Wild-type p53-induced phosphatase 1 (Wip1) forestalls cellular premature senescence at physiological oxygen levels by regulating DNA damage response signaling during DNA replication

Wip1 (protein phosphatase Mg²⁺/Mn²⁺-dependent 1D, Ppm1d) is a nuclear serine/threonine protein phosphatase that is induced by p53 following the activation of DNA damage response (DDR) signaling. Ppm1d^−/− mouse embryonic fibroblasts (MEFs) exhibit premature senescence under conventional culture conditions; however, little is known regarding the role of Wip1 in regulating cellular senescence. In this study, we found that even at a representative physiological concentration of 3% O₂, Ppm1d^−/− MEFs underwent premature cellular senescence that depended on the functional activation of p53. Interestingly, Ppm1d^−/− MEFs showed increased H2AX phosphorylation levels without increased levels of reactive oxygen species (ROS) or DNA base damage compared with wild-type (Wt) MEFs, suggesting a decreased threshold for DDR activation or sustained DDR activation during recovery. Notably, the increased H2AX phosphorylation levels observed in Ppm1d^−/− MEFs were primarily associated with S-phase cells and predominantly dependent on the activation of ATM. Moreover, these same phenotypes were observed when Wt and Ppm1d^−/− MEFs were either transiently or chronically exposed to low levels of agents that induce replication-mediated double-stranded breaks. These findings suggest that Wip1 prevents the induction of cellular senescence at physiological oxygen levels by attenuating DDR signaling in response to endogenous double-stranded breaks that form during DNA replication.     

The Arginine Methyltransferase PRMT6 Cooperates with Polycomb Proteins in Regulating HOXA Gene Expression

Protein arginine methyltransferase 6 (PRMT6) catalyses asymmetric dimethylation of histone H3 at arginine 2 (H3R2me2a), which has been shown to impede the deposition of histone H3 lysine 4 trimethylation (H3K4me3) by blocking the binding and activity of the MLL1 complex. Importantly, the genomic occurrence of H3R2me2a has been found to coincide with histone H3 lysine 27 trimethylation (H3K27me3), a repressive histone mark generated by the Polycomb repressive complex 2 (PRC2). Therefore, we investigate here a putative crosstalk between PRMT6- and PRC-mediated repression in a cellular model of neuronal differentiation. We show that PRMT6 and subunits of PRC2 as well as PRC1 are bound to the same regulatory regions of rostral HOXA genes and that they control the differentiation-associated activation of these genes. Furthermore, we find that PRMT6 interacts with subunits of PRC1 and PRC2 and that depletion of PRMT6 results in diminished PRC1/PRC2 and H3K27me3 occupancy and in increased H3K4me3 levels at these target genes. Taken together, our data uncover a novel, additional mechanism of how PRMT6 contributes to gene repression by cooperating with Polycomb proteins.     

Cellular senescence and organismal ageing in the absence of p21CIP1/WAF1 in ku80−/− mice

Ku80 is important in the repair of DNA double-strand breaks by its essential function in non-homologous end-joining. The absence of Ku80 causes the accumulation of DNA damage and leads to premature ageing in mice. We showed that mouse embryonic fibroblasts (MEFs) from ku80^−/− mice senesced rapidly with elevated levels of p53 and p21. Deletion of p21 delayed the early senescence phenotype in ku80^−/− MEFs, despite an otherwise intact response of p53. In contrast to ku80^−/−p53^−/− mice, which die rapidly primarily from lymphomas, there was no significant increase in tumorigenesis in ku80^−/−p21^−/− mice. However, ku80^−/−p21^−/− mice showed no improvement with respect to rough fur coat or osteopaenia, and even showed a shortened lifespan compared with ku80^−/− mice. These results show that the increased lifespan of ku80^−/− MEFs owing to the loss of p21 is not associated with an improvement of the premature ageing phenotypes of ku80^−/− mice observed at the organismal level.     

HP1α mediates defective heterochromatin repair and accelerates senescence in Zmpste24-deficient cells

Heterochromatin protein 1 (HP1) interacts with various proteins, including lamins, to play versatile functions within nuclei, such as chromatin remodeling and DNA repair. Accumulation of prelamin A leads to misshapen nuclei, heterochromatin disorganization, genomic instability, and premature aging in Zmpste24-null mice. Here, we investigated the effects of prelamin A on HP1α homeostasis, subcellular distribution, phosphorylation, and their contribution to accelerated senescence in mouse embryonic fibroblasts (MEFs) derived from Zmpste24^−/− mice. The results showed that the level of HP1α was significantly increased in Zmpste24^−/− cells. Although prelamin A interacted with HP1α in a manner similar to lamin A, HP1α associated with the nuclease-resistant nuclear matrix fraction was remarkably increased in Zmpste24^−/− MEFs compared with that in wild-type littermate controls. In wild-type cells, HP1α was phosphorylated at Thr50, and the phosphorylation was maximized around 30 min, gradually dispersed 2 h after DNA damage induced by camptothecin. However, the peak of HP1α phosphorylation was significantly compromised and appeared until 2 h, which is correlated with the delayed maximal formation of γ-H2AX foci in Zmpste24^−/− MEFs. Furthermore, knocking down HP1α by siRNA alleviated the delayed DNA damage response and accelerated senescence in Zmpste24^−/− MEFs, evidenced by the rescue of the delayed γ-H2AX foci formation, downregulation of p16, and reduction of senescence-associated β-galactosidase activity. Taken together, these findings establish a functional link between prelamin A, HP1α, chromatin remodeling, DNA repair, and early senescence in Zmpste24-deficient mice, suggesting a potential therapeutic strategy for laminopathy-based premature aging via the intervention of HP1α.     

Absence of p53-dependent apoptosis leads to UV radiation hypersensitivity,enhanced immunosuppression and cellular senescence

Genotoxic stress triggers the p53 tumor suppressor network to activate cellular responses that lead to cell cycle arrest, DNA repair, apoptosis or senescence. This network functions mainly through transactivation of different downstream targets, including cell cycle inhibitor p21, which is required for short-term cell cycle arrest or long-term cellular senescence, or proapoptotic genes such as p53 upregulated modulator of apoptosis (PUMA) and Noxa. However, the mechanism that switches from cell cycle arrest to apoptosis is still unknown. In this study, we found that mice harboring a hypomorphic mutant p53, R172P, a mutation that abrogates p53-mediated apoptosis while keeping cell cycle control mostly intact, are more susceptible to ultraviolet-B (UVB)-induced skin damage, inflammation, and immunosuppression than wild-type mice. p53^R172P embryonic fibroblasts (MEFs) are hypersensitive to UVB and prematurely senesce after UVB exposure, in stark contrast to wild-type MEFs, which undergo apoptosis. However, these mutant cells are able to repair UV-induced DNA lesions, indicating that the UV hypersensitive phenotype results from the subsequent damage response. Mutant MEFs show an induction of p53 and p21 after UVR, while wild-type MEFs additionally induce PUMA and Noxa. Importantly, p53^R172P MEFs failed to downregulate anti-apoptotic protein Bcl-2, which has been shown to play an important role in p53-dependent apoptosis. Taken together, these data demonstrate that in the absence of p53-mediated apoptosis, cells undergo cellular senescence to prevent genomic instability. Our results also indicate that p53-dependent apoptosis may play an active role in balancing cellular growth.     

Involvement of p53 and p21 in Cellular Defects and Tumorigenesis in Atm−/− Mice

Disruption of the mouse Atm gene, whose human counterpart is consistently mutated in ataxia-telangiectasia (A-T) patients, creates an A-T mouse model exhibiting most of the A-T-related systematic and cellular defects. While ATM plays a major role in signaling the p53 response to DNA strand break damage, Atm^−/− p53^−/− mice develop lymphomas earlier than Atm^−/− or p53^−/− mice, indicating that mutations in these two genes lead to synergy in tumorigenesis. The cell cycle G₁/S checkpoint is abolished in Atm^−/− p53^−/− mouse embryonic fibroblasts (MEFs) following γ-irradiation, suggesting that the partial G₁ cell cycle arrest in Atm^−/− cells following γ-irradiation is due to the residual p53 response in these cells. In addition, the Atm^−/− p21^−/− MEFs are more severely defective in their cell cycle G₁ arrest following γ-irradiation than Atm^−/− and p21^−/− MEFs. The Atm^−/− MEFs exhibit multiple cellular proliferative defects in culture, and an increased constitutive level of p21 in these cells might account for these cellular proliferation defects. Consistent with this notion, Atm^−/− p21^−/− MEFs proliferate similarly to wild-type MEFs and exhibit no premature senescence. These cellular proliferative defects are also rescued in Atm^−/− p53^−/− MEFs and little p21 can be detected in these cells, indicating that the abnormal p21 protein level in Atm^−/− cells is also p53 dependent and leads to the cellular proliferative defects in these cells. However, the p21 mRNA level in Atm^−/− MEFs is lower than that in Atm^+/+ MEFs, suggesting that the higher level of constitutive p21 protein in Atm^−/− MEFs is likely due to increased stability of the p21 protein.     

PRMT5 determines the pattern of polyploidization and prevents liver from cirrhosis and carcinogenesis

Human hepatocellular carcinoma(HCC) occurs almost exclusively in cirrhotic livers. Here, we report that hepatic loss of protein arginine methyltransferase 5(PRMT5) in mice is sufficient to cause cirrhosis and HCC in a clinically relevant way. Furthermore, pathological polyploidization induced by hepatic loss of PRMT5 promotes liver cirrhosis and hepatic tumorigenesis in aged liver. The loss of PRMT5 leads to hyperaccumulation of P21 and endoreplication-dependent formation of pathological mono-nu...     

Absence of p53-dependent apoptosis leads to UV radiation hypersensitivity,enhanced immunosuppression and cellular senescence

Genotoxic stress triggers the p53 tumor suppressor network to activate cellular responses that lead to cell cycle arrest, DNA repair, apoptosis or senescence. This network functions mainly through transactivation of different downstream targets, including cell cycle inhibitor p21, which is required for short-term cell cycle arrest or long-term cellular senescence, or proapoptotic genes such as p53 upregulated modulator of apoptosis (PUMA) and Noxa. However, the mechanism that switches from cell cycle arrest to apoptosis is still unknown. In this study, we found that mice harboring a hypomorphic mutant p53, R172P, a mutation that abrogates p53-mediated apoptosis while keeping cell cycle control mostly intact, are more susceptible to ultraviolet-B (UVB)-induced skin damage, inflammation and immunosuppression than wild-type mice. p53^R172P embryonic fibroblasts (MEFs) are hypersensitive to UVB and prematurely senesce after UVB exposure, in stark contrast to wild-type MEFs, which undergo apoptosis. However, these mutant cells are able to repair UV-induced DNA lesions, indicating that the UV-hypersensitive phenotype results from the subsequent damage response. Mutant MEFs show an induction of p53 and p21 after UVR, while wild-type MEFs additionally induce PUMA and Noxa. Importantly, p53^R172P MEFs failed to downregulate anti-apoptotic protein Bcl-2, which has been shown to play an important role in p53-dependent apoptosis. Taken together, these data demonstrate that in the absence of p53-mediated apoptosis, cells undergo cellular senescence to prevent genomic instability. Our results also indicate that p53-dependent apoptosis may play an active role in balancing cellular growth.Key words: UVB irradiation, p53, DNA damage, DNA damage responses, apoptosis, senescence     

TIS21/BTG2/PC3 accelerates the repair of DNA double strand breaks by enhancing Mre11 methylation and blocking damage signal transfer to the Chk2T68–p53S20 pathway

DNA double strand breaks (DSBs) occur more frequently in TIS21^?/? mouse embryo fibroblasts than that in wild type MEFs (wt-MEFs). Therefore, the role TIS21 plays in the DNA damage response was investigated. Adenoviral transduction of Huh7 tumor cells with the TIS21 gene accelerated the repair of DSBs induced by etoposide treatment as evaluated by clearance of γH2AX foci and the Comet assay. TIS21 increased methylation of Mre11 and protein arginine methyltransferase 1 (PRMT1) activity, leading to Mre11 activation in vitro and in vivo, as determined by immunoprecipitation and radiolabeling analyses. When downstream DNA damage response mediators were evaluated in various human cancer cells lines, TIS21 was found to strongly inhibit Chk2^T68 and p53^S20 phosphorylation by p-ATM^S1981 but not p53^S15. The loss of Chk2 activation after etoposide treatment reduced apoptosis in the cells by downregulating the expression of E2F1 and Bax. These data suggest that TIS21 regulates DSB repair and apoptosis. Expression of TIS21 promoted the repair of DSBs and reduced apoptosis by blocking the damage signal from p-ATM^S1981 to Chk2^T68–p53^S20 via the activation of Mre11 and PRMT1.     

Identification of a Novel Inhibitor of Coactivator-associated Arginine Methyltransferase 1 (CARM1)-mediated Methylation of Histone H3 Arg-17

Methylation of the arginine residues of histones by methyltransferases has important consequences for chromatin structure and gene regulation; however, the molecular mechanism(s) of methyltransferase regulation is still unclear, as is the biological significance of methylation at particular arginine residues. Here, we report a novel specific inhibitor of coactivator-associated arginine methyltransferase 1 (CARM1; also known as PRMT4) that selectively inhibits methylation at arginine 17 of histone H3 (H3R17). Remarkably, this plant-derived inhibitor, called TBBD (ellagic acid), binds to the substrate (histone) preferentially at the signature motif, “KAPRK,” where the proline residue (Pro-16) plays a critical role for interaction and subsequent enzyme inhibition. In a promoter-specific context, inhibition of H3R17 methylation represses expression of p21, a p53-responsive gene, thus implicating a possible role for H3 Arg-17 methylation in tumor suppressor function. These data establish TBBD as a novel specific inhibitor of arginine methylation and demonstrate substrate sequence-directed inhibition of enzyme activity by a small molecule and its physiological consequence.