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Mol Cell Proteomics. 2016 Mar;15(3):918-31. doi: 10.1074/mcp.M115.053710. Epub 2015 Dec 18.

Identification of the Post-translational Modifications Present in Centromeric Chromatin.

Author information

1
From the ‡Department of Cell Biology, University of Virginia, Charlottesville, Virginia, 22908;
2
§Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, 19104-6059;
3
¶Department of Chemistry, University of Virginia, Charlottesville, Virginia, 22908;
4
§Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, 19104-6059; dfoltz@northwestern.edu blackbe@mail.med.upe.
5
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.

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.

PMID:
26685127
PMCID:
PMC4813710
[Available on 2017-03-01]
DOI:
10.1074/mcp.M115.053710
[Indexed for MEDLINE]
Free PMC Article

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