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First published online 12 December 2006
doi: 10.1242/jcs.03326

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DNA methylation promotes Aurora-B-driven phosphorylation of histone H3 in chromosomal subdomains

Karine Monier^1,2,*, Sandrine Mouradian² and Kevin F. Sullivan^1,

¹ The Scripps Research Institute, Department of Cell Biology, CB163, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA
² Group Epigenetics of Pericentromeres, Laboratory of Molecular Biology of the Cell, CNRS UMR 5161, INRA U 1237, IFR 128, Ecole Normale Supérieure de Lyon, 46 allée d'Italie, 69364 Lyon CEDEX 07, France

View larger version (70K): [in this window] [in a new window]   Fig. 1. Pericentromeric foci of phosphorylated histone H3S10 colocalize with Aurora-B and fail to appear upon kinase inhibition. (A) Images of an interphase NT2 nucleus counterstained with DAPI (a) and labelled with the H3S10P (b, green), Aur-B (c, red) and centromeric proteins (d, blue). A merged image generated with a green mask to visualize H3S10P foci is shown (e). (B-D) A peptide competition assay was performed before immunodetection with H3S10P peptide (B), H3K9me3-S10P peptide (C) and an irrelevant H3S31P peptide (D). (E-F) Control (E) and VX-680 treated NT2 cells (F) were counterstained with DAPI (a,c) and labelled with H3S10P and Aur-B (b,c, green and red respectively). Percentages of mitotic cells (labeled M) and interphase nuclei positive for H3S10P are indicated below. DAPI-M, DAPI mask. Bars, 10 µm.

View larger version (44K): [in this window] [in a new window]   Fig. 2. H3S10P foci are enriched in late S-phase cells and correlate with cyclin B1 expression. (A) NT2 cells submitted to a short BrdU pulse were counterstained with DAPI (grey) and labelled with BrdU (green) and H3S10P (red). Merged images illustrate the four classes of nuclei analyzed: BrdU-/H3S10P- (c), BrdU+/H3S10P- (f), BrdU+/H3S10P+ (i) and BrdU-/H3S10P+ (l). The percentage of nuclei falling into each category is indicated on the right, as well as the number of H3S10P foci (mean ± s.d.). (B) NT2 cells were counterstained with DAPI (grey) and labelled with cyclin B1 (Cyc B1, green) and H3S10P (red). Merged images illustrate the four classes of nuclei analyzed: Cyc B1-/H3S10P- (c), Cyc B1+/H3S10P- (f), Cyc B1-/H3S10P+ (i), Cyc B1+/H3S10P+ (l). The percentage of nuclei falling into each category is indicated on the right. Bars, 10 µm.

View larger version (41K): [in this window] [in a new window]   Fig. 3. Large pericentromeres colocalize more frequently with H3S10P foci than small pericentromeres. (A-C) Immuno-FISH images display pericentromeres of human chromosome 1 (A), chromosome 2 (B) and chromosomes 14 and 22 (C) in NT2 nuclei exhibiting fewer than ten (a-d), or more than ten H3S10P foci (e-h). Bar, 10 µm. (D) Percentage of phospho-H3 positive pericentromeres in populations of NT2 nuclei exhibiting phospho-H3 foci is shown for seven human chromosomes, as indicated. The number of FISH signals analyzed is indicated for each chromosome (with legends).

View larger version (55K): [in this window] [in a new window]   Fig. 4. Aur-B foci colocalize more frequently with large pericentromeres and other passenger proteins in interphase nuclei. (A,B) Aur-B colocalizes frequently with pericentromeres of chromosome 1 in interphase NT2 nuclei. Aur-B (c, blue) was immunodetected with pericentromeres of chromosome 1 (b, pC1, red) in NT2 nuclei, counterstained with DAPI (a). Percentages of Aur-B positive pericentromeres in populations of NT2 nuclei exhibiting Aur-B foci are shown for chromosomes 1 and 2 (B). The number of FISH signals analyzed is indicated for each chromosome (n). (C,D) NT2 cells counterstained with DAPI (a) were detected separately with survivin (Cb, green) and INCENP (Db, green). Both passenger proteins labelled foci in a subset of interphase nuclei. (E-F) NT2 cells counterstained with DAPI (a) were co-detected with survivin and Aur-B (E) or INCENP and Aur-B (F). Merged images show that the great majority of foci detected with survivin or INCENP colocalize with Aur-B (d). Detection of survivin and INCENP in mitotic cells follow the typical passenger protein pattern (see Fig. S1 in supplementary material for survivin). Bars, 10 µm.

View larger version (69K): [in this window] [in a new window]   Fig. 5. Heterochromatic nature of large pericentromeres in nuclei exhibiting no more than ten H3S10P or Aur-B foci. (A-B) NT2 nuclei immunodetected with H3S10P (yellow) and 5-meC (red in A) or HP1 (red in B), exhibiting fewer than ten (a-d) or more than ten H3S10P foci (e-h). (C) Histogram of the total number of 5-meC accumulations, positive (red) or negative (yellow) for H3S10P according to the number of foci per NT2 nucleus, as displayed in A. (D) Histogram of the percentage of 94 randomly taken NT2 cells exhibiting HP1 accumulations, as shown in B, according to the presence of pericentromeric H3S10P foci (no foci, fewer than ten foci, at least ten foci). (E,F) H3K9me (red in E) or H3K9me3-S10P (red in F) foci colocalize with Aur-B (yellow) in NT2 nuclei exhibiting fewer than ten (a-d) or more than ten nuclear foci (e-h). (G,H) H3S10P-A594 foci (red) colocalize with H3S10P (yellow in G) and H3K9me3S10P (yellow in H) in NT2 nuclei exhibiting fewer than ten (a-d) or more than ten nuclear foci (e-h). Bars, 10 µm.

View larger version (48K): [in this window] [in a new window]   Fig. 6. 5-Azacytidine-induced DNA demethylation partially inhibits Aur-B driven phosphorylation of histone H3 at pericentromeres in interphase nuclei. (A) Images of NT2 nuclei, control (a) or treated with 5-azacytidin (b), counterstained with DAPI and immunodetected with 5-methylcytosine (green). For comparison purposes, 5-methylcytosine images are displayed with the same dynamic scale. Nuclear intensity of 5-methylcytosine signals was quantified on control and 5-AzaC-treated populations (c). Histogram of the integrated intensity per nucleus for both populations normalized by the nuclear integrated intensity of negative control population (c, third column, no HCl). Error bars correspond to s.d. of the analyzed populations and n corresponds to the number of analyzed nuclei. (B) Cell-cycle analysis of exponentially growing NT2 cells by flow cytometry. The ModFit LT software was used to estimate the percentage of cells with a G1, S and G2-M content in control (a) and 5-azacytidin treated populations (b). (C) Western blot analysis of whole NT2 extracts detected with Aur-B (top) and H3S10P (bottom), in equally loaded populations of control (lane 1) and 5-AzaC-treated cells (lanes 2 and 3). (D,E) Analysis of cyclin B1 expression in NT2 cells treated with 5-AzaC. Representative fields of views of control (Da) and 5-AzaC treated NT2 cells (Db) counterstained with DAPI (insets, grey) and labelled with cyclin B1 (green) and H3S10P (red). Table shows the percentages of cyclin B1 and H3S10P positive nuclei (E, second and third row, respectively) and of doubly labelled nuclei (E, fourth row) obtained in control (E, second column) and in 5-AzaC treated NT2 cells (E, third column). The total number of analyzed cells, n, is indicated. (F,G) Analysis of HS10P and Aur-B expression in NT2 cells treated with 5-AzaC. Representative field of views of NT2 cells, control (Fa) or treated with 5-AzaC (Fb), counterstained with DAPI (insets, grey) and immunodetected with H3S10P (green) and Aur-B (red). (G) Table of the percentage of cells exhibiting H3S10P foci and uniform H3S10P labelling obtained in control and in 5-AzaC-treated NT2 cells. The total number of analyzed cells, n, is indicated. Bars, 10 µm.

View larger version (53K): [in this window] [in a new window]   Fig. 7. DNMT1 depletion inhibits Aur-B-mediated histone H3 phosphorylation at pericentromeres. (A) Transfection with antisense oligonucleotide (AS) resulted in >90% depletion of DNMT1 compared with a mismatched oligonucleotide control (MM). Vimentin was detected as a loading control in western blots. (B) Reduction in Aur-B and phospho-H3 is not greater than 50% in these populations. (C-N) Microscopic analysis of Aur-B, phospho-H3 and HP1 in cells transfected with DNMT1-AS and DNMT1-MM oligonucleotides. (C-G) Aur-B and centromere colocalization in cells transfected with DNMT-MM (C,D) and DNMT1-AS (E-G). Examples of cells without (C,E labelled -) and with (D,G labelled +) focal accumulations of Aur-B are shown, as was a third population seen in DNMT1-AS-treated cells that showed a diffuse localization of Aur-B (F, labelled +/-). (H) Histogram summarizing quantification of Aur-B and H3S10P pericentromeric foci in oligonucleotides-treated cells. In each case the DNMT1-MM population was used as a standard, revealing a 62% and 38% decrease in DNMT1-AS cells exhibiting Aur-B and phospho-H3 pericentromeric foci, respectively. (I-N) HP1 and centromere colocalization in cells transfected with DNMT1-MM (I,J) and DNMT1-AS (K,L). Examples of cells exhibiting no focal accumulations of HP1 (I,K labelled -) and with focal accumulations (J,L labelled +). Bars, 10 µm. (M) Histogram represents quantification of cells with focal accumulations of HP1 with DNMT1-MM (blue), set as the standard in comparison with DNMT1-AS (red). Number of analyzed nuclei is reported above. (N) Histogram of percentage of mitotic cells scored in the DNMT1-MM (blue) and the DNMT1-AS (red) populations. Number of analyzed nuclei is reported above.