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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 918-931 |
| Number of pages | 14 |
| Journal | Molecular and Cellular Proteomics |
| Volume | 15 |
| Issue number | 3 |
| DOIs | |
| State | Published - Mar 2016 |
Funding
We are grateful for access to Isotope Pattern Calculator software, developed by Pacific Northwest National Laboratories. This work was supported by National Institutes of Health (NIH) Grants R01 GM037537 (to D.F.H.), R01 GM082989 (to B.E.B.), R01 GM111907 (to D.R.F.), and the American Cancer Society (D.R.F.). A.O.B. and T.P. were supported by the following training grants: T32 GM08715 T32 (to A.O.B.) GM08275 (to T.P.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
ASJC Scopus subject areas
- Analytical Chemistry
- Biochemistry
- Molecular Biology