Phosphorylation of histone H3 Thr-45 is linked to apoptosis

Abstract

Numerous post-translational modifications have been identified in histones. Most of these occur within the histone tails, but a few have been identified within the histone core sequences. Histone core post-translational modifications have the potential to directly modulate nucleosome structure and consequently DNA accessibility. Here, we identify threonine 45 of histone H3 (H3T45) as a site of phosphorylation in vivo. We find that phosphorylation of H3T45 (H3T45ph) increases dramatically in apoptotic cells, around the time of DNA nicking. To further explore this connection, we analyzed human neutrophil cells because they are short-lived cells that undergo apoptosis in vivo. Freshly isolated neutrophils contain very little H3T45ph, whereas cells cultured for 20 h possess significant amounts; the kinetics of H3T45ph induction closely parallel those of caspase-3 activation. Cytokine inhibition of neutrophil apoptosis leads to reduced levels of H3T45ph. We identify protein kinase C-delta as the kinase responsible for H3T45ph in vitro and in vivo. Given the nucleosomal position of H3T45, we postulate that H3T45ph induces structural change within the nucleosome to facilitate DNA nicking and/or fragmentation.

FIGURE 1.

Identification of H3T45ph in chromatin. A, MS/MS identifies H3T45ph in vivo. HL60 cells were treated with calyculin A. Proteins were prepared as described and resolved by SDS-PAGE. Proteins corresponding to the size of H3 were isolated from the acrylamide gel and fragmented by in-gel tryptic digestion. Phosphopeptides were enriched using titanium oxide beads, and after elution, injected into the Orbitrap mass spectrometer. The inset at the top shows the spectrum of the intact peptide of interest encompassing H3T45. This peptide was then subjected to MS/MS shown in the main part of the panel. B, the position of histone H3 (red) within the nucleosome is indicated. The location of H3T45 (yellow) is indicated by the arrows; H3T45 is positioned at the N terminus of the first helix of H3 (the αN1-helix), a position close to where the DNA both enters and exits the nucleosome. Images were constructed using the MacPyMOL software. C, peptide competition analyses confirm the specificity of the anti-H3T45ph antibody. The specificity of the anti-H3T45ph antibody was validated by peptide competition using an ELISA plate assay. The antibody raised to an H3T45ph peptide specifically recognizes only this peptide and fails to recognize the identical but unmodified H3 peptide. Similarly, the anti-H3T45ph antibody does not recognize other H3 phospho-threonine peptides that have previously been shown to be modified on histone H3. OD, optical density. D, the indicated amounts of various phosphorylated histone peptides were spotted to polyvinylidene difluoride membrane and probed with the anti-H3T45ph antibody. The anti-H3T45ph antibody only recognizes the peptide with Thr-45 phosphorylated. E, HL60 cells were incubated with 20 nm calyculin A (Calbiochem 208851) for the times indicated. Whole cell extracts were analyzed by Western blotting using the anti-H3T45ph antibody, and an anti-H3K4me3 (Abcam ab8580) antibody was used as a loading control. F, HL60 cells were treated with (+) or without (−) calyculin A. Protein extracts were prepared by cellular fractionation and analyzed by Western blotting using the anti-H3T45ph, anti-β-tubulin (Sigma), or anti-H3K4me3 (Abcam ab8580) antibodies.

FIGURE 1.

Identification of H3T45ph in chromatin. A, MS/MS identifies H3T45ph in vivo. HL60 cells were treated with calyculin A. Proteins were prepared as described and resolved by SDS-PAGE. Proteins corresponding to the size of H3 were isolated from the acrylamide gel and fragmented by in-gel tryptic digestion. Phosphopeptides were enriched using titanium oxide beads, and after elution, injected into the Orbitrap mass spectrometer. The inset at the top shows the spectrum of the intact peptide of interest encompassing H3T45. This peptide was then subjected to MS/MS shown in the main part of the panel. B, the position of histone H3 (red) within the nucleosome is indicated. The location of H3T45 (yellow) is indicated by the arrows; H3T45 is positioned at the N terminus of the first helix of H3 (the αN1-helix), a position close to where the DNA both enters and exits the nucleosome. Images were constructed using the MacPyMOL software. C, peptide competition analyses confirm the specificity of the anti-H3T45ph antibody. The specificity of the anti-H3T45ph antibody was validated by peptide competition using an ELISA plate assay. The antibody raised to an H3T45ph peptide specifically recognizes only this peptide and fails to recognize the identical but unmodified H3 peptide. Similarly, the anti-H3T45ph antibody does not recognize other H3 phospho-threonine peptides that have previously been shown to be modified on histone H3. OD, optical density. D, the indicated amounts of various phosphorylated histone peptides were spotted to polyvinylidene difluoride membrane and probed with the anti-H3T45ph antibody. The anti-H3T45ph antibody only recognizes the peptide with Thr-45 phosphorylated. E, HL60 cells were incubated with 20 nm calyculin A (Calbiochem 208851) for the times indicated. Whole cell extracts were analyzed by Western blotting using the anti-H3T45ph antibody, and an anti-H3K4me3 (Abcam ab8580) antibody was used as a loading control. F, HL60 cells were treated with (+) or without (−) calyculin A. Protein extracts were prepared by cellular fractionation and analyzed by Western blotting using the anti-H3T45ph, anti-β-tubulin (Sigma), or anti-H3K4me3 (Abcam ab8580) antibodies.

FIGURE 2.

H3T45ph is induced as HL60 cells differentiate. A, HL60 cells were treated with 1.3% (v/v) DMSO for 6 days to induce granulocytic differentiation, and samples were taken daily. ND represents the non-differentiated control cells. Whole cell extracts were analyzed by Western blotting using antibodies against H3T45ph, H3T6ph (Abcam ab14102), or anti-H3K4me3 (Abcam ab8580). B, DMSO-induced increase in cd11b mRNA confirms HL60 cell differentiation. mRNA levels from HL60 cells described in panel A were measured using primers specific to the granulocytic marker, cd11b, and to β-microglobulin (for normalization). Error bars represent the S.D. C, confocal microscopy. Cells were fixed with 4% paraformaldehyde and stained with anti-H3T45ph and anti-rabbit labeled with Alexa 594 (Molecular Probes A11012) antibodies. 4′,6-Diamidino-2-phenylindole (DAPI) was included to identify DNA.

FIGURE 3.

H3T45ph is within apoptotic cells. A, HL60 differentiation activates caspase-3. Cleaved caspase-3, an apoptotic marker, was detected in HL60 cells treated with 1.3% (v/v) DMSO over 6 days. ND represents the non-differentiated control cells. The same whole cell extracts as used in Fig. 2A were analyzed by Western blotting using anti-cleaved caspase-3 (CST 9664) antibodies. B, apoptotic cells were identified using the in situ cell death detection kit (Roche Applied Science). Cells were co-stained with anti-H3T45ph and anti-rabbit labeled with Texas Red 594 (Invitrogen T2767) antibodies and identified by confocal microscopy. C, cells were scored for fluorescein, H3T45ph, and both fluorescein and H3T45ph staining. 4′,6-Diamidino-2-phenylindole (DAPI) was included to identify DNA. A number of random fields of view were scored, and the results shown represent the mean average.

FIGURE 4.

H3T45 is phosphorylated by PKCδ. A, neutrophils were isolated and cultured for the indicated times in the presence or absence of the following protein kinase inhibitors: 200 nm InSolution^TM staurosporine (Calbiochem, 569396), 25 μm InSolution^TM caspase-3 inhibitor I (Calbiochem, 235427), and protein kinase C inhibitor set (Calbiochem, 539573). Whole cell extracts were analyzed by Western blot using anti-H3T45ph, anti-cleaved caspase-3, or anti-H3K4me3. B, radioactive in vitro kinase assay. A mixture of core histones (2 mg), recombinant WT H3 (500 ng), and recombinant H3T45A (500 ng) was used as substrate in the in vitro kinase assay using [γ-³²P]ATP and recombinant PKCδ (7342, Cell Signaling Technology). Equal loading of histones is demonstrated by the Coomassie Blue-stained gel. WT, wild type. C, in vitro kinase assay. Recombinant WT H3 (500 ng) and H3T45A (500 ng) were used as substrates in the in vitro kinase assay using non-radioactive ATP. The reaction products were resolved by SDS-PAGE, and phosphorylation was then analyzed by Western blotting with the anti-H3T45ph antibody. D, human neutrophils were isolated and cultured for the indicated times in the presence or absence of 100 ng/ml granulocyte colony-stimulating factor (GCSF) (G0407 Sigma). Whole cell extracts were then analyzed by Western blotting using anti-H3T45ph, anti-PKCδ, or anti-H3K4me3 (as a loading control) antibodies.