Identification of novel Drosophila centromere‐associated proteins

Abstract

Centromeres are chromosomal regions crucial for correct chromosome segregation during mitosis and meiosis. They are epigenetically defined by centromeric proteins such as the centromere-specific histone H3-variant centromere protein A (CENP-A). In humans, 16 additional proteins have been described to be constitutively associated with centromeres throughout the cell cycle, known as the constitutive centromere-associated network (CCAN). In contrast, only one additional constitutive centromeric protein is known in Drosophila melanogaster (D.mel), the conserved CCAN member CENP-C. To gain further insights into D.mel centromere composition and biology, we analyzed affinity-purified chromatin prepared from D.mel cell lines expressing green fluorescent protein tagged histone three variants by MS. In addition to already-known centromeric proteins, we identified novel factors that were repeatedly enriched in affinity purification-MS experiments. We analyzed the cellular localization of selected candidates by immunocytochemistry and confirmed localization to the centromere and other genomic regions for ten factors. Furthermore, RNA interference mediated depletion of CG2051, CG14480, and hyperplastic discs, three of our strongest candidates, leads to elevated mitotic defects. Knockdowns of these candidates neither impair the localization of several known kinetochore proteins nor CENP-A(CID) loading, suggesting their involvement in alternative pathways that contribute to proper centromere function. In summary, we provide a comprehensive analysis of the proteomic composition of Drosophila centromeres. All MS data have been deposited in the ProteomeXchange with identifier PXD000758 (http://proteomecentral.proteomexchange.org/dataset/PXD000758).

Spt6 is a maintenance factor for centromeric CENP-A

“…Intriguingly, the specific loss of previously deposited dCENP-A during the loading process of new dCENP-A in Spt6-depleted cells suggests that Spt6 binds and reincorporates not only H3 (Kato et al 2013), but also dCENP-A/H4-tetramers. An association of Spt6 with dCENP-A might be expected due to our previously published mass spectroscopy of dCENP-A interactors (Barth et al 2014). Although the exact binding interface is not known, binding of basic histones has been demonstrated for the unstructured highly acidic N-terminus of Spt6 in S. cerevisiae (Fig.…”

“…While a role for FACT at the centromere and its importance for CENP-A deposition has already been demonstrated in numerous organism (Foltz et al 2006;Izuta et al 2006;Okada et al 2009;Chen et al 2015;Choi et al 2012;Prendergast et al 2016) , little is known about a centromeric function of Spt6. In budding yeast and flies, Spt6 was detected in a CENP-A pull-down and mass-spectrometry experiment (Barth et al 2014;Ranjitkar et al 2010). Mutants of Spt6 in budding yeast show segregation defects for a chromosome fragment (Basrai et al 1996), whereas mutants in S. pombe exhibit CENP-A misincorporation genome-wide (Choi et al 2012).…”

Structure of the MIS12 Complex and Molecular Basis of Its Interaction with CENP-C at Human Kinetochores

“…Besides DSN1 (as discussed in Results), all CCAN subunits, except CENP-C, seem to have been lost from the genomes of Drosophila melanogaster and of a few other organisms (Barth et al., 2014, Drinnenberg et al., 2014, Meraldi et al., 2006, Przewloka et al., 2007, Westermann and Schleiffer, 2013). As in humans and yeast, the interaction of MIS12C with CENP-C in Drosophila engages the N-terminal region of CENP-C (Hornung et al., 2014, Liu et al., 2016, Przewloka et al., 2011, Richter et al., 2016, Screpanti et al., 2011).…”

Evolutionary Turnover of Kinetochore Proteins: A Ship of Theseus?

“…However, analysis of kinetochore composition in other eukaryotes revealed an unexpected degree of variation. For instance, the D. melanogaster kinetochore, which has also been well dissected via biochemical and genetic means [7–10], is missing many of the inner kinetochore components found in vertebrate and budding yeast cells (Figure 1C, Key Figure). Similarly, the kinetochores of the holocentric Bombyx mori are distinct from those in vertebrate and budding yeast [11] (Figure 1D, Key Figure).…”