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. 2019 Nov 5;8(11):1391.
doi: 10.3390/cells8111391.

Extrachromosomal Histone H2B Contributes to the Formation of the Abscission Site for Cell Division

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Extrachromosomal Histone H2B Contributes to the Formation of the Abscission Site for Cell Division

Laura Monteonofrio et al. Cells. .
Free PMC article

Abstract

Histones are constitutive components of nucleosomes and key regulators of chromatin structure. We previously observed that an extrachromosomal histone H2B (ecH2B) localizes at the intercellular bridge (ICB) connecting the two daughter cells during cytokinesis independently of DNA and RNA. Here, we show that ecH2B binds and colocalizes with CHMP4B, a key component of the ESCRT-III machinery responsible for abscission, the final step of cell division. Abscission requires the formation of an abscission site at the ICB where the ESCRT-III complex organizes into narrowing cortical helices that drive the physical separation of sibling cells. ecH2B depletion does not prevent membrane cleavage rather results in abscission delay and accumulation of abnormally long and thin ICBs. In the absence of ecH2B, CHMP4B and other components of the fission machinery, such as IST1 and Spastin, are recruited to the ICB and localize at the midbody. However, in the late stage of abscission, these fission factors fail to re-localize at the periphery of the midbody and the abscission site fails to form. These results show that extrachromosomal activity of histone H2B is required in the formation of the abscission site and the proper localization of the fission machinery.

Keywords: CHMP4B; ESCRT-III fission machinery; abscission site; extrachromosomal histone H2B.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Depletion of ecH2B with siRNAs. (a) Schematic representation of H2B isoforms (gene symbol, chromosomal location, and protein name are reported). In red are indicated the isoforms recognized by the nine variant-specific siRNAs. (bd) HeLa cells were depleted for histone H2B with a combination of the nine variant-specific siRNAs (H2Bi) or with universal negative control (CTRi). Representative WB for the indicated proteins is shown. Cells were subdivided in two aliquots and cell extracts obtained with lysis buffer and centrifugation to eliminate chromatin or with acid extraction to purify nucleosome histones. (c) Representative IF imagines of CTRi and H2Bi HeLa cells—transfected with the indicated molarity of siRNAs—stained with Hoechst (blue) and anti-β-Tubulin Ab (red) to visualize nuclear DNA and cytoplasm. Scale bar is 10 μm. (d) Representative IF imagines of CTRi and H2Bi HeLa cells stained with anti-β-Tubulin Ab (red), anti-phospho-H2B-Ser14 Ab (green) and Hoechst (blue) to visualize nuclei. Representative images of cells in anaphase and cytokinesis are shown. At least 30 anaphase and 100 cytokinetic cells form three independent experiments were analyzed. Scale bar is 10 μm.
Figure 2
Figure 2
ecH2B depletion in HeLa cells induces cytokinesis defects. Proliferating, asynchronous HeLa cells were depleted for histone H2B, stained for β-Tubulin and DNA as described in Figure 1, and analyzed in panels A to F. (a) Representative IF images of cytokinetic cells. Compared to normal cytokinesis in CTRi (upper panels), the presence of long and thin (middle panels) or long and broken (lower panels) ICBs are shown in the H2Bi cells. Scale bar is 10 μm. (b) The percentage of cells in cytokinesis was measured by scoring at least 1000 cells per IF sample in four independent experiments. (c,d) The relative amount of normal and aberrant ICB was measured by scoring at least 1000 cells per sample and pooling (c) or subdividing (d) cytokinetic cells based on the defect type. (e) The length of each ICB was measured in three independent experiments. (f) The presence of cells with two or more nuclei and (g) ICBs with DNA (i.e., chromosome bridges, lagging chromosomes) was measured by scoring the same samples described in (b). (h) H2Bi and CTRi HeLa cells were transfected with GFP-H2B or GFP-empty vector and stained for β-Tubulin and DNA as above. The amount of long, aberrant ICB was measured by scoring at least 100 GFP-positive cells per sample. Data are reported as mean ± SD. ns p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.
Figure 3
Figure 3
ecH2B depletion does not affect the recruitment of cytokinetic proteins. Proliferating, asynchronous HeLa cells were depleted for histone H2B, as described above, and analyzed by double IF for β-Tubulin (red) and the indicated proteins (green); DNA was marked with Hoechst (Blue). The analyzed proteins are associated with (a) central spindle formation and cleavage furrow ingression, (b) midbody formation, and (c) abscission machinery. Representative images of CTRi and H2Bi HeLa cells are shown. At least 20 metaphases and anaphases in (a) and 50 cytokinetic cells in (b,c) from two independent experiments have been scored. For each of the analyzed proteins, no significant difference was observed in the percentage of positive ICBs that was > 98% in both H2Bi and CTRi cells. Scale bar is 10 μm.
Figure 4
Figure 4
H2B depletion is associated with abscission delay. CTRi and H2Bi HeLa cells were obtained as above and were followed through division by time-lapse microscopy. (a) Representative stills from time-lapse recording of HeLa CTRi and H2Bi cells. The time (in hrs:min) since the beginning of the round up is shown. Scale bar is 10 μm. The relative videos are Videos S1 and S2. CTRi cells divide and return mononucleated in about 3 h while H2Bi cells remain connected by intercellular bridge for several hours before abscission occurs. (b) The time from round up to cleavage furrow ingression was determined in CTRi HeLa cells (n = 150) and in H2Bi (n = 100). (c) The time from cleavage furrow ingression to abscission (abscission time) was determined in CTRi HeLa cells (n = 81) and in H2Bi (n = 107). In (b) and (c), the cumulative percentage of the analyzed cells from four independent transfections is plotted as function of time.
Figure 5
Figure 5
ecH2B binds and colocalizes with CHMP4B at the intercellular bridge. (a) GST-pull down was performed to analyze the binding between recombinant GST-CHMP4B and H2B. GST alone was used as negative control. WBs were performed to detect the indicated proteins. Red asterisks (*) indicate GST-CHMP4B sub-products consistently present in the commercial preparation. (b) Recombinant His-H2B was phosphorylated at Ser14 by incubation with HIPK2 Kinase Domain (HIPK2 KD) in the presence of cold γ-ATP. GST-pull down was performed to compare the binding between recombinant GST-CHMP4B and His-H2B or His-H2B-S14P. GST alone was used as negative control. WBs were performed to detect the indicated proteins. Red asterisks (*) indicate GST-CHMP4B sub-products. (c,d) Proliferating, asynchronous HeLa cells were fixed and analyzed by proximity ligation assay (PLA) with anti-CHMP4B Ab and anti-phospho-H2B-Ser14 in two independent experiments (c) or anti-phospho-H2B-Ser32 Abs in two other independent experiments (d). Representative images of cells in early and late cytokinesis and abscission sites are shown. 30 ICBs per sample were analyzed and overall PLA positivity was observed in 37.7 ± 2.5% of the ICBs. Scale bar is 10 μm.
Figure 6
Figure 6
ecH2B contributes to abscission site. (a) Proliferating, asynchronous HeLa cells were fixed and analyzed by double IF with anti-β-Tubulin Ab (red) and anti-phospho-H2B-Ser14 Ab (green) in more than five independent experiments. Representative images of early and late cytokinesis and abscission sites are shown. Fifty ICBs per sample were analyzed. Scale bar is 10 μm. (b) Proliferating, asynchronous HeLa cells were fixed and analyzed by double IF with anti-phospho-H2B-Ser14 Ab (green) and anti-CHMP4B Ab. Two representative images for late cytokinesis, abscission site and the relative 3D reconstructions are shown. (c,d) CTRi and H2Bi HeLa cells were obtained as above and analyzed by double IF for β-Tubulin (red) and the indicated proteins (green); DNA was marked with Hoechst (Blue). Representative images (c) and quantification (d) from three independent experiments of abscission site formation based on the three abscission factors are reported. Data are reported as mean ± SD. ** p < 0.01; *** p < 0.001. (e) Graphical abstract is reported. Microtubules are indicated in red, ecH2B is represented in blue and CHMP4B in green.

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