Phosphorylation-induced Rearrangement of the Histone H3 NH2-terminal Domain during Mitotic Chromosome Condensation

Abstract

The NH2-terminal domain (N-tail) of histone H3 has been implicated in chromatin compaction and its phosphorylation at Ser10 is tightly correlated with mitotic chromosome condensation. We have developed one mAb that specifically recognizes histone H3 N-tails phosphorylated at Ser10 (H3P Ab) and another that recognizes phosphorylated and unphosphorylated H3 N-tails equally well (H3 Ab). Immunocytochemistry with the H3P Ab shows that Ser10 phosphorylation begins in early prophase, peaks before metaphase, and decreases during anaphase and telophase. Unexpectedly, the H3 Ab shows stronger immunofluorescence in mitosis than interphase, indicating that the H3 N-tail is more accessible in condensed mitotic chromatin than in decondensed interphase chromatin. In vivo ultraviolet laser cross-linking indicates that the H3 N-tail is bound to DNA in interphase cells and that binding is reduced in mitotic cells. Treatment of mitotic cells with the protein kinase inhibitor staurosporine causes histone H3 dephosphorylation and chromosome decondensation. It also decreases the accessibility of the H3 N-tail to H3 Ab and increases the binding of the N-tail to DNA. These results indicate that a phosphorylation-dependent weakening of the association between the H3 N-tail and DNA plays a role in mitotic chromosome condensation.

Analysis of cyclin B1 and CDK activity during apoptosis induced by camptothecin treatment

“…We directly observed CPT treated cells by confocal microscopy and detected cells with chromosomes that were poorly organized relative to non-treated cells (Figure 8). These cells also expressed phosphohistone H3, a phosphorylated epitope that is expressed during mitosis in proliferating cells (Sauve et al, 1999). The coexpression of phospho-histone H3 in cells with condensed chromosomes, at the time when cyclin B1 is active, strongly supports the idea that these cells have entered a mitotic catastrophe.…”

An embarrassment of niches: the many covalent modifications of histones in transcriptional regulation

“…Several studies have indicated that histone H3 phosphorylation on serine 10 and serine 28 correlates with mitotic chromosome condensation (Van Hooser et al, 1998;de la Barre et al, 2000;Goto et al, 1999;Sauve et al, 1999). The role of histone phosphorylation during the cell cycle was clearly established in Tetrahymena, where mitosis is restricted to the germ line micronuclei, and transcription is limited to the amitotic macronuclei.…”

ATM-dependent phosphorylation and accumulation of endogenous BLM protein in response to ionizing radiation

“…They were then incubated for 2 h at room temperature with a polyclonal antibody speci®c for the phosphorylated Ser 10 of the histone H3 (Euromedex), at a ®nal concentration of 5 mg/ml. This antibody labels mitotic cells with speci®cally increased intensity because all H3 molecules become phosphorylated at Ser 10 during mitosis, whereas few H3 molecules are phosphorylated in interphase (Gurley et al, 1978;Sauve et al, 1999). Cells were then rinsed and incubated with an FITC-conjugated goat anti-rabbit antibody (Jackson) (45 min at room temperature), resuspended in PBS containing 100 mg/ ml RNase A and 10 mg/ml propidium iodide, and mitotic cells were scored (on a total of 10 000 cells) by two-color FACScalibur (Becton Dickinson) analysis, gating for phospho-Histone H3 positive cell populations.…”