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Mdm4 supports DNA replication in a p53-independent fashion

Abstract

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.

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Fig. 1: Loss of Mdm4 impairs DNA replication fork progression.
Fig. 2: Mdm4 and Mdm2 act through partially distinct mechanisms.
Fig. 3: Full-length Mdm4 is required for supporting DNA replication.
Fig. 4: Mdm4 depletion increases replicative stress, but overall DNA synthesis is maintained.
Fig. 5: Mdm4 depletion sensitizes cells to gemcitabine.
Fig. 6: Mdm4 cooperates with Polycomb Repressor Complex members and regulates H2A ubiquitination.
Fig. 7: Removal of accumulated DNA/RNA-hybrids allows replication fork progression despite the loss of Mdm4.

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Acknowledgements

We thank Guillermina Lozano for the MEFs with p53/Mdm4/Mdm2 deletions. pCMV-Flag-Mdm4 was a gift from Zhi-Min Yuan. pCMV-MDM2 was a gift from Bert Vogelstein (Addgene plasmid #16441), pCMV-MDM2(C464A) was provided by Tyler Jacks (Addgene plasmid #12086), pICE-RNaseHI-WT-NLS-mCherry (Addgene plasmid #60365) as well as pICE-RNaseHI-D10R-E48R-NLS-mCherry (Addgene plasmid #60367) were obtained from Patrick Calsou. H2A and EZH2 expression plasmids were from Titia Sixma (Addgene plasmids #63561 and #63564) and Kristian Helin (Addgene plasmid #24230), respectively. pLenti6/V5-DEST-RNF2 was a gift from Lynda Chin (Addgene plasmid #31216). This work was supported by the Deutsche Krebshilfe (to MD and KW), the Wilhelm Sander Stiftung, the Else Kröner Fresenius Stiftung, the Deutsche José Carreras Leukämie Stiftung, the Deutsche Forschungsgemeinschaft, the Boehringer Ingelheim Fonds (to IK) and the German Academic Scholarship Foundation (to KW). IK, PD, and VM were members of the IMPRS/MSc/PhD program Molecular Biology and IK, VM, CG and JC also of the Göttingen Graduate School GGNB Göttingen.

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KW, IK, and MD designed research; KW, IK, PD, KH, JC, AM, VM, and CG performed research; CME contributed expression constructs for Mdm4 mutants and DNA replication expertise; AGJ performed immunoprecipitation; KW and IK analyzed data; KW, IK, and MD wrote the manuscript.

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Correspondence to Matthias Dobbelstein.

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Wohlberedt, K., Klusmann, I., Derevyanko, P.K. et al. Mdm4 supports DNA replication in a p53-independent fashion. Oncogene 39, 4828–4843 (2020). https://doi.org/10.1038/s41388-020-1325-1

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