Previously, we showed that, in primary human WI38 cells, HIRA localizes to PML nuclear bodies prior to senescence induced by either activated oncogenes or extended growth in culture (69
). In WI38 cells grown under standard culture conditions and ambient (20%) oxygen, senescence after extended growth in culture is thought to occur due to a cell stress response rather than to critically short telomeres (20
). Previous studies showed that ectopic expression of hTERT in WI38 fibroblasts maintains telomere length, but this is insufficient for immortalization (20
). Therefore, to test whether relocalization of HIRA in WI38 cells under these conditions is linked to cell stress or shortening of telomeres, we suppressed the latter by infecting the cells with a virus encoding the catalytic subunit of human telomerase, hTERT. In young mock-infected WI38 cells, HIRA was predominantly diffused throughout the nucleoplasm, although a small proportion of cells did contain HIRA in PML bodies, as usual. As the cells aged in culture, the proportion of cells with HIRA in PML bodies steadily increased, until 90% of the cells contained HIRA in PML bodies (Fig. ). Expression of hTERT did not abolish relocalization of HIRA to PML bodies although it did delay it. The observation that HIRA relocalizes to PML bodies even in WI38 cells expressing hTERT supports the idea that relocalization of HIRA to PML bodies occurs even in cells whose telomeres are not short enough to trigger senescence. Consistent with the idea that there is not an obligatory link between short telomeres and the SAHF-assembly pathway, we confirmed the previous observations of Narita and coworkers that SAHF are spatially distinct from telomeres (45
), this time using a telomeric DNA FISH probe (Fig. , inset). In WI38 cells ectopically expressing hTERT, senescence is thought to be due to upregulation of p16INK4a by the stress of extended cell culture (4
). Indeed, expression of p16INK4a was upregulated during prolonged cell culture of WI38 cells, regardless of ectopic expression of hTERT (Fig. ).
FIG. 1. 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 (more ...)
However, the fact that hTERT expression delayed localization of HIRA to PML bodies suggests that relocalization is also linked to telomere shortening to some extent. In contrast to WI38 cells, BJ fibroblasts grown in ambient oxygen are thought to undergo senescence solely due to shortened telomeres. Indeed, in contrast to WI38 cells, these cells can be immortalized by hTERT expression and do not upregulate p16INK4a after extended cell culture (Fig. ) (4
). Therefore, to directly test whether HIRA also relocalizes to PML bodies in response to short telomeres, we examined HIRA localization as BJ cells were passaged toward senescence. In noninfected and mock-infected BJ cells, HIRA also relocalized to PML bodies as the cells approached senescence (Fig. and data not shown). However, in striking contrast to WI38 cells, in BJ cells relocalization of HIRA to PML bodies was abolished by ectopic expression of hTERT. Together, these data indicate that relocalization of HIRA to PML bodies is linked to senescence triggered by short telomeres in WI38 and BJ cells and by stress-induced expression of p16INK4a in WI38 cells. Previously, we also showed that relocalization of HIRA to PML bodies is induced by an activated Ras oncogene (69
). We conclude that a wide range of senescence triggers causes HIRA's localization to PML bodies.
We previously reported that in WI38 cells, localization of HIRA to PML bodies is a precursor to formation of SAHF (69
). However, as originally reported by Narita and coworkers, BJ cells do not form SAHF during replicative senescence (45
). This indicates that localization of HIRA to PML bodies is not a sufficient trigger for formation of SAHF. Therefore, we probed the relationship between HIRA's localization to PML bodies and formation of SAHF in WI38 cells in more detail. To do this, we sought a way to block HIRA's localization to PML bodies in WI38 cells. Assuming that HIRA is specifically targeted to PML bodies, we reasoned that we ought to be able to identify a mutant of HIRA that is unable to form SAHF but competes out localization of the endogenous protein to PML bodies. As reported previously, wild-type HA-tagged HIRA efficiently localized to PML bodies when ectopically expressed in cells by retrovirus infection (69
) (Fig. ). Next, we tested a panel of HIRA deletion mutants in this assay. The results are summarized in Fig. . Significantly, we identified two HIRA mutants which we previously showed are unable to bind to ASF1a and induce SAHF (69
) but which are still recruited normally to PML bodies (Fig. and data not shown). One HIRA mutant consists of residues 1 to 1017 with a 37-amino-acid deletion of the ASF1a-binding B domain (consisting of residues 439 to 475) [HIRA(1-1017)ΔB], and the other, HIRA(520-1017), carries a large N-terminal truncation and also lacks the B domain. We reasoned that these mutants might compete out the localization of endogenous HIRA to PML bodies and, because the mutant itself cannot induce SAHF, would block the induction of SAHF by endogenous HIRA. Indeed, both mutants blocked localization of endogenous HIRA to PML bodies and formation of SAHF, induced by an activated Ras oncogene (Fig. and data not shown). In contrast, another mutant HIRA(1-400), which does not bind to ASF1a or induce SAHF and did not block localization of endogenous HIRA to PML bodies, did not affect formation of SAHF (Fig. ).
FIG. 2. 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 (more ...)
Concordant with these results, we showed previously by shRNA knock-down that HIRA's binding partner, ASF1a, is required for formation of SAHF (69
). We next attempted to confirm a role for HIRA in formation of SAHF using lentivirus-expressed shRNAs to knock down its expression. However, we were unable to identify shRNAs that stably knocked down HIRA for up to 8 days but did not kill the cells (see Fig. S1 in the supplemental material). Regardless of this result, the data obtained with the HIRA dominant negative mutant, HIRA(520-1017), support the idea that, in WI38 cells, localization of HIRA to PML bodies is necessary for formation of SAHF.
To directly test a role for PML bodies in formation of SAHF, we used a PML-RARα fusion protein to inactivate PML bodies. The PML-RARα fusion protein is expressed in many acute PML cells due to a chromosomal translocation that fuses the two proteins (55
). Expression of PML-RARα causes dispersal of the normally 20 to 30 PML nuclear bodies into a large number of much smaller PML-containing foci or speckles, and this is thought to inactivate PML bodies (55
). As expected, ectopic retrovirus-mediated expression of PML-RARα in WI38 cells disrupted PML bodies (Fig. ). Interestingly, the HIRA foci characteristic of senescent cells were also disrupted, and HIRA was distributed in speckles throughout the nucleus that colocalized with PML. Strikingly, PML-RARα virtually eliminated formation of SAHF in WI38 cells expressing an activated Ras oncogene (Fig. ). These results suggest that the normal undisrupted integrity of PML bodies is essential for formation of SAHF.
FIG. 3. 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 (more ...)
Taken together, these results and results published previously indicate a key role for HIRA's translocation to intact PML bodies in induction of SAHF in senescent WI38 cells (69
). On the other hand, results presented here and published previously also indicate that in BJ fibroblasts translocation of HIRA to PML bodies is not a sufficient trigger for SAHF (45
). Beausejour and coworkers have suggested that at least one difference between WI38 cells and BJ cells is in the activity of the pRB tumor suppressor pathway. Specifically, BJ cells express low levels of the pRB activator, p16INK4a, and so have a less active pRB pathway (4
) (Fig. ). Based on this idea, we hypothesized that the pRB pathway might cooperate with HIRA and ASF1a in induction of SAHF at a point downstream of HIRA's translocation to PML nuclear bodies.
To test this, we analyzed HIRA's localization to PML bodies and formation of SAHF in cells lacking functional p53 and/or pRB. WI38 cells were infected with retroviruses encoding hTERT alone, the ER of the SV40 virus, hTERT plus SV40 ER, or a mock virus. SV40 ER encodes two oncoproteins, small t antigen and large T antigen. SV40 large T antigen binds to and inactivates both the pRB and p53 tumor suppressor proteins (3
). In additional experiments, cells were infected with a virus expressing SV40 large T antigen alone instead of SV40 ER. After infection, the cells were drug selected to enrich for the infected cells and cultured over subsequent passages, and the percentage of cells with HIRA localized to PML bodies was determined by immunofluorescence at each passage. As before (Fig. ), expression of hTERT delayed, but did not abolish, localization of HIRA to PML bodies (Fig. ). These hTERT-expressing cells exhibited an extended life span in culture but ultimately became senescent, as expected, and assumed a large flat morphology similar to the control infected cells (Fig. ). Senescent control and hTERT-infected cells did not incorporate 5′ BrdU, an indicator of DNA synthesis in proliferating cells (Fig. ). Strikingly, infection with SV40 ER or SV40 large T antigen did not abrogate recruitment of HIRA to PML bodies (Fig. ) in either the absence or presence of hTERT. As shown previously, human WI38 fibroblasts expressing both hTERT and SV40 ER or large T antigen were immortal; they never assumed a senescent morphology and could be maintained indefinitely as a culture that readily labels with 5′ BrdU (29
) (Fig. ). In contrast, WI38 cells expressing either SV40 ER or SV40 large T-antigen alone entered crisis, characterized by a large number of rounded dead cells and many cells that incorporate 5′ BrdU (54
) (Fig. ). The ability of SV40 ER and large T antigen to drive cells into crisis or immortalization, in the absence and presence of hTERT, respectively, confirms the biological activity of large T antigen in these cells. To further verify the activity of ectopically expressed SV40 large T antigen in these cells, we tested it for binding to the p53 tumor suppressor protein. Immunoprecipitation of SV40 large T antigen coprecipitated p53 and vice versa, showing that large T antigen is functional in these cells and physically bound to p53 and, presumably, pRB (see Fig. S2 in the supplemental material). Together, these results show that HIRA is still localized to PML bodies in cells in which pRB and p53 are inactivated by SV40 large T antigen. In the experiments shown in Fig. , extended growth in culture acted as the trigger for HIRA's relocalization to PML bodies. Previously, we showed that HIRA also relocalizes to PML bodies during senescence triggered by an activated Ras oncogene (45
). To test whether relocalization of HIRA under these conditions is also independent of pRB and p53, cells were coinfected with retroviruses encoding activated Ras and SV40 large T antigen, and the cells were then drug selected for the infected cells. Immunofluorescence was performed to determine HIRA's localization. Large T antigen did not affect localization of HIRA to PML bodies, even though Western blotting confirmed that both T antigen and activated Ras were expressed as expected (Fig. ). Finally, to confirm by another method that the pRB pathway is not required for HIRA's recruitment to PML bodies, we used a retrovirus to deliver an shRNA that knocks down p16INK4a. Like ectopic expression of large T antigen, this had no significant effect on HIRA's localization to PML bodies compared to activated Ras alone or activated Ras plus a control shRNA to luciferase (Fig. ). In sum, these experiments show that recruitment of HIRA to PML bodies does not require functional pRB or p53 tumor suppressor pathways.
FIG. 4. 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 (more ...)
Next, we asked whether pRB and p53 are required for formation of SAHF. Previously, we showed that ectopic expression of wild-type HIRA and some mutants, e.g., HIRA(421-729), accelerates formation of SAHF (69
). WI38 cells were infected with retroviruses encoding wild-type HIRA or HIRA(421-729), together with a virus encoding SV40 large T antigen. The cells were drug selected for infection by the HIRA virus and scored for expression of SV40 large T antigen and formation of SAHF by immunofluorescence. Ectopic expression of wild-type HIRA or HIRA(421-729) increased the number of cells with SAHF (Fig. ). Expression of a mutant lacking the ASF1a-binding B domain, HIRA(421-729)ΔB, did not induce SAHF. Coexpression of SV40 large T antigen largely abolished formation of SAHF induced by HIRA (Fig. ). Likewise, large T antigen largely abolished formation of SAHF induced by an activated Ras oncogene (Fig. ). Since SV40 large T antigen binds to and inactivates at least two key tumor suppressor proteins, pRB and p53, we tested a pair of T-antigen mutants to determine which pathway, if either, is responsible for formation of SAHF. As before, wild-type large T antigen efficiently blocked formation of SAHF, this time induced by ectopic expression of ASF1a (Fig. ). By comparison, the SV40 large T-antigen K1 mutant that is specifically impaired in binding to pRB (and related proteins p107 and p130) was impaired in its ability to block SAHF. Likewise, another mutant, T-antigen Δ434, which is impaired in its ability to bind to p53, was also impaired (Fig. ). This indicates that both the pRB and p53 pathways contribute to formation of SAHF. Because SV40 T-antigen Δ434 was underexpressed compared to wild-type T antigen, to confirm a role for the pRB pathway we used a retrovirus-encoded shRNA to knock down p16INK4a. Comparable to the large T-antigen Δ434 mutant that also inactivates the pRB pathway but not the p53 pathway, knock-down of p16INK4a reduced SAHF by about 50% (Fig. ). We conclude that efficient formation of SAHF depends on active pRB and p53 pathways in WI38 cells.
FIG. 5. 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 (more ...)
In light of the possibility that differential formation of SAHF in WI38 cells and BJ cells might depend on the relative activity of the pRB pathway in the two cell types, we were particularly interested in the role of this pathway. Based on our results so far, the pRB pathway might be required for formation of SAHF because the HIRA/ASF1a pathway activates the pRB pathway and this, in turn, makes SAHF. Consistent with this idea, pRB is known to recruit heterochromatin-forming enzymes to chromatin (70
). Alternatively, the HIRA/ASF1a pathway and the pRB pathway might converge and cooperate to make SAHF at a point downstream of HIRA's localization to PML bodies. To differentiate between these two models, we asked whether SAHF induced by forced activation of the pRB pathway requires the histone chaperone, ASF1a. Lowe and coworkers previously showed that ectopic expression of the pRB activator, 16INK4a, induces SAHF. This is consistent with either of the two models outlined above (45
). However, only the second “convergent/cooperating pathways” model predicts that inactivation of ASF1a will prevent induction of SAHF in cells in which the pRB pathway is activated. According to this second model, formation of SAHF requires simultaneous activity of both pathways.
As shown previously, forced expression of p16INK4a in WI38 cells induced formation of SAHF, judged by 4′,6′-diamidino-2-phenylindole (DAPI) staining and visualization of condensed chromatin (Fig. ). However, coinfection of the p16INK4a-encoding virus with a virus encoding an shRNA targeted to ASF1a reduced formation of SAHF (Fig. ). Interestingly, ectopic expression of p16INK4a also induced localization of HIRA to PML bodies (Fig. ). The mechanism behind this is not clear. However, we observed that shRNA-mediated knock-down of ASF1a did not affect HIRA localization (Fig. ). This indicates that in cells ectopically expressing p16INK4a but lacking ASF1a due to its shRNA-mediated knock-down, p16INK4a activity is still elevated. In sum, these data show that even in the presence of elevated p16INK4a/pRB activity, formation of SAHF requires ASF1a activity. This supports the “convergent/cooperating pathways” model (see Fig. ).
FIG. 6. 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 (more ...)
FIG. 9. 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 (more ...)
Based on these data, we hypothesized that the HIRA/ASF1a and pRB pathways converge downstream of HIRA's localization to PML bodies. Remarkably, a single protein, DNAJA2, has been independently cloned as a HIRA and pRB binding protein in two-hybrid screens performed by two different laboratories. Specifically, Lorain and coworkers identified DNAJA2 as an HIRA-binding protein (40
). Subsequently, Benevolenskaya and coworkers identified DNAJA2 in a differential two-hybrid screen intended to identify proteins that contribute to pRB-mediated cell differentiation (6
). Importantly, the BIND database (www.bind.ca/Action
) lists no other protein-protein interactions for DNAJA2, suggesting that the reported interactions with HIRA and pRB are relatively specific.
To be able to ask whether DNAJA2 physically interacts with pRB and HIRA in vivo, we raised a rabbit polyclonal antibody to DNAJA2. The purified antibody specifically detected DNAJA2 in Western blot analysis, as judged by reaction with a single polypeptide close to the predicted molecular mass of 46 kDa, whose expression was knocked down by specific small interfering RNA toward DNAJA2 (see Fig. S3 in the supplemental material). Unfortunately, we were unable to immunoprecipitate DNAJA2 from cell extracts, unless the extracts were first boiled in denaturing buffer containing 1% sodium dodecyl sulfate and then diluted (see Fig. S3 in the supplemental material). This indicates that DNAJA2 is probably contained in protein complexes and not accessible to antibodies, therefore preventing us from asking whether endogenous DNAJA2 is bound to HIRA and pRB in cells.
As an alternative approach to ask whether DNAJA2 physically interacts with HIRA and pRB in vivo, we ectopically expressed Flag-tagged DNAJA2 with either HA-tagged HIRA or HA-pRB in U2OS cells and tested for interactions by coimmunoprecipitation analysis. When coexpressed with Flag-DNAJA2, both HA-HIRA and HA-pRB efficiently coprecipitated in an anti-Flag immunoprecipitation but not when ectopically expressed Flag-DNAJA2 was omitted from the reaction (Fig. ). Similarly, Flag-DNAJA2 coimmunoprecipitated in an anti-HA immunoprecipitation when coexpressed with either HA-pRB or HA-HIRA (Fig. ). As an additional specificity control, Flag-DNAJA2 did not bind to the largest subunit of the chromatin assembly factor-I complex, p150CAF-I, when it was overexpressed as an HA-tagged protein (see Fig. S3 in the supplemental material). We conclude that DNAJA2 specifically binds to pRB and HIRA when the proteins are coexpressed in U2OS cells.
FIG. 7. 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 (more ...)
Next, we wanted to ask whether DNAJA2 plays a role in formation of SAHF. First, we set out to ask whether DNAJA2 is necessary for formation of SAHF. Since formation of SAHF takes several days or weeks, depending on whether it is induced by activated oncogenes or extended growth in culture, we attempted to generate retroviruses encoding shRNAs to stably knock down DNAJA2. Unfortunately, we were unable to generate a virus-encoded shRNA that stably knocks down DNAJA2 without killing the infected cells (data not shown). Thus, DNAJA2 appears to have one or more essential functions that preclude us from specifically testing a requirement for SAHF formation. Therefore, to address a role for DNAJA2 in formation of SAHF, we asked whether ectopic expression of DNAJA2 is sufficient to trigger formation of SAHF. As shown in Fig. , ectopic expression of DNAJA2 efficiently promoted formation of SAHF, as indicated by formation of DAPI foci enriched in HP1 proteins and macroH2A. DNAJA2-induced SAHF was only partially abolished by inactivation of pRB and p53 by SV40 large T antigen (Fig. ) or by shRNA-mediated knock-down of ASF1a (Fig. ), suggesting that DNAJA2 does not act exclusively up- or downstream of HIRA and pRB. In sum, DNAJA2 physically interacts with pRB and HIRA, and its ectopic expression accelerates formation of SAHF. These data strongly suggest that DNAJA2 plays a role in formation of SAHF, perhaps by integrating signals from the pRB and HIRA/ASF1a pathways (Fig. ).
FIG. 8. 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 (more ...)