title: The CENP-T C-terminus is exclusively proximal to H3.1 and not to H3.2 or H3.3.
abstract: The kinetochore proteins assemble onto centromeric chromatin and regulate DNA segregation during cell division. The inner kinetochore proteins bind centromeres while most outer kinetochore proteins assemble at centromeres during mitosis, connecting the complex to microtubules. The centromere-kinetochore complex contains specific nucleosomes and nucleosomal particles. CENP-A replaces canonical H3 in centromeric nucleosomes, defining centromeric chromatin. Next to CENP-A, the CCAN multi-protein complex settles which contains CENP-T/W/S/X. These four proteins are described to form a nucleosomal particle at centromeres. We had found the CENP-T C-terminus and the CENP-S termini next to histone H3.1 but not to CENP-A, suggesting that the Constitutive Centromere-Associated Network (CCAN) bridges a CENP-A- and a H3-containing nucleosome. Here, we show by in vivo FRET that this proximity between CENP-T and H3 is specific for H3.1 but neither for the H3.1 mutants H3.1(C96A) and H3.1(C110A) nor for H3.2 or H3.3. We also found CENP-M next to H3.1 but not to these H3.1 mutants. Consistently, we detected CENP-M next to CENP-S. These data elucidate the local molecular neighborhood of CCAN proteins next to a H3.1-containing centromeric nucleosome. They also indicate an exclusive position of H3.1 clearly distinct from H3.2, thus documenting a local, and potentially also functional, difference between H3.1 and H3.2.
authors.affiliation: Molecular Biology, Fritz Lipmann Institute, Beutenbergstr. 11, D-07745 Jena, Germany. christian_abendroth@gmx.de.
authors.forename: Christian
authors.initials: C
authors.lastname: Abendroth
authors.affiliation: Molecular Biology, Fritz Lipmann Institute, Beutenbergstr. 11, D-07745 Jena, Germany. hofi.antje@googlemail.com.
authors.forename: Antje
authors.initials: A
authors.lastname: Hofmeister
authors.affiliation: Department of Molecular Biology, Center for Integrated Protein Science Munich (CIPSM), Adolf-Butenandt-Institute, Ludwig-Maximilians-Universität Munich, Schillerstr. 44, D-80336 Munich, Germany. sandra.hake@med.uni-muenchen.de.
authors.forename: Sandra B
authors.initials: SB
authors.lastname: Hake
authors.affiliation: Molecular Biology, Fritz Lipmann Institute, Beutenbergstr. 11, D-07745 Jena, Germany. pkamweru@fli-leibniz.de.
authors.forename: Paul K
authors.initials: PK
authors.lastname: Kamweru
authors.affiliation: Molecular Biology, Fritz Lipmann Institute, Beutenbergstr. 11, D-07745 Jena, Germany. elke.miess@med.uni-jena.de.
authors.forename: Elke
authors.initials: E
authors.lastname: Miess
authors.affiliation: Molecular Biology, Fritz Lipmann Institute, Beutenbergstr. 11, D-07745 Jena, Germany. cdornblut@fli-leibniz.de.
authors.forename: Carsten
authors.initials: C
authors.lastname: Dornblut
authors.affiliation: Molecular Biology, Fritz Lipmann Institute, Beutenbergstr. 11, D-07745 Jena, Germany. isabell.kueffner@uni-jena.de.
authors.forename: Isabell
authors.initials: I
authors.lastname: Küffner
authors.affiliation: Department of Biology II, Center for Integrated Protein Science, Ludwig-Maximilians-Universität Munich, Planegg-Martinsried, D-82152 Munich, Germany. dengwen001@gmail.com.
authors.forename: Wen
authors.initials: W
authors.lastname: Deng
authors.affiliation: Department of Biology II, Center for Integrated Protein Science, Ludwig-Maximilians-Universität Munich, Planegg-Martinsried, D-82152 Munich, Germany. h.leonhardt@lmu.de.
authors.forename: Heinrich
authors.initials: H
authors.lastname: Leonhardt
authors.affiliation: PicoQuant GmbH, Kekulestr. 7, D-12489 Berlin, Germany. sandra.orthaus@googlemail.com.
authors.forename: Sandra
authors.initials: S
authors.lastname: Orthaus
authors.affiliation: Molecular Biology, Fritz Lipmann Institute, Beutenbergstr. 11, D-07745 Jena, Germany. hoischen@fli-leibniz.de.
authors.forename: Christian
authors.initials: C
authors.lastname: Hoischen
authors.affiliation: Molecular Biology, Fritz Lipmann Institute, Beutenbergstr. 11, D-07745 Jena, Germany. diekmann@fli-leibniz.de.
authors.forename: Stephan
authors.initials: S
authors.lastname: Diekmann
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006657:Histones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018386:Kinetochores / Q000737:chemistry / Q000378:metabolism; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D016415:Sequence Alignment; D000097573:Red Fluorescent Protein
chemical_list: D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C534001:CENPT protein, human