pRB- and p53-dependent and -independent steps in formation of SAHF. We propose that DNAJA2 facilitates the cooperative formation of SAHF by the pRB and HIRA/ASF1a pathways. pRB, DNAJA2, and HIRA/ASF1a are enclosed within a dashed box to indicate the activity of these proteins in a protein complex and/or a mutually interdependent network. Dashed lines indicate that the molecular details of SAHF formation remain to be determined. Data in the text also indicate that p16INK4a, pRB, and p53 also contribute to HIRA's localization to PML bodies. However, because these proteins are not essential for this relocalization, these functional interactions have been omitted from the figure for simplicity. See text for details.

PML-RARα disrupts PML bodies and formation of SAHF. (A) WI38 cells were infected with a retrovirus expressing activated Ras, together with a control virus or a virus encoding PML-RARα. Eight days later the cells were stained with antibodies to HIRA and PML and with DAPI to visualize DNA. (B) A total of 100 cells from panel A were scored for the absence or presence of DAPI foci (SAHF). (C) Lysates were prepared from the cells in panel A and subjected to Western blotting to detect expression of activated Ras. Con, control.

Formation of SAHF induced by p16INK4a requires ASF1a. (A) WI38 cells were infected with a retrovirus encoding p16INK4a or a control virus, the cells were selected for infected cells in puromycin for 8 days, and then cells were stained with antibodies to p16INK4a and HIRA and with DAPI to visualize DNA. (B) A total of 100 cells from panel A were scored for localization of HIRA to nuclear foci (PML bodies) and formation of SAHF. (C) WI38 cells were infected with retroviruses encoding p16INK4a and/or an shRNA targeted to ASF1a and/or appropriate control viruses. The virus encoding shRNA to ASF1a also encodes green fluorescent protein and resistance to hygromycin. The cells were then selected in puromycin (for p16INK4a) for 8 days. The cells were stained with antibodies to HIRA and with DAPI to visualize DNA. The percentage of cells with HIRA foci (in PML bodies) and DAPI foci was determined by scoring 100 green fluorescent protein-positive cells. Another aliquot of the infected cells was selected in puromycin and hygromycin for 8 days, and whole-cell extracts were prepared and subjected to Western blotting with antibodies to ASF1a and p16INK4a.

DNAJA2-induced SAHF is partially abolished by SV40 large T-antigen or knock-down of ASF1a. (A) WI38 cells were infected with retroviruses encoding DNAJA2, SV40 large T antigen, or control viruses as indicated. The infected cells were selected in puromycin (for DNAJA2 and control) and neomycin (for SV40 large T antigen and control). Eight days later the cells were stained with DAPI, and 100 cells were scored for formation SAHF (DAPI foci). (B) Lysates were prepared from the cells in panel A and subjected to Western blotting to detect expression of SV40 large T antigen and DNAJA2. (C) WI38 cells were infected with retroviruses encoding DNAJA2, an shRNA to ASF1a, or control viruses as indicated. The cells were selected in puromycin (for DNAJA2 or the control). The virus encoding shRNA to ASF1a (or the control) also encodes green fluorescent protein and resistance to hygromycin. The cells were stained with DAPI to visualize DNA. A total of 100 green fluorescent protein-expressing cells were scored for formation of SAHF. (D) Another aliquot of the infected cells was selected in puromycin and hygromycin for 8 days, and whole-cell extracts were prepared and subjected to Western blotting with antibodies to ASF1a, ASF1b, and DNAJA2. Con, control; Tag, large T antigen.

DNAJA2 is a candidate effector of pRB- and HIRA-mediated SAHF formation. (A) U2OS cells were transfected with plasmids encoding the indicated epitope-tagged proteins. Anti-Flag and anti-HA immunoprecipitations were performed and subjected to Western blotting with anti-HA or anti-Flag to detect the coimmunoprecipitating proteins. (B) WI38 fibroblasts were infected with a retrovirus encoding DNAJA2 or a control virus, and the infected cells were selected in puromycin. Eight days later, the cells were stained with antibodies to HP1β and macroH2A and with DAPI to visualize DNA. A total of 100 cells were scored for presence or absence of SAHF (DAPI foci), macroH2A in SAHF, and HP1β in SAHF, and the results are expressed as a histogram. (C) Representative cells from panel B, infected with control and DNAJA2-expressing retroviruses. The single large domain of intense macroH2A staining in control cells is the inactive X chromosome. IP, immunoprecipitation; Con, control.

HIRA mutants that block localization of endogenous HIRA to PML bodies also prevent formation of SAHF. (A) WI38 cells were infected with retroviruses encoding the indicated HA-tagged wild-type and mutant HIRA proteins and then selected in puromycin for 8 days. Localization of the HA-tagged protein to PML bodies and formation of SAHF was scored by immunofluorescence and DAPI staining, respectively, and positive (+) or negative (−) results are summarized. ASF1a binding data are taken from Zhang et al. (69). (B) WI38 cells were infected with a retrovirus encoding HA-HIRA(520-1017) or a control virus and stained with antibodies to PML and HA and with DAPI to visualize DNA (cells were preextracted with EBC buffer prior to fixation [1]). (C) WI38 cells were infected with the indicated retroviruses; the infected cells were selected by growth in puromycin (HIRA) and neomycin (Ras) and then stained with antibodies to HIRA and PML and with DAPI to visualize DNA. The antibody WC119 used to detect endogenous HIRA does not detect HIRA(520-1017) (data not shown). (D) Results from panel C were quantitated in terms of cells with DAPI foci (SAHF), HIRA localized to PML bodies, and macroH2A and HP1β localized to SAHF. Results are representative of more than three independent experiments. Cont, control.

Recruitment of HIRA to PML bodies is linked to senescence induced by short telomeres and cell stress. (A) WI38 cells were infected with a retrovirus encoding hTERT or a control virus and selected for infected cells with puromycin, and the number of cells with HIRA localized to PML bodies was determined by immunofluorescence at subsequent culture passages. Expression of hTERT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were assayed by RT-PCR. (Inset) A confocal image of a WI38 cell stained by FISH to detect telomeres (red) and with DAPI to detect DNA (blue). (B) Immunofluorescence images of control or hTERT-infected WI38 cells of indicated passage number (pN) from panel A stained with antibodies to HIRA and PML. (C) Lysates were prepared from WI38 and BJ fibroblasts infected with a retrovirus encoding hTERT or a control virus and subjected to Western blotting to detect expression of p16INK4a. “Young” lysates were all prepared at PD2 after virus infection. “Old” lysates were prepared when the cells became senescent (20PD after infection in case of WI38-hTERT) or 20PD after infection in the case of immortal BJ-hTERT. (D) Results of the same experiment as in panel A but performed in BJ fibroblasts. Con, control.

pRB and p53 tumor suppressor proteins are not required for recruitment of HIRA to PML bodies. (A) WI38 cells were infected with retroviruses encoding SV40 ER, hTERT, both, or a control virus, as indicated. The infected cells were selected in puromycin (for hTERT and control) and zeocin (SV40 ER), and the number of cells with HIRA localized to PML bodies was determined by immunofluorescence over the subsequent passages. (B) WI38 cells were infected with a control retrovirus or viruses expressing SV40 large T antigen, hTERT, or SV40 large T antigen and hTERT. The infected cells were selected in puromycin (for control and hTERT) and neomycin (for control and SV40 large T antigen) and then passaged until senescence (control and hTERT) or crisis (SV40 large T antigen) or more than PD150 in case of immortal SV40 plus hTERT. (C) Cells from panel B were pulse-labeled with 5′ BrdU for 2 h and then immunofluorescence stained to detect 5′ BrdU and HIRA. A total of 100 cells were scored for the absence or presence of 5′ BrdU incorporation or HIRA foci (in PML bodies). (D) WI38 cells were infected with retroviruses encoding activated Ras, SV40 large T antigen, or control viruses as indicated, and the infected cells were selected in puromycin (for Ras and control) and neomycin (for SV40 large T antigen and control) for 8 days. The cells were stained with antibodies to HIRA and PML, and the percentage of cells with HIRA localized to PML bodies was determined by scoring 100 cells for colocalization of HIRA and PML. Results are the mean of three independent experiments. Whole-cell lysates were prepared and subjected to Western blotting to detect Ras and SV40 large T-antigen (TAg). (E) WI38 cells were infected with control, activated Ras, and shp16 and shLuc retroviruses; cells were then selected in puromycin (for Ras) for 8 days and stained with antibodies to HIRA and PML (shp16 and shLuc are retroviruses encoding shRNAs to p16INK4a and firefly luciferase, respectively, as well as green fluorescent protein and resistance to hygromycin). The percentage of cells with HIRA localized to PML bodies was determined by scoring 100 green fluorescent protein-positive cells for colocalization of HIRA and PML. Results are the mean of three independent experiments. P values obtained with Student's t test are shown. Another aliquot of the infected cells was selected in puromycin and hygromycin for 8 days; whole-cell extracts were prepared and subjected to Western blotting with antibodies to Ras and p16INK4a. Con, control.

pRB and p53 proteins are required for formation of SAHF. (A) WI38 cells were infected with retroviruses encoding HIRA(421-729) and SV40 large T antigen, selected by growth in puromycin for infection with the HIRA virus, and then stained with antibodies to SV40 large T antigen and with DAPI to visualize DNA. The white arrow indicates a cell with DAPI foci that does not express SV40 large T antigen. (B) WI38 cells were infected with retroviruses encoding SV40 large T antigen in the absence or presence of the indicated HA-tagged HIRA mutants. Cells were selected in puromycin for infection by the HIRA or corresponding control virus, and then 100 individual cells were scored as positive or negative for expression of SV40 T antigen and formation of SAHF, judged by DAPI foci. Data are the percentages of SAHF-positive cells in both the T-antigen-positive and -negative populations of cells. Results are representative of at least three independent experiments. (C) WI38 cells were infected with retroviruses encoding activated Ras, SV40 large T antigen, or control as indicated, and then selected for 8 days in puromycin (for Ras) and neomycin (for SV40 large T antigen). Cells were stained with DAPI to visualize formation of SAHF, and 100 individual cells were scored for SAHF. Results are the mean of three independent experiments. Whole-cell extracts were subjected to Western blotting with antibodies to Ras and SV40 T antigen. This experiment was done in parallel with the experiment described in the legend of Fig. 4D, so the Western blot is the same. (D) WI38 cells were infected with retroviruses encoding SV40 large T antigen or the indicated mutant (K1 and Δ434) and/or ASF1a and/or control vectors. The cells were double-drug selected in puromycin (for ASF1a) and neomycin (for SV40 large T antigen), and 100 cells were scored for the absence or presence of SAHF. Whole-cell extracts were prepared and subjected to Western blotting to detect ASF1a and SV40 large T antigen. (E) WI38 cells were infected with control, activated Ras, shp16, and shLuc retroviruses; cells were selected in puromycin (for Ras) for 8 days and then stained with DAPI to visualize DNA. Shp16 and shLuc are retroviruses encoding shRNAs to p16INK4a and firefly luciferase, respectively, as well as green fluorescent protein and resistance to hygromycin. The percentage of cells with SAHF was determined by scoring 100 green fluorescent protein-positive cells for condensed chromatin by DAPI stain. Results are the mean of three independent experiments. Another aliquot of the infected cells was selected in puromycin and hygromycin for 8 days; whole-cell extracts were prepared and subjected to Western blotting with antibodies to Ras and p16INK4a. This experiment was done in parallel with the experiment described in the legend of Fig. 4E, so the Western blot is the same. LT, large T antigen; Con, control.