Volume 47, Issue 1, Pages 87-98 (July 2012) HIPK2 Controls Cytokinesis and Prevents Tetraploidization by Phosphorylating Histone H2B at the Midbody  Cinzia Rinaldo, Alice Moncada, Alessandra Gradi, Laura Ciuffini, Donatella D'Eliseo, Francesca Siepi, Andrea Prodosmo, Alessandra Giorgi, Giovanna M. Pierantoni, Francesco Trapasso, Giulia Guarguaglini, Armando Bartolazzi, Enrico Cundari, M. Eugenia Schininà, Alfredo Fusco, Silvia Soddu  Molecular Cell  Volume 47, Issue 1, Pages 87-98 (July 2012) DOI: 10.1016/j.molcel.2012.04.029 Copyright © 2012 Elsevier Inc. Terms and Conditions

Molecular Cell 2012 47, 87-98DOI: (10.1016/j.molcel.2012.04.029) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 HIPK2 Depletion Is Associated with Cytokinesis Failures (A) MEFs from littermate Hipk2+/+ and Hipk2−/− embryos were stained, at the indicated days (d) after plating, with DAPI and anti-β-tubulin Ab (green) to identify the cytoplasm in interphase and the LIBs in telophase. The percentage (left panel) and the morphology (right panel) of mono-, bi-, and multinucleated cells are reported. About 1,000 cells per sample were scored for the presence of one, two, or more nuclei/cell, and the data are represented as mean ± standard deviation (SD). Here and in the following figures, bar is 10 μm. (B and C) Control (Ctri) and HIPK2-depleted (HIPK2i) HeLa cells (transfection efficiency 80%) were employed as total populations. Stills from Movies S1 and S2 of Ctri (B) and HIPK2i (C) HeLa cells are shown. Ctri cells divided and remained mononucleated while HIPK2i cells became binucleated after failing abscission. Open arrowheads indicate the cells to follow; closed arrowheads indicate the end-up into binucleated cells. (D–G) Hipk2−/− MEFs were transfected with EGFP-control vector (Ctr) or EGFP-HIPK2 expression vector (HIPK2). Apoptosis in mono- and binucleated cells was evaluated by TUNEL at the indicated times (D). Cells were stained with DAPI and anti-β-tubulin Ab 48 hr posttransfection; EGFP-positive cells were analyzed for the percentages of mono-, bi-, and multinucleated cells and the percentage of LIBs (E and F). Starting at 16 hr posttransfection, the numbers of EGFP-positive cells were measured by direct counting of each dish (G). Data represent mean ± SD. See also Movies S1–S6 and Figure S1. Molecular Cell 2012 47, 87-98DOI: (10.1016/j.molcel.2012.04.029) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 Localization of Endogenous HIPK2 and H2B-S14P at the Midbody (A and B) Representative immunostaining of HeLa cells with anti-HIPK2(1) and anti-β-tubulin (A) or anti-Aurora B (B). (C) HeLa cells were transfected with EGFP-HIPK2 expression vector and γ-irradiated (5 Gy) at 20 hr posttransfection. Cells were stained with anti-p-Histone H2A.X-S139 (anti-γ-H2AX) or anti-p-Histone H2B-S14(1) Abs and DAPI at the indicated times after irradiation. Multiple stacks were recorded, and representative deconvolution images are shown. (D) HeLa cells were transfected with EGFP-HIPK2 expression vector and immunostained 20 hr posttransfection. Representative immunostaining with anti-p-Histone H2B-S14(1) Ab is reported. (E and F) Representative immunostaining of HeLa cells with anti-p-Histone H2B-S14(1) and anti-β-tubulin (E) or anti-Aurora B (F). See also Figures S2 and S3 and Table S1. Molecular Cell 2012 47, 87-98DOI: (10.1016/j.molcel.2012.04.029) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 HIPK2, H2B, and H2B-S14P Are Enriched in Midbody Fractions (A) Representative image of an enrichment of cells at telophase (approximately 70%) from which midbodies were isolated and extracted. (B) Indicated fractions of HeLa cells were analyzed by WB for the indicated proteins. A representative WB is shown. Ponceau shows the relative loading quantity. (C) Densitometric values of the WB shown in (B) were first normalized according to protein amount, then calculated taking the I-TCE level as reference value. (D) Representative fields of cells enriched at telophase and stained as in Figure 2E. Molecular Cell 2012 47, 87-98DOI: (10.1016/j.molcel.2012.04.029) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 H2B Localizes at the Midbody Independently of the Presence of Chromosome Bridges (A) HeLa cells stably expressing H2B-GFP were stained with anti-β-tubulin Ab to mark midbodies and DAPI to visualize DNA. Autofluorescence of H2B-GFP was detected in all the DAPI-positive chromosome bridges (5% of the total population; n = 50) (top panel, open arrowhead). No GFP autofluorescence was detectable at the midbodies without chromosome bridges (bottom panels; n = 300). (B) HeLa H2B-GFP cells were stained without Ag retrieval with anti-GFP Ab and DAPI. Anti-GFP shows a GFP staining (red) at the midbody comparable to the GFP autofluorescence shown in (A), i.e., only in cells with chromosome bridges. Representative images of telophases with (top panels, open arrowhead) or without (bottom panels) chromosome bridges are reported (BF, bright-field). (C) HeLa H2B-GFP and parental HeLa cells were stained with anti-GFP Ab upon Ag retrieval. Anti-GFP shows a GFP staining (red) comparable to the localization of endogenous H2B (Figures 2D, 2E, and S3C), i.e., at all the midbodies, independently of the presence of chromosomal DNA (n = 120). Representative images of a telophase without chromosome bridge are reported in the top panels. No staining with anti-GFP Ab was observed in untransfected HeLa cells (bottom panels). See also Figures S4 and S5 and Table S1. Molecular Cell 2012 47, 87-98DOI: (10.1016/j.molcel.2012.04.029) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 HIPK2 Binds and Phosphorylates H2B at S14 (A) TCEs from HeLa cells were obtained by acid condition or extraction buffer and analyzed by WB with anti-H2B Ab. Ponceau shows the relative loading quantity. (B) eGST, eGST-HIPK2, and its indicated derivatives were produced by T7-vaccinia system in H1299 cells, purified by GST pull-down, and incubated with an equal amount of human recombinant H2B. H2B binding was detected by WB (lower panel). (C) HeLa cells were transfected with EGFP- or EGFP-HIPK2(1-622)-encoding vectors. TCEs were obtained and proteins immunoprecipitated with anti-GFP Ab and analyzed by WB with the indicated Abs. TCE-input lane is loaded with 20% of the immunoprecipitated extract. (D) TCE was obtained from HeLa cells, and proteins were immunoprecipitated with anti-HIPK2 or anti-IgG Abs and analyzed by WB with the indicated Abs. TCE-input lane is loaded with 6% of the immunoprecipitated extract. (E) The indicated purified proteins were prepared as in (B) and incubated with recombinant H2B in the presence of [γ-32P]ATP. Kinase reaction products were resolved by SDS-PAGE and analyzed by autoradiography (left panel) and Coomassie blue staining (right panel). (F) Cold kinase assay was performed as in (E). Kinase reaction products were resolved by SDS-PAGE and analyzed by WB with the indicated Abs. See also Figure S6. Molecular Cell 2012 47, 87-98DOI: (10.1016/j.molcel.2012.04.029) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 HIPK2 Knockout Impairs H2B-S14P but Does Not Affect H2B Localization at the Midbody (A) MEFs were stained with anti-H2B(2) Ab, which preferentially recognizes extranucleosomal H2B, anti-β-tubulin Ab, and DAPI. H2B is detectable at midbody in MEFs from both Hipk2+/+ (top panels) and Hipk2−/− (bottom panels). (B) MEFs were stained with anti-p-Histone H2B-S14(1) Ab, anti-β-tubulin Ab, and DAPI. H2B-S14P is detectable at midbody in Hipk2+/+ MEFs (top panels), but not in Hipk2−/− MEFs (bottom panels). (C) HeLa cells were transfected with HIPK2-HA or HIPK2-K221A-HA expression vectors. Representative images of cells stained with anti-HA and anti-β-tubulin Abs 24 hr posttransfection are reported. (D and E) Hipk2−/− MEFs were transfected with EGFP control vector (Ctr), EGFP-HIPK2 (HIPK2), or EGFP-HIPK2-K221R (K221R) expression vectors and analyzed as described in Figures 1E and 1F. Data represent mean ± SD. (F) Representative images of indicated MEFs stained with anti-p-Histone H2B-S14(1) Ab and DAPI 16 hr after UV irradiation. Molecular Cell 2012 47, 87-98DOI: (10.1016/j.molcel.2012.04.029) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 7 H2B-S14 Phosphorylation by HIPK2 Is Required for Cytokinesis (A and B) HeLa cells stably expressing GFP-tagged wild-type H2B (H2B) or phosphomimetic H2B-S14D (S14D) were obtained. Representative WB showing the expression level of exogenous proteins is reported (A). The levels of the indicated mRNAs were analyzed by real-time RT-PCR (B). Data represent mean ± SD. (C–E) Hipk2−/− MEFs were transfected with control vector (Ctr) or expression vectors encoding GFP-tagged H2B (H2B), H2B-S14A (S14A), and H2B-S14D (S14D). Three days posttransfection GFP-positive cells were stained and analyzed for the percentages of mono-, bi-, and multinucleated cells (C) and LIBs (D). Hipk2−/− MEFs (E) were transfected as in (C). Starting at 16 hr posttransfection, the numbers of GFP-positive cells were measured by direct counting of each dish. EGFP-HIPK2 transfected MEFs were used as control. Data represent mean ± SD. (F) The levels of the indicated mRNAs were analyzed in HeLa cells stably expressing H2B or H2B-S14D as in (B). Molecular Cell 2012 47, 87-98DOI: (10.1016/j.molcel.2012.04.029) Copyright © 2012 Elsevier Inc. Terms and Conditions