Cell-cycle-dependent structural transitions in the human CENP-A nucleosome in vivo

Abstract

In eukaryotes, DNA is packaged into chromatin by canonical histone proteins. The specialized histone H3 variant CENP-A provides an epigenetic and structural basis for chromosome segregation by replacing H3 at centromeres. Unlike exclusively octameric canonical H3 nucleosomes, CENP-A nucleosomes have been shown to exist as octamers, hexamers, and tetramers. An intriguing possibility reconciling these observations is that CENP-A nucleosomes cycle between octamers and tetramers in vivo. We tested this hypothesis by tracking CENP-A nucleosomal components, structure, chromatin folding, and covalent modifications across the human cell cycle. We report that CENP-A nucleosomes alter from tetramers to octamers before replication and revert to tetramers after replication. These structural transitions are accompanied by reversible chaperone binding, chromatin fiber folding changes, and previously undescribed modifications within the histone fold domains of CENP-A and H4. Our results reveal a cyclical nature to CENP-A nucleosome structure and have implications for the maintenance of epigenetic memory after centromere replication.

Figure 1. CENP-A Nucleosomes Are Heterotypic throughout the Cell Cycle and Bind the Chaperone HJURP at G1, G1/S, and G2 Phases but Not at S Phase

Denaturing protein gel analysis of (A) native CENP-A IP from long chromatin arrays shows CENP-A contains H2A, H2B, and H4 at all points of the cell cycle and binds kinetochore proteins CENP-C and CENP-N (WB panel). (B and C) Native CENP-A IP from short chromatin arrays (B) and mononucleosomal input (C) demonstrate that CENP-A is always associated with H2A, H2B, and H4, but not with H3. (D) Western blot of HJURP within the CENP-A IP from (B) demonstrates loss of the HJURP at S phase, and return of HJURP at G2 phase. (E) Extracted chromatin fibers stained for HJURP (green) and CENP-A as centromeric marker (red) demonstrate that HJURP is lost from centromeric fibers at S phase but enriched in G2 and G1 phases. Scale bars, 1 µm. See also Figure S2.

Figure 2. CENP-A Nucleosomes Alter from Tetramers to Octamers at S Phase

Single-molecule AFM measurements depicting (A) heights and (B) volumes of purified H3 and bulk chromatin (BC) nucleosomes show they have octameric dimensions. In contrast CENP-A nucleosomes are tetramers at G2/M, M, early G1, and G1/S but are octamers in early and late S phases. Red dotted line indicates octameric particle values. BC values are in gray, H3 values are in green, and CENP-A values are in blue. Average values for heights and volumes are indicated under each graph. Table 1 gives a detailed overview of the data. See also Figures S3, S4A, and S4B.

Figure 3. FRET Measurements Reveals the CENP-A Chromatin Fiber Undergoes a Transition at G1/S Phase

FRET measurements between CENP-A C termini and H3 C termini across the cell cycle. H3 FRET is constant across the cell cycle (red), whereas CENP-A FRET shows high FRET in G1 (Black bars) but no FRET from S through M (mitosis) or C (Cytokinesis) phase (Grey bars). Black or red bars, significant FRET (p < 0.001); gray bars, no FRET. See also Table S1, S2, and Figure S5.

Figure 4. CENP-A Lys124 and H4 Lys79 Are Acetylated at G1/S Phase

(A) MS/MS spectrum showing CENP-A K124 is acetylated in the peptide “VTLFPK(acetyl)DVQLAR”. Location of the parent peptide ion prior to fragmentation is indicated with a blue diamond. Peptide fragmentation ions identified are indicated in the spectra and on the peptide sequence. The masses of ions b9, y8, y9, y10 are diagnostic of K124 acetylation. See also Figure S6B. (B) MS/MS spectrum showing fragmentation of H4 “K(acetyl)TVTAM(oxi)DVVYALK” as a doubly charged, monoisotopic ion, m/z 748.90 (−0.83 ppm difference from the theoretical m/z). Location of the parent ions prior to fragmentation is indicated with a green diamond. Peptide fragmentation ions identified are indicated in the spectra and on the peptide sequence. All fragment ions of the b series have masses diagnostic of K79 acetylation. See also Figure S6C. (C) Model of the CENP-A octamer showing two CENP-As (red), DNA (blue), core histones (gray), CENP-A K124 (yellow), and CENP-A H115, R118, and D125 (green). Dotted lines represent distances (Å) between residues. (D) Distance (Å) between H4K79 and DNA and H4K79 and CENP-A F84. Models were generated in PyMol by using published crystallographic coordinates (Tachiwana et al., 2011). See also Figure S6.

Figure 5. Cyclical Oscillations of HJURP and CENP-A Drive Centromere Dynamics

Model depicting how cyclical oscillations in CENP-A nucleosomal structure could derive from changes in HJURP chaperone binding mediated by chromatin remodeling, histone modifications, and cell-cycle events such as replication.