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. 1999 Apr 19;145(2):225-35.
doi: 10.1083/jcb.145.2.225.

Phosphorylation-induced rearrangement of the histone H3 NH2-terminal domain during mitotic chromosome condensation

D M Sauvé  1 , H J Anderson, J M Ray, W M James, M Roberge
Affiliations

Phosphorylation-induced rearrangement of the histone H3 NH2-terminal domain during mitotic chromosome condensation

D M Sauvé et al. J Cell Biol. .

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.

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Figures

Figure 1
Figure 1
Specificity of H3P and H3 antibodies. (A) ELISA of H3P Ab binding to a peptide corresponding to residues 1–17 of H3 unphosphorylated (open square) or phosphorylated at Ser10 (closed square). (B) Western blot analysis of H3P Ab binding to total cellular proteins obtained from cycling cells (−N, containing 3% mitotic cells) or cells treated with nocodazole (+N, 60% mitotic cells). The unfilled arrow indicates the minor cross-reactive band. Molecular mass standards are shown on the left. (C) ELISA of H3 Ab binding to the unphosphorylated H3 peptide (open square), phosphorylated H3 peptide (closed square), total cellular proteins from cycling cells (open circle) or from cells treated with nocodazole (closed circle). (D) Western blot analysis of H3 Ab binding to cellular proteins performed as in B.
Figure 2
Figure 2
Phosphorylation of histone H3 Ser10 at mitosis. Paired photographs of cells showing bisbenzimide fluorescence of DNA (left) or H3P Ab immunofluorescence (right). Cells in interphase (indicated by arrowheads), prophase (A and B), metaphase (C, D, G, and H), anaphase (E and F), and telophase (G and H) are shown. Bar, 20 μm.
Figure 3
Figure 3
Accessibility of the H3 N-tail during mitosis. Paired photographs of cells showing DNA fluorescence (left), and immunofluorescence (right) using the H3 Ab (B, D, and F) or mAb to all histones (H). The panels show cells in interphase as well as prophase (A and B, arrows), metaphase (C–H, arrows), anaphase (C and D, double arrows), and telophase (E and F, right angled arrow). Bar, 20 μm.
Figure 4
Figure 4
Quantitation of H3 Ab immunofluorescence associated with interphase and mitotic cells. (A) The H3 Ab immunofluorescence associated with interphase cells (n = 60), prophase cells (n = 4), metaphase cells (n = 14), anaphase cells (n = 5), and telophase (n = 1) measured by scanning densitometry as described in Materials and Methods and expressed relative to that of interphase cells. (B) The total histone immunofluorescence associated with interphase cells (n = 6) or mitotic cells (n = 7) was measured and expressed as in A. Bars and error bars show mean and standard deviation.
Figure 5
Figure 5
Association of the H3 N-tail with DNA in vivo. (A) Cells were treated or not with nocodazole (N), staurosporine (Stsp), and irradiation using a single 5-ns 50-mJ pulse of UV light as indicated. H3 was immunoprecipitated and cross-linked DNA was end-labeled with phosphorus-32. Cross-linked H3–DNA complexes were separated by SDS-PAGE and detected using a PhosphorImager. The extent of cross-linking expressed as a percentage of that observed in interphase cells is shown at the bottom for one representative experiment. (B) Phosphorus-32– labeled cross-linked H3–DNA complexes obtained from interphase cells were treated or not with SV8 protease to cleave the N-tail and were analyzed as in A. The positions of H3 and of the H3 N-tail are indicated.
Figure 6
Figure 6
Effect of staurosporine on chromosome condensation and on the accessibility of the H3 N-tail at mitosis. (A and B) Mitotic spreads of cells arrested in mitosis with nocodazole and treated with (B) or without (A) staurosporine for 15 min and stained with bisbenzimide are shown. (C–H) Paired photographs of cells arrested in mitosis with nocodazole and treated without (C, D) or with (E–H) staurosporine, showing DNA fluorescence (left column) or H3 Ab immunofluorescence (right). (I and J) Paired photographs of cells arrested in mitosis with nocodazole and treated with staurosporine, showing DNA fluorescence (left) or H3P Ab immunofluorescence (right). The arrows indicate mitotic cells with decondensed chromosomes as a result of staurosporine treatment. Bar, 20 μm.
Figure 7
Figure 7
Intracellular distribution of [3H]spermidine in interphase (−N), mitotic (+N) and mitotic cells treated with staurosporine (+N + Stsp).
Figure 8
Figure 8
Effect of staurosporine on the distribution of polyamines in mitotic cells. SPM8-2 antibody immunofluorescence shows that the predominantly nuclear localization of spermine and spermidine in cells arrested in mitosis with nocodazole (A) is lost after 15 min of staurosporine treatment (B). Bar, 20 μm.
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