title: Identification of the Post-translational Modifications Present in Centromeric Chromatin.
abstract: The centromere is the locus on the chromosome that acts as the essential connection point between the chromosome and the mitotic spindle. A histone H3 variant, CENP-A, defines the location of the centromere, but centromeric chromatin consists of a mixture of both CENP-A-containing and H3-containing nucleosomes. We report a surprisingly uniform pattern of primarily monomethylation on lysine 20 of histone H4 present in short polynucleosomes mixtures of CENP-A and H3 nucleosomes isolated from functional centromeres. Canonical H3 is not a component of CENP-A-containing nucleosomes at centromeres, so the H3 we copurify from these preparations comes exclusively from adjacent nucleosomes. We find that CENP-A-proximal H3 nucleosomes are not uniformly modified but contain a complex set of PTMs. Dually modified K9me2-K27me2 H3 nucleosomes are observed at the centromere. Side-chain acetylation of both histone H3 and histone H4 is low at the centromere. Prior to assembly at centromeres, newly expressed CENP-A is sequestered for a large portion of the cell cycle (late S-phase, G2, and most of mitosis) in a complex that contains its partner, H4, and its chaperone, HJURP. In contrast to chromatin associated centromeric histone H4, we show that prenucleosomal CENP-A-associated histone H4 lacks K20 methylation and contains side-chain and α-amino acetylation. We show HJURP displays a complex set of serine phosphorylation that may potentially regulate the deposition of CENP-A. Taken together, our findings provide key information regarding some of the key components of functional centromeric chromatin.
authors.affiliation: From the ‡Department of Cell Biology, University of Virginia, Charlottesville, Virginia, 22908;
authors.forename: Aaron O
authors.initials: AO
authors.lastname: Bailey
authors.affiliation: §Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, 19104-6059;
authors.forename: Tanya
authors.initials: T
authors.lastname: Panchenko
authors.affiliation: ¶Department of Chemistry, University of Virginia, Charlottesville, Virginia, 22908;
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Shabanowitz
authors.affiliation: ¶Department of Chemistry, University of Virginia, Charlottesville, Virginia, 22908;
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: Lehman
authors.affiliation: ¶Department of Chemistry, University of Virginia, Charlottesville, Virginia, 22908;
authors.forename: Dina L
authors.initials: DL
authors.lastname: Bai
authors.affiliation: ¶Department of Chemistry, University of Virginia, Charlottesville, Virginia, 22908;
authors.forename: Donald F
authors.initials: DF
authors.lastname: Hunt
authors.affiliation: §Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, 19104-6059; dfoltz@northwestern.edu blackbe@mail.med.upe.
authors.forename: Ben E
authors.initials: BE
authors.lastname: Black
authors.affiliation: From the ‡Department of Cell Biology, University of Virginia, Charlottesville, Virginia, 22908; ‖Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia, 22908; **Department of Biochemistry and Molecular Genetics, Northwestern University, Feinberg School of Medicine, Chicago Illinois 60611 dfoltz@northwestern.edu blackbe@mail.med.upe.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Foltz
mesh_terms: D001324:Autoantigens / Q000378:metabolism*; D002453:Cell Cycle; D002503:Centromere / Q000378:metabolism*; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008745:Methylation; D009707:Nucleosomes / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012694:Serine / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D001324:Autoantigens; C000619700:CENPA protein, human; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; C524371:HJURP protein, human; D006657:Histones; D009707:Nucleosomes; D012694:Serine; D008239:Lysine