Figure 1.
Presence of ASF1a and HIRA inside PML bodies in ALT cells and normal fibroblasts.
(A) p21 was upregulated in C7 and C8 cells, that express a p53-ER fusion protein, after 4 days of 4OHT-treatment. The western blot was probed with the indicated antibodies. (B) Untreated, proliferating C7 cells were stained for ASF1a and HIRA. No ASF1a and HIRA foci were present in these nuclei. (C) Triple immunofluorescence of ASF1a, HIRA and PML in C8 cells treated with 4OHT for 4 days, and in senescent IMR-90 fibroblasts, showed colocalization of ASF1a and HIRA in PML bodies. (D) Immunostaining showed colocalization of ALT-associated PML bodies (APBs; visualized here as large TRF2 foci) and ASF1a or HIRA in C7 and C8 cells treated with 4OHT for 4 days. The antibodies used for this figure and subsequent figures are described in Materials and Methods and are only specified in the figure legends when Materials and Methods lists more than one antibody against that protein; here the mouse anti-HIRA (B, C), rabbit anti-HIRA (D), mouse anti-p21 (A), goat anti-p21 (D) and goat anti-PML (C) antibodies were used. Bars, 20 µm.
Figure 2.
Localization of HIRA in PML bodies is independent of ASF1a.
(A) C7 cells were triple stained for ASF1a, HIRA and PML 24 h after treatment with 4OHT. At this early time point, HIRA was more readily detected inside PML bodies than ASF1a. (B) The effectiveness of ASF1a siRNAs (ASF1a and ASF1a-2) and HIRA siRNAs (HIRA-2 and HIRA-4) was demonstrated in IIICF-T/B3 cells by Western blots, that were probed with the indicated antibodies. (C) Triple immunostaining of ASF1a, HIRA and PML in C7 cells after treatment with the indicated combinations of siRNAs for 5 days, and with 4OHT for 3 days before the end of siRNA-treatment. Distinct HIRA foci colocalized with PML bodies in cells depleted of ASF1a, while ASF1a was absent from PML bodies in cells depleted of HIRA. The arrows indicate cells with knockdown of ASF1a (top row) or HIRA (bottom row). Antibodies used here included mouse anti-HIRA (A, C), rabbit anti-HIRA (B) and goat anti-PML (A, C). Bars, 20 µm.
Figure 3.
HIRA is required for p53/p21-mediated induction of APBs.
(A) Triple immunostaining with the indicated antibodies in C7 cells after treatment with HIRA or ASF1a siRNA for 5 days, and with 4OHT for 3 days before the end of siRNA-treatment. Knockdown of HIRA, but not ASF1a, largely prevented p53/p21-mediated induction of APBs. The arrows indicate cells with knockdown of HIRA (top row) or ASF1a (bottom row). (B) IIICF-T/B3 cells were stained for SV40T, p21 and TRF1 4 days after transfection of the indicated combinations of siRNAs. HIRA siRNA (HIRA-2), but not ASF1a siRNA (ASF1a-2), significantly suppressed SV40T siRNA-mediated induction of APBs (visualized here as large TRF1 foci). Antibodies used here included rabbit anti-HIRA (A), goat anti-p21 (B) and goat anti-PML (A). Bars, 20 µm.
Table 1.
Proportion of APB-positive C7 cells after treatment with siRNAs and 4-hydroxytamoxifen (4OHT).
Table 2.
Proportion of APB-positive IIICF-T/B3 cells after siRNA-treatment.
Table 3.
Proportion of APB-positive IIICF-T/B3 cells after siRNA-treatment.
Figure 4.
HIRA is required for p53/p21-mediated formation of large HP1 foci.
(A) Triple immunostaining showed colocalization between HIRA and HP1α inside PML bodies in C7 cells treated with 4OHT for 2 days. (B) IIICF-T/B3 cells were stained for SV40T, HP1α and PML 4 days after transfection of the indicated combinations of siRNAs (T, SV40T; C, control). HIRA siRNA (HIRA-4), but not ASF1a siRNA (ASF1a), largely blocked SV40T siRNA-mediated induction of large HP1α foci. Antibodies used here included rabbit anti-HIRA (A), mouse anti-HP1α (A), rabbit anti-HP1α (B) and goat anti-PML (A, B). Bars, 20 µm.
Table 4.
Proportion of IIICF-T/B3 cells with large HP1α-foci after siRNA-treatment.
Figure 5.
ASF1a and ASF1b are both dispensable for APB formation.
(A) Triple immunostaining of ASF1b, TRF2 and PML in C7 cells treated with 4OHT for 4 days. No ASF1b foci were observed inside APBs. (B) The effectiveness of ASF1b siRNAs (ASF1b and ASF1b-2) was demonstrated in IIICF-T/B3 cells by immunoblotting. The blots were probed with the indicated antibodies. (C) Triple staining of SV40T, p21 and TRF1 in IIICF-T/B3 cells treated with the indicated combination of siRNAs (T, SV40T; A2, ASF1a-2; B2, ASF1b-2) for 4 days. APBs (visualized here as large TRF1 foci) were still detected in cells that were negative for SV40T. Antibodies used here included rabbit mAb anti-ASF1b (A), rabbit anti-ASF1b (B), goat anti-p21 (C) and goat anti-PML (A). Bars, 20 µm.
Figure 6.
Accumulation of ASF1a inside nucleoli.
(A) Immunostaining of proliferating C7 cells showed colocalization between ASF1a and the nucleolar marker B23. (B) Triple staining of ASF1a, Ki67 and DNA in C7 cells treated with 4OHT for 4 days. ASF1a was largely absent from the nucleoli of Ki67-negative cells. (C) ASF1a-staining of proliferating IIICF/c, Hela and IMR-90 cells showed nucleolar localization of ASF1a in all three cell lines. Bars, 20 µm.
Figure 7.
A hypothetical model for HIRA and ASF1a in APB formation and senescence in ALT cells.
Growth arrest/senescence signals trigger relocalization of HIRA to PML bodies, which leads to the subsequent translocation of HP1α/γ, telomeric DNA and its associated shelterin proteins, each of which is essential for APB formation. The dashed line indicates that translocation of ASF1a to PML bodies and its colocalization there with HIRA is not essential for APB formation, even though HIRA and ASF1a are both required for the establishment of the senescent phenotype in ALT cells.