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Genes to Cells (2007) 12, 1371-1382. doi:10.1111/j.1365-2443.2007.01135.x
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Polycomb complexes regulate cellular senescence by repression of ARF in cooperation with E2F3

 Jun Miki1, Yu-ichi Fujimura2, Haruhiko Koseki2 and Takehiko Kamijo1,3,*

1 Department of Pediatrics, Shinshu University School of Medicine, Matsumoto, Nagano 390-8621, Japan
2 Developmental Genetics Group, RIKEN Research Center for Allergy and Immunology, Tsurumi-ku, Yokohama 230-0045, Japan
3 Division of Biochemistry, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuoh-ku, Chiba 260-8717, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Cellular senescence is a program in normal cells triggered in response to various types of stress that cells experience when they are explanted into culture. In this study, functional analyses on the role of the class II polycomb complex in cellular senescence were performed using mouse embryo fibroblasts (MEFs) with a genetically deleted member of the complex, Mel18. Mel18-null MEFs undergo typical premature senescence accompanied by the up-regulation of ARF/p53/p16INK4a and decrease of Ring1b/Bmi1. Our results demonstrated that ARF or p53 deletion cancels the senescence in Mel18-null MEFs, and the fact that p16INK4a is up-regulated in double-null MEFs suggests that the ARF/p53 pathway plays a central role in stress-induced senescence. The in vivo binding of Ring1b and E2F3b to the ARF promoter decreased progressively in senescence, and Mel18 inactivation accelerated the exfoliation of Ring1b/E2F3b from the promoter sequence, indicating the cooperation of polycombs/E2F3b on ARF expression and cellular senescence. Taken together, it seems that class II polycomb proteins and E2F3b dually control cellular senescence via the ARF/p53 pathway.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Cellular senescence is a fundamental cellular program that is activated in various stress situations and acts to prevent further cell proliferation. As a population of cells is propagated in culture, cells are exposed to various extrinsic and intrinsic stresses, and the cell population gradually stops dividing. In contrast to ‘replicative senescence’ which stands for the widely accepted model of a terminal growth arrest due to telomere attrition, it is now clear that many stimuli, including oxidative stress, DNA damage and oncogene activation cause an acutely inducible, ‘premature’ form of cellular senescence which is independent of telomere attrition. Cellular senescence is considered to play an important role in tumor suppression and to contribute to organismal aging. In fact, two definitive tumor suppressor pathways, ARF/MDM2/p53 and p16INK4a/Rb, have been shown to play critical roles in the induction of cellular senescence (Sherr & DePinho 2000).

Polycomb group (PcG) genes were first identified in Drosophila as a group of genes required to maintain the stable repression of Hox cluster genes during development. An increasing number of mammalian genes structurally and functionally related to Drosophila PcG genes have been identified, including Mel18, Bmi1, M33 and rae28, and shown to form multimeric protein complexes associated with chromatin (Levine et al. 2004). There are increasing lines of evidence that PcG proteins themselves affect cellular proliferation and cellular senescence. Targeted disruption of Bmi1, Mel18, rae28 and M33, members of the class II PcG complex, leads to proliferation defects in hematopoietic stem cells (Lessard et al. 1999; Ohta et al. 2002; Park et al. 2003; Iwama et al. 2004) and mouse embryo fibroblasts (MEF) (Bmi1, Jacobs et al. 1999; M33, Core et al. 1997; Phc2, Isono et al. 2005), indicating that inactivation of these PcGs results in cell proliferation failure. Premature senescence of MEFs derived from Bmi1-, M33- and Phc2-null mice has been shown to be mediated by de-repression of the central mediators of senescence signals, p19ARF and p16INK4a, which are encoded by the p15INK4b/p19ARF/p16INK4a genomic region (INK4/ARF region). The molecular mechanism underlying the transcriptional regulation of these genes by mammalian PcG complexes, however, has not yet been appropriately addressed, except for physical interactions of Bmi1 and Phc2 gene products with p19ARF and p16INK4a genomic regions (Jacobs et al. 1999; Itahana et al. 2003; Isono et al. 2005). The Mel18 component of PcG complexes shares homologous amino acid sequences with Bmi1 proteins and interacts with almost the same set of mammalian PcG proteins (Jacobs & van Lohuizen 2002). Accordingly, Mel18- and Bmi1-knockout mice exhibited similar phenotypes in axial and hematopoietic systems, and phenotypic analyses of Mel18/Bmi1 double-knockout mice revealed their functional redundancy. However, it is also true that the molecular functions of Mel18 and Bmi1 are not totally identical since their deficient phenotypes were found to be mutually distinct in various aspects (van der Lugt et al. 1994; Akasaka et al. 1996). This was supported by recent biochemical analysis that Bmi1 enhances Ring1b-mediated ubiquitinylation of histone H2A more efficiently than Mel18 (Cao et al. 2005). Although it has been reported that Mel18-null MEFs undergo premature senescence similar to Bmi1 mutants, the molecular mechanisms underlying Mel18-mediated regulation of senescence remain to be elucidated (Kanno et al. 1995; Jacobs et al. 1999), particularly, whether its action involves the transcriptional regulation of p19ARF and p16INK4a, as in Bmi1.

E2F proteins have been shown to control the expression of a large number of genes involved in DNA replication, cell cycle progression and cell fate determination (Sears & Nevins 2002). The E2F family is composed of six distinct gene products (E2F1–E2F6) that form heterodimeric complexes with partners of the DP family, DP-1 and DP-2. E2F1–E2F3 act as positive regulators of transcription, whereas E2F4–E2F6 function primarily as transcriptional repressors. E2F3 protein appears to be particularly important for cell proliferation, as seen from the inhibition of E2F3 activity by antibody microinjection (Leone et al. 1998). Furthermore, the loss of E2F3a+b is shown to de-repress ARF, triggering the activation of p53 and expression of p21Cip1/Waf1 (Humbert et al. 1998). ARF mutation in E2F3a+b mutants suppresses p21Cip1/Waf1, and rescues the known cell cycle re-entry defect of mutant MEFs. Moreover, in wild-type MEFs, the ARF promoter is predominantly occupied by E2F3b, which differs from E2F3a in its N-terminal sequence, suggesting that transcription of ARF is negatively controlled primarily by E2F3b in cell culture (Aslanian et al. 2004). In that study, the authors suggested the presence of E2F3 co-repressors cooperating in the regulation of ARF transcription. A recent study by Core et al. (2004) has shown that the premature senescence of M33-null MEFs is canceled by a transdominant negative form of E2F (E2F-DB). This observation suggests that E2F family proteins mediate de-repression of ARF/p16INK4a expression in MEFs deficient in genes encoding components of class II PcG complexes; however, the molecular mechanisms underlying the functional correlation between PcG complexes and E2F3s are not fully known.

In the present study, we first addressed the role of Mel18 in the cellular senescence of MEFs. Severe proliferation disturbance, up-regulation of p19ARF/p53/p16INK4a and decrease of Ring1b/Bmi1 were observed in Mel18-null MEFs. Genetic deletion of ARF or p53 cancelled premature senescence, confirming the importance of the ARF/p53 pathway in senescence. We further analyzed the physical associations of PcG and E2F3 proteins with the INK4/ARF region in wild-type and Mel18-null MEFs by chromatin immunoprecipitation (ChIP) assays. Associations of Ring1b components with class II PcG complexes and E2F3b with the ARF promoter region were found to be collinearly impaired in Mel18-null MEFs. It is thus presumed that the lack of a single PcG component may affect the amount of class II PcG complex and the binding of class II complexes with the ARF promoter region, which may in turn affect the association of E2F3b and result in de-repression of the ARF gene. Taken together, the association of E2F3b with the ARF promoter, which is regulated at least in part by PcG complexes, may be one of the essential parameters that mediate cellular proliferation and senescence in MEFs.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Mel18 inactivation impairs cell growth of MEFs

Normally, the growth of passaged wild-type MEFs gradually decreases in sequential in vitro culture (Kamijo et al. 1997), but Mel18-null MEFs grew very slowly from the beginning of culture and soon stopped dividing, as reported previously (Fig. 1a; Jacobs et al. 1999). Consistent with this, the proliferation of Mel18-null MEFs was significantly slower than their wild-type littermate-derived MEFs in growth speed assays at passage-3 (Fig. 1b). Indicative of premature senescence, we found that Mel18-null MEFs exhibited strong positive staining for the SA-β-Gal enzyme as well as Bmi1-null MEFs, whereas only a few cells stained positive for the SA-β-Gal enzyme in wild-type MEF controls (Fig. 1c). In addition, Mel18-null and Bmi1-null MEFs exhibited enlarged nuclei and cell bodies.


Figure 1
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  		Figure 1  Mel18-null MEFs showed proliferation failure accompanied by ARF/p53/p16INK4a activation. (a) Each data point represents the average of a minimum of three separate culture experiments as indicated in Experimental procedures. (b) Growth speeds were determined by growth speed assays for cells taken at passage-3. The values represent average and standard deviations of triplicate cultures. (c) Passage-5 MEFs, which had been cultured by the protocol used in Fig. 1a, were counted and seeded on a 3-cm diameter culture dish. Senescence-associated β-galactosidase enzyme activity was studied as indicated in Experimental procedures. (d) Cells were treated with indicated stresses (low serum, UV and VP-16) for 24 h and then % of sub-G0/G1 was determined by FACScan.

 
To examine whether the proliferation failure of Mel18-null cells was not caused by the acceleration of apoptotic cell death, we treated Mel18-null and wild-type MEFs with serum starvation, ionizing irradiation and DNA topoisomerase II inhibitor, VP-16 and then assessed apoptotic cell death by FACS. There was no significant difference in the rate of apoptosis against these stresses between Mel18-null cells and controls (Fig. 1d); therefore, mitotic failure of Mel18-null MEFs was shown to not involve the acceleration of apoptotic outbursts.

Effects of Mel18 inactivation on INK4/ARF/p53 pathways and class II polycomb proteins

We then performed Western blot analysis of the molecules negatively controlling cell cycle progression. In wild-type MEFs, proliferative capacity was found to dwindle and eventually cease, and the cells expressed elevated levels of negative cell cycle regulators, including the CDK inhibitors p16INK4a and p21Cip1/Waf1, the p53 inducer p19ARF and p53 itself (Kamijo et al. 1997; Zindy et al. 1998). In the passaged cells, p19ARF, p53, p16INK4a and p53-downstream p21Cip1/Waf1/MDM2 were already up-regulated in Mel18-null MEFs compared with their wild-type littermate-derived MEFs (Fig. 2a). We studied the expression of ARF mRNA and p16INK4a mRNA by RT-PCR assay (Fig. 2b). Up-regulation of ARF and p16INK4a mRNA expression was clearly observed in Mel18-null MEFs compared with wild-type littermate MEFs, indicating that the transcription of cell cycle-negative regulators was increased in Mel18-null MEFs.


Figure 2
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  		Figure 2  Mel18 deficiency induces ARF/p16INK4a transcription and reduces class II PcG protein amounts. (a and c) MEFs grown by the indicated culture protocol used in Fig. 1a were sampled at each passage for the indicated protein expression by SDS PAGE/direct Western blotting. (b) RT-PCR amplification of p16INK4a and p19ARF RNAs. Lane 1 shows the products recovered from equal amounts of p53-mutated MEFs-derived RNA used as a positive control. Lane 2 shows the results with no templates. Lanes 3 and 4 show results of wild-type MEFs and Mel18-null MEFs, respectively, at passage-6. ARF, p16INK4a and G3PDH were amplified as described in Experimental procedures. (d) RT-PCR amplification of PcGs. Total RNAs were extracted from wild-type or Mel18-null MEFs and then the expressions of Bmi1, Ring1b and Mel18 were studied by RT-PCR.

 
Next, we analyzed the effect of Mel18 knockout on class II PcG proteins and E2F3 proteins. Class II PcG proteins, Ring1b and Bmi1 were clearly down-regulated in Mel18-null MEFs although E2F3a and E2F3b were hardly modulated (Fig. 2c). The down-regulation of PcG proteins seemed to be caused at the protein level since mRNA expression of Ring1b and Bmi1 was similar in both wild-type and Mel18-null MEFs (Fig. 2d).

Loss of ARF or p53 reverses proliferation failure of Mel18-null MEFs

We generated and analyzed Mel18/ARF- and Mel18/p53-knockout mice as described in Experimental procedures to address the effects of ARF or p53 on premature senescence in Mel18-null MEFs. Deletion of ARF or p53 with the Mel18-null background rescued premature growth arrest in modified 3T9 assays (Fig. 3a) and growth speed retardation of passage-6 MEFs (Fig. 3c) due to Mel18 loss. Since the proliferation of ARF/Mel18 and p53/Mel18 double-null MEFs was arrested by re-induction of ARF and p53 by retroviruses, respectively, as observed to a similar extent in ARF and p53 single-null MEFs, activation of ARF and p53 may be a rate-limiting process to mediate growth arrest in Mel18-null MEFs (Fig. 3c-1 and c-2). Intriguingly, the proliferation of ARF- or p53-single-null MEFs was faster than those of ARF/Mel18- or p53/Mel18-double-null MEFs, respectively (Fig. 3c-1 and c-2, mock transfection).


Figure 3
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  		Figure 3  ARF or p53 inactivation cancels cellular senescence in Mel18-null MEFs. (a) Cells were passaged at 3-day intervals, counted, and re-plated according to the modified 3T9 protocol. Each data point represents the average of a minimum of three separate assays. Genotypes: Mel18-null (filled diamond), ARF/Mel18-null (filled circle) and p53/Mel18-null (filled triangle). (b) MEFs cultured by the modified 3T9 protocol were sampled at passage-12 for the indicated protein expression by direct Western blotting. Equal quantities of total cell lysate (30 µg of protein) were loaded per lane of mini-SDS-PAGE gel, transferred onto PVDF membranes, and probed with the antibodies indicated in the left margin. Lane 1, wild-type; lane 2, Mel18-null; lane 3, ARF/Mel18-null; lane 4, p53/Mel18-null MEFs. (c-1) ARF-null (square) and ARF/Mel18-null (circle) MEFs (passage-7) were infected with mock virus (closed) or ARF virus (open), and then diluted to 2 x 104 per 60-mm diameter dish and replica plated. Individual cultures harvested every 2 days thereafter were counted. Values represent average and standard deviations of triplicate cultures. (c-2) p53-null (diamond) and p53/Mel18-null (triangle) MEFs (passage-7) were infected with mock virus (closed) or wild-type p53 virus (open). Cell proliferation analysis was then performed, as in Fig. 3c-1. Primer sequences for RT-PCR analysis are listed in Table 1.

 
Western blot analysis of passage-12 MEFs in modified 3T9 experiments indicated a significant reduction of p53-downstream molecules, p21Cip1/Waf1 and MDM2, in both double-null MEFs. In contrast, the expression of p16INK4a was even increased in double-null cells compared to Mel18 single mutants (Fig. 3b; Kamijo et al. 1997; Zindy et al. 1998). These findings suggest that ARF and p53 are key players in the premature senescence of Mel18-null cells, and p21Cip1/Waf1 may be an especially important effector in arrested Mel18-null cells.

Binding of PcG proteins to p15INK4b/p19ARF/p16INK4a genomic locus

Recently, we reported the binding of Phc2, which interacts with Mel18, to p16INK4a exons 1 and 2 genomic sequences in developing embryos (Isono et al. 2005); however, it has not been addressed whether this association involves PcG complexes. We thus examined the binding of Mel18 and Ring1b, which interact with both Mel18 and Phc2, with ChIP assays (Suzuki et al. 2002). Immunoprecipitated genomic DNA fragments from 11.5 dpc embryos were subjected to PCR reactions using primer pairs (Table 2), which amplified the genomic regions schematically indicated in Fig. 4a. The region including the E2F site in the ARF promoter region was characterized by strong binding with Ring1b, although there was weak binding with Mel18 (Fig. 4b). In contrast, the p16 promoter sequence was characterized by abundantly bound Ring1b and Mel18 (Fig. 4c). Species-matched immunoglobulins were negative controls for ChIP experiments (Fig. 4b–d). In summary, although Ring1b association was seen in all regions examined, Mel18 binding was missing from several regions (Fig. 4a). This may localize the physical association of PcG complexes to an approximately 20 kb genomic region in and around the p15INK4b/p19ARF/p16INK4a locus, and suggest a model of compositional heterogeneity of PcG complexes.


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  		Table 2  Primer sequences used in Fig. 4
 

Figure 4
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  		Figure 4  Analysis of the in vivo binding of polycomb proteins in the INK4/ARF region. (a) 11.5-dpc embryos were subjected to ChIP assays as described in Experimental procedures with anti-Ring1b mouse monoclonal antibody and anti-Mel18 goat serum. The squares represent the degree of enrichment of immunoprecipitated genomic DNA. Each square means twofold enrichment in comparison with un-fractionated ‘input’ DNA. (b–d) Representative results of three ChIP experiments which indicated the association of these polycomb proteins with the INK4/ARF locus: E2F site (b), p16 promoter (c) and p19ARF exon 1 (d). ‘Ig’ means species-matched immunoglobulin as a negative control for immunoprecipitation (for Ring1b: mouse Ig, for Mel18: goat Ig).

 
Collinear decrease of Ring1b and E2F3b binding to the E2F site upon synergic effects of culture stress and Mel18 deficiency

We went on to examine the impact of replicative stress and Mel18 deficiency on Ring1b binding at the E2F binding site in the ARF promoter, 1st exon of p19ARF (1st exon) and p16INK4a promoter (Fig. 5a). Although all these regions bound Ring1b, the functional E2F site was only seen to bind to the ARF promoter. Wild-type and Mel18-null MEFs cultured according to the modified 3T9 protocol were subjected to ChIP assays. Both wild-type and Mel18-null MEFs showed growth retardation at passage-6 in the modified 3T9 assay. In particular, proliferation ability was apparently impaired in Mel18-null MEFs (Fig. 1a,b). In wild-type MEFs, Ring1b association in these three regions was decreased in passage-6 MEFs compared with passage-2. In Mel18-null MEFs, Ring1b association was more strongly decreased than in the wild-type, which was highest in the region including an E2F site in comparison with the 1st exon of ARF (1st exon) and p16INK4a promoter; therefore, Ring1b binding to the region including an E2F site was synergistically impaired by culture stress and Mel18 deficiency, although a weak association of Mel18 with this region in 11.5 dpc embryos was observed (Fig. 4b). We thus re-examined Mel18 association with the E2F site using MEFs (Fig. 5b). We found Mel18 association with the p16 promoter but to neither the E2F site nor the 1st exonic region of the ARF gene. Mel18-null MEFs provided negative controls for this experiment; therefore, although Mel18 itself does not directly bind to the genomic region around the E2F site of the ARF promoter, Mel18 seems to regulate the binding of class II PcG complexes to the p15INK4b/p19ARF/p16INK4a genomic locus by forming a complex with at least Ring1b.


Figure 5
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  		Figure 5  In vivo binding of polycomb proteins and E2F3s are attenuated in Mel18-null MEFs. MEFs derived from Mel18-null embryo and wild-type littermates were cultured by the modified 3T9 protocol and the passaged cells were analyzed by ChIP assays with the indicated antibodies (a, anti-Ring1b; b, anti-Mel18 goat serum; c, anti-E2F3a carboxyl terminus rabbit serum, which recognizes both E2F3a and E2F3b; d, anti-E2F3a amino terminus rabbit serum). Purified DNA was analyzed by PCR with primers specific for the E2F site and exon 1 for ARF and the p16 promoter region, as described in Experimental procedures. These are representative results of at least three independent experiments using different embryos. In (c) and (d), ‘e’,11.5 dpc-embryo; ‘p1’, passage-1; ‘p2’, passage-2; ‘p6’, passage-6. For E2F3a (d) and E2F3a+b (c) antibodies, experiments including rabbit immunoglobulin (Ig) were presented as negative controls.

 
The reduction of Ring1b binding to the E2F site in Mel18-null MEFs prompted us to examine E2F3b association with this region since E2F3b has been shown to act as a repressor of ARF transcription (Aslanian et al. 2004). Since a specific antibody against E2F3b is not yet available to the best of our knowledge, we used antibodies that recognize E2F3a+b or E2F3a for ChIP assays and estimated E2F3b binding based on the differences of these two antibodies. Binding of E2F3a+b was seen at the E2F site and the 1st exonic region in 11.5 dpc embryos and passage-1 MEFs, but not at the p16 promoter, irrespective of Mel18 genotype (Fig. 5c), which is consistent with a previous report (Aslanian et al. 2004). A significant difference was seen in passage-2 MEFs between wild-type and Mel18 mutants in that E2F3a+b binding was exclusively seen in the wild-type. In passage-6 MEFs, E2F3a+b binding was seen in neither wild-type nor Mel18 mutants. E2F3a binding to the E2F site and 1st exonic region of ARF was seen in 11.5 dpc embryos, but not in MEFs (Fig. 5d), suggesting that the signals obtained by the anti-E2F3a+b antibody were derived from E2F3b. E2F3b may bind to the E2F site and 1st exonic region in MEFs and be exfoliated upon culture stress, which is exaggerated by Mel18 deficiency. Together, this implies that E2F3b and Ring1b binding to the E2F site are collinearly decreased upon culture stress and Mel18 deficiency. It is also noteworthy that culture stress was suggested to alter the balance of E2F3a and E2F3b at the E2F site and 1st exonic region.

PcG proteins and E2F3s form binary complexes

The above-mentioned results of ChIP assays, which indicate that Ring1b and E2F3b bind to the E2F site in the ARF promoter, prompted us to study whether PcG complexes can form binary complexes with E2F3b. We examined Mel18/E2F3s interaction in whole-cell extracts from 11.5 dpc embryos in which PcG complexes have been shown to act as repressors of Hox gene expression (Akasaka et al. 1996) and from passage-5 MEFs (Fig. 6). In 11.5 dpc embryos, significant amounts of Mel18 were immunoprecipitated by both anti-E2F3a and -E2F3a+b, although the signal given by anti-E2F3a+b was stronger than that by anti-E2F3a (Fig. 6a); however, Mel18 bound only to E2F3b and not to E2F3a in passage-5 MEFs (Fig. 6b). These observations are consistent with the results of ChIP assays that the E2F3a+b binding to p19 genomic regions seen in 11.5 dpc embryos rapidly disappeared after cells were explanted into culture (Fig. 5c,d). Meanwhile, Ring1b was shown to form complexes with both E2F3a and E2F3b in passage-5 MEFs (Fig. 6c,d). This may imply that PcG proteins interact with E2F3a outside INK4/ARF genomic regions in MEFs. These results suggest the presence of PcG/E2F3s binary complexes, and that the role of E2F3b in the transcriptional regulation of ARF may involve direct interactions with PcG complexes.


Figure 6
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  		Figure 6  Polycomb proteins bind to E2F3s in embryos and MEFs. In Fig. 6a, lysate of a wild-type mouse embryo at 11.5 dpc (500 µg protein) was immunoprecipitated with goat immunoglobulin (lanes 2, 4 and 6) or Mel18 antibody (lanes 3, 5 and 7). In Fig. 6b–d, lysate from wild-type MEFs at passage-5 was analyzed. Western blot analysis was performed using Mel18 antibody (Fig. 6a,b) and E2F3a+b antibody (Fig. 6c,d). In lane 1, 20 µg protein of total cell lysate was analyzed as a control. Equal amounts of total cell lysates (500 µg protein) were subjected to IP with control IgG (lane 2), anti-E2F3a ab (b, lane 3), anti-E2F3a+b ab (b, lane 4), and anti-Ring1b ab (c and d, lane 3). In panels c and d, E2F3a and E2F3b were discriminated by their differences in relative molecular weights and by blotting with anti-E2F3a ab.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In the present study, we first demonstrated that deficiency of the Mel18 component of class II PcG complexes results in premature senescence via de-repression of ARF expression and subsequent stabilization of p53, as well as Bmi1, M33, Phc1 and Phc2 components. ChIP analyses indicated that transcriptional repression of p16INK4/ARF by PcG gene products may involve direct interactions between PcG complexes and genomic regions. This is consistent with another observation that the reduction of Ring1b binding at the ARF promoter in MEFs is correlated with the degree of premature senescence of MEFs, which is exaggerated by Mel18 deficiency, although it is possible that the accelerated exfoliation of PcGs was caused by enhanced senescence. Since genetic depletion of Mel18, Bmi1, M33, Phc1 or Phc2 uniformly exhibits premature senescence of MEFs accompanied by de-repression of ARF, all components of the class II complex may be indispensable to maintain fully functional PcG complexes at the ARF promoter under replicative stress. We thus postulate that the quantity of class II PcG complexes physically associated with the INK4/ARF region may be involved in limiting the transcriptional status of ARF and p16INK4a. The molecular mechanisms regulating the quantity of class II PcG complexes are not known yet, although intrinsic mechanisms could at least be involved in maintaining the stability of complexes as manifested by mutant phenotypes of respective components, for example, the decrease of class II PcGs in Mel18-null MEFs (Fig. 2c). Moreover, Ring1b and Bmi1 components of class II PcG components have been shown to be linked to mono- and poly-ubiquitinylation of their targets (Wang et al. 2004; Cao et al. 2005; Hernandez-Munoz et al. 2005). These observations suggest that replicative stress is at least partly mediated by the binding of class II PcG complexes to the INK4/ARF region, and that ARF in particular regulates cellular senescence in MEFs.

It is noteworthy that although Mel18 deficiency impacted ARF transcription, no significant association of Mel18 with the ARF promoter region was observed in wild-type MEFs, but was seen in p16INK4a-promoter and exonic regions (Fig. 4,5). This again implies that Mel18 mediates transcriptional regulation of the INK4/ARF region by forming multimeric protein complexes, in which the Mel18 component may not be juxtaposed to ARF-promoter/1st exon regions. Another possibility involves other multimeric complexes, including Ring1b, but not Mel18, which bind to ARF-specific regions, such as E2F6 complexes (Ogawa et al. 2002). This latter possibility is supported by our unpublished observation that mutant allele products of the MBLR gene, which are components of E2F6 complexes and interact with Ring1b, exhibit genetic interactions with Mel18 mutations, at least at Hox cluster loci (J. Shinga and H.K., unpubl. obs.). The binding of the E2F6 complex components and mutant interactions between Mel18 and MBLR genes in the INK4/ARF region should be addressed in future.

Similarly to Ring1b binding, the association of E2F3b, which is also essential for transcriptional repression of ARF, with the ARF promoter region was correlated with the degree of cellular senescence of MEFs. Moreover, associations of E2F3b and Ring1b with the ARF promoter were collinearly decreased with synergistic effects of culture stress and Mel18 deficiency. E2F3b and Ring1b are capable of interacting directly upon mammalian cells (Fig. 6). This implies that the association of E2F3b with the ARF promoter may be at least partly dependent on class II PcG complexes at that locus. The convergence of class II complexes and E2F3b at the ARF promoter may be required to repress ARF transcription in MEFs, and binary complexes are presumably exfoliated by the stress that cells experience when explanted into culture.

Intriguingly, although significant binding of E2F3b or E2F3a was not seen at the p16INK4a promoter, p16INK4a has been shown to be de-repressed in E2F3a+b -null MEFs, as well as ARF (Aslanian et al. 2004). This implies that repression of the p16INK4a promoter is also impacted by E2F3s, which may bind to the ARF promoter and/or 1st exonic regions. Since the p16INK4a promoter is bound by class II PcG complexes, class II complexes may require E2F3s for repressive functions at the p16INK4a locus in MEFs. From these results, we can surmise that class II PcG complexes and E2F3s act in mutually dependent manners in the INK4/ARF region. The formation of binary complexes may mediate various signals to the locus, since both oncogenic signals and culture stress were shown to alter the binding of E2F3s to the E2F site of the ARF promoter. It will be important to further address the binding of class II PcG components to the INK4/ARF region of E2F3a+b -null MEFs.

Moreover, it is notable that E2F3b bound to the ARF promoter/1st exon irrespective of the E2F site, which is required to mediate transcriptional regulation by E2F family proteins. Since E2F3b is capable of interacting with Ring1b, it is possible that the binding of E2F3b to the first exonic region of ARF is mediated by its interaction with class II PcG complexes rather than with genomic components. It may also be true that there are some discriminating activities to recruit E2F3b between ARF- and p16INK4a-genomic regions, although both are bound by Ring1b. Since ARF and p16INK4a genomic regions are quite different in terms of Mel18 binding in MEFs, it is possible that different interactions for Ring1b in respective regions could play a role. We thus suggest that class II PcG complexes and E2F3b interact in the INK4/ARF region and mutually regulate each other's function to limit cellular senescence.

It is notable that another E2F family protein, E2F6, is reported to interact with PcG and its related complexes, which exert a significant impact on the transcription of cell cycle-related genes (Trimarchi et al. 2001; Ogawa et al. 2002); however, E2F6 is only distantly related to other E2Fs and lacks the sequences responsible for both transactivation and binding to retinoblastoma protein. In support of this, E2F6-deficient mouse experiments indicate that E2F6 is essential for the long-term somatic silencing of certain male germ-cell-specific genes, but it is dispensable for cell-cycle regulation (Pohlers et al. 2005). Meanwhile, E2F3a/E2F3b double-null MEFs showed severe retardation in cell cycle progression (Aslanian et al. 2004) and our present study indicates that class II PcGs and E3F3s act in a mutually dependent manner in the regulation of p16INK4a/ARF transcription. Taken together, it is possible that, in concert with PcG proteins, different E2F family proteins may be utilized to mediate transcriptional regulation in a locus- or cell type-specific manner.

We are currently planning to study the impact of oncogenic stresses, for example, Myc and E2Fs, on the binding of class II PcG proteins and E2F3s via the E2F site, 1st exon of ARF and p16 promoter regions. This will be informative to address the role of PcGs and E2F3s in tumor suppression by the control of stress-induced cellular arrest or apoptotic cell death induced by oncogene products via ARF/p16INK4a.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Reagents and antibodies

Anti-p21Cip1/Waf1 mouse monoclonal antibody (clone F-5), anti-E2F3a rabbit serum (antibody against N-terminal of E2F3a: sc-879) and anti-E2F3a+b rabbit serum (antibody against common C-terminal of E2F3a and E2F3b: sc-878) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-p19ARF rabbit polyclonal antibody (R562) and anti-Mel18 goat serum (ab5267) were purchased from Abcam (Cambridge, UK). Anti-HA antibody (clone 12CA5) and anti-β-tubulin mouse monoclonal antibody were purchased from Boehringer Mannheim K.K. (Tokyo, Japan). Anti-p53 mouse monoclonal antibody (clone pAb421) was from Oncogene Research Products (Cambridge, MA). Anti-mouse p16 monoclonal antibody (16P04) was from NeoMarkers (Fremont, CA). Anti-MDM2 mouse monoclonal antibody (clone 2A10) was kindly provided by Dr A.J. Levine. Anti-Ring1b mouse monoclonal antibodies were as described in a previous report (Isono et al. 2005). Anti-Bmi1 antibody (clone 229F6) was from Upstate (Lake Placid, NY). Other biochemical reagents were purchased from Sigma-Aldrich Japan (Tokyo, Japan), or Wako (Osaka, Japan).

Interbreeding of mice

The generation of Mel18-null (Akasaka et al. 1996), ARF-null (Kamijo et al. 1997) and p53-null (Gondo et al. 1994), each with a 129/svj x C57BL/6 background, was described in previous reports. These mice had been backcrossed to a C57BL/6 background more than 8 times, and were interbred to yield animals lacking Mel18/p53 or Mel18/ARF.

Cells and cell culture

MEFs were prepared as described previously (Kamijo et al. 1997). Mel18-null, Mel18/ARF and Mel18/p53 double-null MEFs were routinely maintained with DMEM supplemented with 20% fetal bovine serum (FBS), 1x non-essential amino acid (GibcoBRL®) and 1x penicillin–streptomycin (GibcoBRL®).

To induce senescence effectively, 3 x 106 cells were plated in a 10-cm diameter dish, passaged at 3-day intervals, counted after trypsinization, and the number of cells per dish was recorded. One-third of cells were re-plated in a 10-cm diameter dish every 3 days (Fig. 1a).

In modified 3T9 assays, cells were passaged at 3-day intervals and counted after trypsinization, and the number of cells per dish was recorded. Amounts of 1 x 106 cells were re-plated in 60-mm diameter dishes every 3 days (Fig. 3a; Kamijo et al. 1997). In growth-speed assays, cells diluted at 2 x 104 per 60-mm diameter dish were replica plated, and individual cultures harvested every day thereafter were counted (Figs 1b and 3c).

Procedure for senescence-associated β-galactosidase staining of MEFs

In situ SA-β-Gal activity was detected according to the manufacturer's protocol (Senescence Detection Kit, BioVision, Mountain View, CA). Briefly, MEFs were washed with PBS, fixed with fixation solution for 15 min at room temperature, washed twice with PBS and stained for 16 h at 37 °C with 1 mg/mL 5-bromo-4-chloro-3-indolyl-β-galactoside (X-Gal) staining solution. Micrographs of β-galactosidase-stained MEFs were taken at 400x magnification using a phase contrast microscope (Olympus, Japan).

Apoptosis assay

Sub-G0/G1 fractions were analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Samples of 15 000 cells were analyzed for each sample, and quantitation of cell-cycle distribution was performed using CELL QUEST software (Becton Dickinson).

Retrovirus production and infection

A retrovirus vector for virus production, pMSCV-IRES-GFP, was a generous gift from Dr Robert G. Hawley, The George Washington University Medical Center. Virus production and cell infection were performed using retroviral helper and vector plasmids provided by Charles Sawyers (University of California, Los Angeles, CA). Cells from the human kidney 293T cell line were from Dr David Baltimore (California Institute of Technology, Pasadena, CA). The following cDNAs were cloned into pMSCV-IRES-GFP or pSRa-MSV-tk-CD8 plasmids for production of recombinant retroviruses expressing wild-type p53 and ARF. Wild-type p53 and mouse ARF cDNAs were provided by Drs C.J. Sherr and M.F. Roussel, St. Jude Children's Research Hospital. For virus infection of MEFs, three infections at 4-h intervals with high-titer replication-defective viruses were performed. Virus infection efficiency was determined 48 h after infection by fluorescence-activated flow cytometry (FACS) as a percentage of GFP-positive cells compared to non-infected cells.

Analysis of RNA and protein expression

Immunoprecipitation and Western blotting were performed according to previous reports (Kamijo et al. 1998; Nakazawa et al. 2003). Total RNA was extracted from NB cells using Isogen® (Wako, Tokyo, Japan), and cDNA was synthesized from 1 µg of total RNA templates according to the manufacturer's protocol (RiverTra-Ace-{alpha}- RT-PCR kit, TOYOBO, Osaka, Japan). PCR amplification was performed using the primers listed in Table 1. Semi-quantitative RT-PCR analysis was performed according to the previous report (Machida et al. 2006).


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  		Table 1  Primer sequences used in RT-PCR experiments
 
Chromatin-immunoprecipitated (ChIP) assay

ChIP assay was performed as described previously (Fujimura et al. 2006). Briefly, either minced embryos or MEFs were chemically cross-linked in 1% formaldehyde–PBS for 10 min. After washing 3 times with PBS and once with TE (Tris–EDTA pH 8.0), tissues were suspended in a ten fold volume of TE. To solubilize proteins and cleave genomic DNA to average 2 kbp, samples were subjected to repeated sonication and ice-chilling. Insoluble matter was removed by centrifugation at 20 400 g for 5 min. Correct amounts of NaCl and NP-40 were added to the supernatants in order to perform optimal immunoprecipitation for each antibody (100 mM NaCl and 0.4% NP-40 for anti-Ring1b, 200 mM NaCl, and 0.1% NP-40 for anti-Mel18, anti-E2F3a and anti-E2F3a+b). Precleared protein extracts were incubated with the correct amount of antibodies, at 4 °C with rocking, from 2 h to overnight. Immune complexes were captured through 3-h incubation with Protein A Sepharose beads. Beads were washed with the same components as immunoprecipitation 3 times briefly and 7 times for 10 min under intense rotation.

To isolate genomic DNA from immune complexes, beads were treated with 50 µg/mL of RNaseA at 37 °C for 30 min followed by overnight incubation with 500 µg/mL Proteinase K/0.5% SDS at 37 °C. After 3-h heating at 65 °C for reverse cross-linking, supernatants were collected, extracted by phenol–chloroform and concentrated by ethanol precipitation. Genomic DNA was also isolated from the original lysates through the same procedure as described above and designated as ‘Input’ DNA in Figs 4 and 5. To measure the DNA yield after immunoprecipitation, aliquots of immunoprecipitated DNA were electrophoresed for 5 min in an agarose gel, and then serially diluted input DNA and band intensities were compared after ethidium bromide staining.

An equivalent amount of immunoprecipitated DNA to that of ‘Input’ DNA loaded in lane ‘20’ was subjected to PCR reactions. Usually 10–20 ng of genomic DNA was used. Mock-immunoprecipitated DNA (ab-) and species-matched immunoglobulin-immunoprecipitated DNA (Ig), derived from the same volume of the chromatin fraction used for specific antibody immunoprecipitation, were subjected to PCR. To carry out semi-quantitative PCR, serially diluted ‘Input’ DNA and immunoprecipitated DNA were used as templates. The relative quantity of each genomic region in immunoprecipitated genomic DNA was estimated by referring to serial dilutions of ‘Input’ DNA isolated from the initial lysates and an enrichment value was determined. Each series of experiments was performed at least 3 times. Primers used in this study are listed in Table 2.


   Acknowledgements
 
We are deeply indebted to Prof. Kenichi Koike (Department of Pediatrics, Shinshu University School of Medicine) and Prof. Hiroto Okayama (Department of Molecular Biology, The University of Tokyo, Graduate School of Medicine) for their excellent advice, and to Drs C.J. Sherr and M.F. Roussel, Tumor Cell Biology, St. Jude Children's Research Hospital for various cDNAs. We thank Dr Yozo Nakazawa, Dr Kenji Kurata, Dr Ryu Yanagisawa and Kumiko Sakurai for their technical assistance, and Daniel Mrozek for editorial assistance. This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Grant-in-Aid for Scientific Research on Priority Areas (C) (contract grant number: 13043003).


   Footnotes
 
Communicated by: Tetsuya Taga

* Correspondence: E-mail: tkamijo{at}chiba-cc.jp


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Accepted: 24 September 2007





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