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.
Highlights
From the ‡Department of Cell Biology, University of Virginia, Charlottesville, Virginia, 22908; §Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, 19104-6059; ¶Department of Chemistry, University of Virginia, Charlottesville, Virginia, 22908; ʈDepartment 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
Despite the essential presence of the centromere protein-A (CENP-A) nucleosome for centromere formation and maintenance, the centromere-specific nucleosomes are present within a context of chromatin containing post-translational modifications (PTMs) of histones that are thought to be important for centromere function, as outlined below
Nucleosomal CENP-A-bound H3.1 in Asynchronously Cycling Cells—Affinity purification of localization and affinity purification (LAP)-tagged CENP-A from chromatin that had been digested with MNase to produce primarily mono, di- and tri-nucleosome stretches of chromatin was used identify the PTMs present on histone H3 nucleosomes that are interspersed among CENP-A nucleosomes at the centromere (Fig. 1A, 1B, supplemental Fig. S1)
Summary
Stable Cell Culture, Nocodazole Treatment, and Affinity Purification of CENP-A-LAP—Stable HeLa S3 cell lines expressing CENP-A tagged with a localization and affinity purification (LAP) tag were established, as described previously [18, 31]. Nucleosomal CENP-A-LAP protein was eluted from beads by 1 h incubation with 14 g of PreScission® protease in wash buffer, 300 mM KCl, and 0.1% Tween-20. Three missed cleavages were allowed for all searches, and each search was performed with the enzyme appropriate to the proteases used for sample digestion (trypsin, LysC, GluC, or AspN). Pnl.gov/) was used to calculate the expected dominant isotopic peak (based on natural abundance of carbon as 1.070% 13C and nitrogen as 0.368% 15N) for plotting XICs. MS1 spectra of peptides from histones H3 and H4 were analyzed manually using a mass tolerance of 0 –5 ppm to interpret and assign combinations of acetylation and methylation. For H4 S1-R23 N-terminal tail we measured the most abundant charge state (z ϭ ϩ7 or ϩ6) of the isotope peak corresponding to the 13C x 1 (Aϩ1) species
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