RESUMO
The Mdm4 (alias MdmX) oncoprotein, like its paralogue and interaction partner Mdm2, antagonizes the tumor suppressor p53. p53-independent roles of the Mdm proteins are emerging, and we have reported the ability of Mdm2 to modify chromatin and to support DNA replication by suppressing the formation of R-loops (DNA/RNA-hybrids). We show here that the depletion of Mdm4 in p53-deficient cells compromises DNA replication fork progression as well. Among various deletion mutants, only full-length Mdm4 was able to support DNA replication fork progression. Co-depletion of Mdm4 and Mdm2 further impaired DNA replication, and the overexpression of each partially compensated for the other's loss. Despite impairing replication, Mdm4 depletion only marginally hindered cell proliferation, likely due to compensation through increased firing of replication origins. However, depleting Mdm4 sensitized p53-/- cells to the nucleoside analog gemcitabine, raising the future perspective of using Mdm4 inhibitors as chemosensitizers. Mechanistically, Mdm4 interacts with members of the Polycomb Repressor Complexes and supports the ubiquitination of H2A, thereby preventing the accumulation of DNA/RNA-hybrids. Thus, in analogy to previously reported activities of Mdm2, Mdm4 enables unperturbed DNA replication through the avoidance of R-loops.
Assuntos
Replicação do DNA/genética , Fibroblastos/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteína Supressora de Tumor p53/genética , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Humanos , Camundongos Knockout , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Proteína Supressora de Tumor p53/metabolismoRESUMO
The p53-Mdm2 system is key to tumor suppression. We have recently reported that p53 as well as Mdm2 are capable of supporting DNA replication fork progression. On the other hand, we found that Mdm2 is a modifier of chromatin, modulating polycomb repressor complex (PRC)-driven histone modifications. Here we show that, similar to Mdm2 knockdown, the depletion of PRC members impairs DNA synthesis, as determined in fiber assays. In particular, the ubiquitin ligase and PRC1 component RNF2/Ring1B is required to support DNA replication, similar to Mdm2. Moreover, the Ring finger domain of Mdm2 is not only essential for its ubiquitin ligase activity, but also for proper DNA replication. Strikingly, Mdm2 overexpression can rescue RNF2 depletion with regard to DNA replication fork progression, and vice versa, strongly suggesting that the two ubiquitin ligases perform overlapping functions in this context. H2A overexpression also rescues fork progression upon depletion of Mdm2 or RNF2, but only when the ubiquitination sites K118/K119 are present. Depleting the H2A deubiquitinating enzyme BAP1 reduces the fork rate, suggesting that both ubiquitination and deubiquitination of H2A are required to support fork progression. The depletion of Mdm2 elicits the accumulation of RNA/DNA hybrids, suggesting R-loop formation as a mechanism of impaired DNA replication. Accordingly, RNase H overexpression or the inhibition of the transcription elongation kinase CDK9 each rescues DNA replication upon depletion of Mdm2 or RNF2. Taken together, our results suggest that chromatin modification by Mdm2 and PRC1 ensures smooth DNA replication through the avoidance of R-loop formation.
Assuntos
Cromatina/metabolismo , Replicação do DNA , DNA/genética , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA/genética , Linhagem Celular Tumoral , Cromatina/genética , DNA/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Complexo Repressor Polycomb 1/genética , Domínios Proteicos , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/genética , RNA/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , UbiquitinaçãoRESUMO
p53 induces cell death upon DNA damage, but this may not confer all of its tumor suppressor activity. We report that p53 activation enhances the processivity of DNA replication, as monitored by multi-label fiber assays, whereas removal of p53 reduces fork progression. This is observed in tumor-derived U2OS cells but also in murine embryonic fibroblasts with heterozygous or homozygous p53 deletion and in freshly isolated thymocytes from mice with differential p53 status. Mdm2, a p53-inducible gene product, similarly supports DNA replication even in p53-deficient cells, suggesting that sustained Mdm2-expression is at least one of the mechanisms allowing p53 to prevent replicative stress. Thus, p53 helps to protect the genome during S phase, by preventing the occurrence of stalled or collapsed replication forks. These results expand p53's tumor-suppressive functions, adding to the ex-post model (elimination of damaged cells) an ex-ante activity; i.e., the prevention of DNA damage during replication.
