A Switch in Mitotic Histone H4 Lysine 20 Methylation Status Is Linked to M Phase Defects upon Loss of HCF-1  Eric Julien, Winship Herr  Molecular Cell  Volume 14, Issue 6, Pages 713-725 (June 2004) DOI: 10.1016/j.molcel.2004.06.008

Figure 1 HCF-1C Depletion Induces Multinucleation (A) Schematic structure of HCF-1. The HCF-1 mRNA sequence (nucleotides 124–144) targeted by the HCF-1 siRNA duplex and the same sequence with silent mutations (in red) to protect from siRNA silencing are shown. (B) Quantitation of binucleated cells represented with white portions of the bars or cells with more than two nuclei (>binucleated cells) represented with gray portions of the bars in parental HeLa cells after control (Ba) or HCF-1 (Bb) siRNA, in HCF-1 siRNA-treated HCF-1wt/SR (Bc) or HCF-1N/SR (Bd) cells. The percentage of multinucleated cells was obtained by counting 200 cells per sample in each of three independent experiments. (C) HCF-1N/SR cells with micronuclei after HCF-1 siRNA. αHCF-1C (green), α-tubulin (red), and DAPI (blue) staining are shown. Arrowheads indicate the micronuclei in a HCF-1C-depleted binucleated cell. (D) Examples of HCF-1C-depleted HCF-1N/SR cells at metaphase (Ba and Bb) and anaphase (Bc–Be), 3 days after siRNA treatment. Staining with α-tubulin (red) and DAPI (blue). HCF-1C depletion was verified by αHCF-1C staining (data not shown). Arrowhead indicates missegregated and lagging chromosomes. (E) Examples of HCF-1C-depleted HCF-1N/SR cells at metaphase (Ba), anaphase (Bb), and telophase (Bc), 4 to 5 days after HCF-1 siRNA treatment. Staining with α-tubulin (red) and DAPI (blue). HCF-1C depletion was verified by αHCF-1C staining (data not shown). Scale bars, 5 μm. Molecular Cell 2004 14, 713-725DOI: (10.1016/j.molcel.2004.06.008)

Figure 2 Autonomous HCF-1C Subunit Chromatin Association (A) Control (lanes 1–3) or HCF-1 (lanes 4–6) siRNA-treated HCF-1C/SR cells were subjected to biochemical fractionation; soluble fractions S2 and S3 and chromatin-enriched fraction P3 were probed with αHCF-1N antisera (upper), with αHA tag (middle), and with αMEK1 antisera (lower). (B) The P3 fraction of HCF-1 siRNA-treated HCF-1C cells was treated with MNase for the times indicated. The digested suspension was then centrifuged and the supernatant (upper) and pellet (lower) analyzed by immunoblotting with αHA tag antiserum. (C and D) Schematics of the wild-type or truncated HA epitope-tag recombinant HCF-1C subunit proteins are shown. HeLa cells synthesizing the different HCF-1C-truncated proteins were tranfected with control (lanes 1–3) or HCF-1 (lanes 3–6) siRNA, subjected to biochemical fractionation, and probed by immunoblot. For each cell line, endogenous HCF-1 was efficiently depleted after HCF-1 siRNA, and MEK1 was in fraction S3 in both samples (data not shown). Molecular Cell 2004 14, 713-725DOI: (10.1016/j.molcel.2004.06.008)

Figure 3 The ChAD and AD Are Required for Proper Cytokinesis Quantitation of multinucleated cells of parental cells (samples 1 and 2), or cells synthesizing the indicated recombinant HCF-1SR proteins (samples 3–16), treated with control (odd numbered samples) or HCF-1 (even numbered samples) siRNA. The percentage of multinucleated cells was obtained by counting 200 cells per sample in each of three independent experiments. (**), p < 0.08 as determined by Student's t test. Molecular Cell 2004 14, 713-725DOI: (10.1016/j.molcel.2004.06.008)

Figure 4 Unbalanced Mitotic H4-K20 Methylation upon Loss of HCF-1 (A) Immunofluorescence of H3-K9-3xMe methylation in control (Aa–Ac) and HCF-1 (Ad–Af) siRNA-treated cells. DNA (Aa and Ad), αHCF-1 (Ab and Ae), and αH3-K9-3xMe (Ac and Af) staining are shown. Arrows point to HCF-1-positive mitotic cells, whereas arrowheads point to an HCF-1-negative mitotic cell. (B) Immunofluorescence of αH4-K20-2xMe methylation in control (Ba–Bd) and HCF-1 (Be–Bl) siRNA-treated cells. DNA (Ba, Be, and Bi), αHCF-1 (Bb, Bf, and Bj), and αH4-K20-2xMe (Bc, Bd, Bg, Bh, Bk, and Bl) staining is shown. Arrows point to HCF-1-nondepleted mitotic cells, whereas arrowheads point to HCF-1-depleted mitotic cells. (Bd), (Bh), and (Bl) show 50-fold digitally reduced exposures of the corresponding images in (Bc), (Bg), and (Bk). We note in mitotic cells an absence of condensed chromosome staining by the α-HCF-1 antibody (see Bb) consistent with inefficient HCF-1 association with metaphase chromosomes (our unpublished data). (C) Immunofluorescence of αH4-K20-1xMe methylation in control (Ca–Cc) and HCF-1 (Cd–Cf) siRNA-treated cells. DNA (Ca and Cd), αHCF-1 (Cb and Ce), and αH4-K20-1xMe (Cc and Cf) staining are shown. Open arrowheads point to interphase cells with αH4-K20-1xMe staining. (D) Immunofluorescence of αH4-K20-3xMe methylation in control (Da–Dc) and HCF-1 (Dd–Df) siRNA-treated cells. In both panels, HCF-1-positive (arrows) and -negative (arrowheads) mitotic cells are indicated. Scale bar, 5 μm. Molecular Cell 2004 14, 713-725DOI: (10.1016/j.molcel.2004.06.008)

Figure 5 HCF-1C Subunit Chromatin Functions Are Associated with the Regulation of Mitotic H4-K20 Methylation (A) Immunofluorescence analysis of H4-K20-2xMe methylation in control cells (Aa–Ad) and HCF-1 siRNA-treated HeLa (Ae–Ah), HCF-1N/SR (Ai–Al), and HCF-1C/SR (Am–Ap) cells. DAPI staining (Aa, Ae, Ai, and Am), αHCF-1C (Ab, Af, and Aj) and αHCF-1N (An) staining (endogenous HCF-1), and αH4-K20-2xMe methylation staining (Ac, Ad, Ag, Ah, Ak, Al, Ao, and Ap). (Ad), (Ah), (Al), and (Ap), 50-fold digitally reduced exposures of the images in (Ac), (Ag), (Ak), and (Ao). Control cell pictures are the same as in Figure 4B. (B) Quantitation of mitotic cells with enhanced H4-K20-2xMe (upper) or reduced H4-K20-1xMe (lower) methylation in parental HeLa cells (sample 1) or HeLa cells with the indicated recombinant HCF-1SR proteins (samples 2–9) after HCF-1 siRNA. The percentage of mitotic cells with H4-K20 methylation defects was obtained by counting 50 mitotic cells per sample in each of three independent experiments. Scale bar, 5 μm. Molecular Cell 2004 14, 713-725DOI: (10.1016/j.molcel.2004.06.008)

Figure 6 Induction of Improper Mitotic H4-K20 Methylation upon Loss of HCF-1 Coincides with Upregulation of PR-Set7 (A) HCF-1N, cyclin A, SUV39H1, PR-Set7, and β-actin protein levels in cells untreated (lane 1) or treated with control (lane 2) or HCF-1 (lane 3) siRNA. (B) HCF-1 (top), PR-Set7 (middle), and β-actin (lower) mRNA levels in control (lane 1) or HCF-1 siRNA-treated (lane 2) cells. (C) Cyclin A, PR-Set7, and β-actin levels in HCF-1N/SR (lanes 1 and 2) and HCF-1C/SR (lanes 3 and 4) protein levels in control (lanes 1 and 3) or HCF-1 (lanes 2 and 4) siRNA treatment. Efficient depletion of endogenous HCF-1 was verified (data not shown). (D) Cyclin A, PR-Set7, and β-actin in HCF-1C/ΔAD (lanes 1 and 2) and HCF-1C/ΔPOST-AD (lanes 3 and 4) cells 2.5 days after control (lanes 1 and 3) or HCF-1 (lanes 2 and 4) siRNA treatment. Molecular Cell 2004 14, 713-725DOI: (10.1016/j.molcel.2004.06.008)

Figure 7 PR-Set7 Depletion Suppresses H4-K20 Methylation and Cytokinesis Defects Induced by Loss of HCF-1 (A) HCF-1, PR-Set7, and β-actin protein levels in control (lane 1), in HCF-1 (lane 2), PR-Set7 (lane 3), or in HCF-1/PR-Set7 (lane 4) siRNA treatment as indicated in (B). (B) Quantitation of multinucleated cells treated with control (sample 1), HCF-1 (sample 2), PR-Set7 (sample 3), and HCF-1/PR-Set7 (sample 4) siRNA. (C) Quantitation of mitotic cells with reduced H4-K20-1xMe (upper) or enhanced H4-K20-2xMe (lower) methylation in cells treated with HCF-1 (sample 1), PR-Set7 (sample 2), and HCF-1/PR-Set7 (sample 3) siRNA. “*,” PR-Set7 depletion led to reduced mitotic H4-K20-2xMe methylation compared to control cells. Molecular Cell 2004 14, 713-725DOI: (10.1016/j.molcel.2004.06.008)