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Identification of novel Drosophila centromere-associated proteins

Authors

  • Teresa K. Barth,

    1. Munich Center of Integrated Protein Science, Adolf-Butenandt Institute, Ludwig Maximilians University of Munich, Munich, Germany
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    • These authors contributed equally to this work.

  • Georg O. M. Schade,

    1. Max-Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany
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    • These authors contributed equally to this work.

  • Andreas Schmidt,

    1. Munich Center of Integrated Protein Science, Adolf-Butenandt Institute, Ludwig Maximilians University of Munich, Munich, Germany
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  • Irene Vetter,

    1. Munich Center of Integrated Protein Science, Adolf-Butenandt Institute, Ludwig Maximilians University of Munich, Munich, Germany
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  • Marc Wirth,

    1. Munich Center of Integrated Protein Science, Adolf-Butenandt Institute, Ludwig Maximilians University of Munich, Munich, Germany
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  • Patrick Heun,

    Corresponding author
    1. Max-Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany
    • Correspondence: Dr. Axel Imhof, Munich Centre of Integrated Protein Science, Adolf-Butenandt Institute, Ludwig Maximilians University of Munich, Schillerstr. 44, 80336 Munich, Germany

      E-mail: Imhof@lmu.de

      Fax: +49-89-218075440

      Additional corresponding authors: Dr. Andreas W. Thomae,

      E-mail: andreas.thomae@med.uni-muenchen.de

      Dr. Patrick Heun, E-mail: heun@ie-freiburg.mpg.de

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  • Andreas W. Thomae,

    Corresponding author
    1. Munich Center of Integrated Protein Science, Adolf-Butenandt Institute, Ludwig Maximilians University of Munich, Munich, Germany
    • Correspondence: Dr. Axel Imhof, Munich Centre of Integrated Protein Science, Adolf-Butenandt Institute, Ludwig Maximilians University of Munich, Schillerstr. 44, 80336 Munich, Germany

      E-mail: Imhof@lmu.de

      Fax: +49-89-218075440

      Additional corresponding authors: Dr. Andreas W. Thomae,

      E-mail: andreas.thomae@med.uni-muenchen.de

      Dr. Patrick Heun, E-mail: heun@ie-freiburg.mpg.de

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  • Axel Imhof

    Corresponding author
    1. Munich Center of Integrated Protein Science, Adolf-Butenandt Institute, Ludwig Maximilians University of Munich, Munich, Germany
    • Correspondence: Dr. Axel Imhof, Munich Centre of Integrated Protein Science, Adolf-Butenandt Institute, Ludwig Maximilians University of Munich, Schillerstr. 44, 80336 Munich, Germany

      E-mail: Imhof@lmu.de

      Fax: +49-89-218075440

      Additional corresponding authors: Dr. Andreas W. Thomae,

      E-mail: andreas.thomae@med.uni-muenchen.de

      Dr. Patrick Heun, E-mail: heun@ie-freiburg.mpg.de

    Search for more papers by this author

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-ACID 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).

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