recycling are essential for the inheritance of dCENP-A nucleosomes at centromeres (Figure 2)
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.