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. 2014;13(3):440-52.
doi: 10.4161/cc.27269. Epub 2013 Nov 25.

Mitotic Phosphorylation of Histone H3 Threonine 80

Free PMC article

Mitotic Phosphorylation of Histone H3 Threonine 80

Sharra L Hammond et al. Cell Cycle. .
Free PMC article


The onset and regulation of mitosis is dependent on phosphorylation of a wide array of proteins. Among the proteins that are phosphorylated during mitosis is histone H3, which is heavily phosphorylated on its N-terminal tail. In addition, large-scale mass spectrometry screens have revealed that histone H3 phosphorylation can occur at multiple sites within its globular domain, yet detailed analyses of the functions of these phosphorylations are lacking. Here, we explore one such histone H3 phosphorylation site, threonine 80 (H3T80), which is located on the nucleosome surface. Phosphorylated H3T80 (H3T80ph) is enriched in metazoan cells undergoing mitosis. Unlike H3S10 and H3S28, H3T80 is not phosphorylated by the Aurora B kinase. Further, mutations of T80 to either glutamic acid, a phosphomimetic, or to alanine, an unmodifiable residue, result in an increase in cells in prophase and an increase in anaphase/telophase bridges, respectively. SILAC-coupled mass spectrometry shows that phosphorylated H3T80 (H3T80ph) preferentially interacts with histones H2A and H4 relative to non-phosphorylated H3T80, and this result is supported by increased binding of H3T80ph to histone octamers in vitro. These findings support a model where H3T80ph, protruding from the nucleosome surface, promotes interactions between adjacent nucleosomes to promote chromatin compaction during mitosis in metazoan cells.

Keywords: chromatin condensation; histone phosphorylation; mitosis.


Figure 1. The commercial MC491 H3K79me3T80ph antibody recognizes H3K9me3S10ph. (A) Table showing the sequence identity surrounding the H3T80 residue. (B) Crystal structure of the nucleosome core. H3T80 is indicated in red and H3K79 is indicated in green. (C) Dot blot analysis of MC491 using decreasing quantities of the indicated peptides. (D) Western blot analysis of MC491 using wild-type DT40 cells and Dot1L−/− DT40 cells. H3K79me2 and H3S10ph were used as controls. (E) MC491 and H3S10ph staining of HeLa cells with or without Hesperidin treatment.
Figure 2. A new Histone H3T80ph antibody is specific. (A) Serially diluted peptides were dotted on PVDF membrane and probed with anti-H3T80ph. The antibody detects H3T80ph regardless of whether or not the adjacent lysine 79 is methylated. (B) Immunoprecipitation of H3 with H3T80ph antibody from prometaphase-arrested or asynchronous HeLa cells, probed with an anti-histone H3 antibody. (C) Peptide completion assays using the H3T80ph antibody competed against the indicated peptides. Antibody-peptide complexes were removed by centrifugation and the remaining supernatant was used for immunofluorescence of formaldehyde-fixed HeLa cells. Shown are metaphase cells. (D) H3T80ph and H3S10ph staining of HeLa cells treated with 0–100 nM of Hesperidin.
Figure 3. Histone H3T80 phosphorylation is enriched in mitosis. (A) Flow cytometry analysis of HeLa cells stained with anti-H3T80ph antibody using goat anti-rabbit Alexa Fluor® 488 as a secondary antibody and counter stained with propidium iodide (PI). Top left: The cell cycle profile using propidium iodide staining alone. Top right: Scatter plot of anti-H3T80ph staining vs. PI. Bottom: Cell number vs. anti-H3T80ph staining intensity during each phase of the cell cycle. The green arrow in the G2 panel indicates the cells where anti-H3T80ph staining was enriched over background. (B) Flow cytometry analysis of HCT-116, MCF7, and MCF10A cell lines stained with anti-H3T80ph antibody using goat anti-rabbit Alexa Fluor® 488 as a secondary antibody and counter stained with PI. (C) Immunofluorescence analysis of methanol fixed HeLa cells using histone H3T80ph antibody. Arrows indicate centrosomes.
Figure 4. Histone H3T80 phosphorylation is conserved across metazoans. (A) Immunofluorescence analysis of Drosophila S2 cells using anti-H3T80ph antibody. Anti-α-tubulin staining was used to determine mitotic phase and to mark spindles. (B) Top: Immunofluorescence analysis of mouse MM3MG cells using anti-H3T80ph antibody. Middle: Anti-H3T80ph staining was compared with anti-α-tubulin staining. Bottom: Anti-H3T80ph and anti-α-tubulin staining merged with DAPI (C) Top: Immunofluorescence analysis of mouse MM3MG cells using anti-H3T80ph antibody. Middle: Anti-H3T80ph staining was compared with anti-H3S10ph staining. Bottom: Anti-H3T80ph and anti-H3S10ph staining merged with DAPI.
Figure 5. Expression of histone H3T80 mutants results in mitotic phenotypes. HeLa cells transiently expressing either wild type, T80A (unmodifiable), or T80E (phosphomimic) H3.1-V5/6xHis evaluated 48 h post transfection. (A) Cells were fixed and immunofluorescence was performed to detect V5 tagged H3.1 (red) and gamma tubulin (green). A representative cell is shown. Mitotic cells (300–500) were counted in each experiment and were classified based on their mitotic stage. Data shown is the average of 4 independent experiments. (B) The percentage of metaphase cells and anaphase/telophase cells from (A) with mitotic defects. A representative cell is shown with an arrow indicating the corresponding mitotic defect. (C) Representative western analysis of transfected cells using an N-term H3 antibody. The percentage of H3.1-V5/6xHis relative to the amount of total H3 is shown. (D) Immunofluorescence analysis of HeLa cells in prometaphase transiently expressing wild type, T80A, or T80E H3.1-V5/6xHis.
Figure 6. In vitro and structural data suggest that H3T80ph may promote nucleosome-nucleosome interactions. (A) Schematic illustrating SILAC approach to identify H3T80ph interacting proteins. (B) Representative high-resolution mass spectra from histone H2A (top) and histone H4 (bottom) peptides identified as specifically interacting with an H3T80ph peptide. (C) Quantification of the association of H2A and H4 with the H3T80ph peptide. (D) In vitro binding assay using recombinant histone octamers and the indicated peptides. (E) In vitro binding assay using recombinant histone octamers and the indicated peptides. Histogram is the average of 2 experiments. Representative western analysis of bound histone octamers using anti-histone H3. Histogram shows fold change over no peptide. (F) Two histone octamers from the tetranucleosome structure as solved by Schalch et al. H3T80 is highlighted in red and H2AK74 is highlighted in orange.

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