Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Mdm4 supports DNA replication in a p53-independent fashion


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  1. 1.

    Linares LK, Hengstermann A, Ciechanover A, Muller S, Scheffner M. HdmX stimulates Hdm2-mediated ubiquitination and degradation of p53. Proc Natl Acad Sci. 2003;100:12009–14.

    CAS  PubMed  Article  Google Scholar 

  2. 2.

    Danovi D, Meulmeester E, Pasini D, Migliorini D, Capra M, Frenk R, et al. Amplification of Mdmx (or Mdm4) directly contributes to tumor formation by inhibiting p53 tumor suppressor activity. Mol Cell Biol. 2004;24:5835–43.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  3. 3.

    Laurie NA, Donovan SL, Shih CS, Zhang J, Mills N, Fuller C, et al. Inactivation of the p53 pathway in retinoblastoma. Nature. 2006;444:61–6.

    CAS  PubMed  Article  Google Scholar 

  4. 4.

    Gembarska A, Luciani F, Fedele C, Russell EA, Dewaele M, Villar S, et al. MDM4 is a key therapeutic target in cutaneous melanoma. Nat Med. 2012;18:1239–47.

    CAS  PubMed  Article  Google Scholar 

  5. 5.

    Han X, Medeiros LJ, Zhang YH, You MJ, Andreeff M, Konopleva M, et al. High expression of human homologue of murine double minute 4 and the short splicing variant, hdm4-s, in bone marrow in patients with acute myeloid leukemia or myelodysplastic syndrome. Clin Lymphoma Myeloma Leuk. 2016;16:S30–8.

    PubMed  Article  Google Scholar 

  6. 6.

    Matijasevic Z, Krzywicka-Racka A, Sluder G, Jones SN. MdmX regulates transformation and chromosomal stability in p53-deficient cells. Cell Cycle. 2008;7:2967–73.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. 7.

    Carillo Alexia M. Bouska Alzssa, Arrate Maria Pia ECM. NIH Public Access. Oncogene 2015;34:846–56.

    Article  CAS  Google Scholar 

  8. 8.

    Strachan GD, Jordan-Sciutto KL, Rallapalli R, Tuan RS, Hall DJ. The E2F-1 transcription factor is negatively regulated by its interaction with the MDMX protein. J Cell Biochem. 2003;88:557–68.

    CAS  PubMed  Article  Google Scholar 

  9. 9.

    Zhang H, Hu L, Qiu W, Deng T, Zhang Y, Bergholz J, et al. MDMX exerts its oncogenic activity via suppression of retinoblastoma protein. Oncogene. 2015;34:5560–9.

    CAS  PubMed  Article  Google Scholar 

  10. 10.

    Jin Y, Zeng SX, Sun X-X, Lee H, Blattner C, Xiao Z, et al. MDMX promotes proteasomal turnover of p21 at G1 and early S phases independently of, but in cooperation with, MDM2. Mol Cell Biol. 2008;28:1218–29.

    CAS  PubMed  Article  Google Scholar 

  11. 11.

    Bohlman S, Manfredi JJ. p53-independent effects of Mdm2. In: Deb S, Deb S (eds). Mutant p53 and MDM2 in Cancer. Subcellular Biochemistry, vol 85. (Springer, Dordrecht, 2014) pp 235–46.

  12. 12.

    Alt JR, Bouska A, Fernandez MR, Cerny RL, Xiao H, Eischen CM. Mdm2 Binds to Nbs1 at sites of DNA damage and regulates double strand break repair. J Biol Chem. 2005;280:18771–81.

    CAS  PubMed  Article  Google Scholar 

  13. 13.

    Bouska A, Lushnikova T, Plaza S, Eischen CM. Mdm2 promotes genetic instability and transformation independent of p53. Mol Cell Biol. 2008;28:4862–74.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. 14.

    Klusmann I, Rodewald S, Müller L, Friedrich M, Wienken M, Li Y, et al. p53 activity results in DNA replication fork processivity. Cell Rep. 2016;17:1845–57.

    CAS  PubMed  Article  Google Scholar 

  15. 15.

    Wienken M, Dickmanns A, Nemajerova A, Kramer D, Najafova Z, Weiss M, et al. MDM2 associates with polycomb repressor complex 2 and enhances stemness-promoting chromatin modifications independent of p53. Mol Cell 2016;61:68–83.

    CAS  PubMed  Article  Google Scholar 

  16. 16.

    Klusmann I, Wohlberedt K, Magerhans A, Teloni F, Korbel JO, Altmeyer M, et al. Chromatin modifiers Mdm2 and RNF2 prevent RNA:DNA hybrids that impair DNA replication. Proc Natl Acad Sci. 2018;115:E11311–20.

    CAS  PubMed  Article  Google Scholar 

  17. 17.

    Gan W, Guan Z, Liu J, Gui T, Shen K, Manley JL, et al. R-loop-mediated genomic instability is caused by impairment of replication fork progression. Genes Dev. 2011;25:2041–56.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. 18.

    Shvarts A, Bazuine M, Dekker P, Ramos YFM, Steegenga WT, Merckx G, et al. Isolation and identification of the human homolog of a new p53-binding protein, Mdmx. Genomics 1997;43:34–42.

    CAS  PubMed  Article  Google Scholar 

  19. 19.

    Gazdar AF, Gao B, Minna JD. Lung cancer cell lines: useless artifacts or invaluable tools for medical science? Lung Cancer 2010;68:309–18.

    PubMed  PubMed Central  Article  Google Scholar 

  20. 20.

    Gradiz R, Silva HC, Carvalho L, Botelho MF, Mota-Pinto A. MIA PaCa-2 and PANC-1 – pancreas ductal adenocarcinoma cell lines with neuroendocrine differentiation and somatostatin receptors. Sci Rep. 2016;6:21648.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. 21.

    Parant J, Chavez-Reyes A, Little NA, Yan W, Reinke V, Jochemsen AG, et al. Rescue of embryonic lethality in Mdm4-null mice by loss of Trp53 suggests a nonoverlapping pathway with MDM2 to regulate p53. Nat Genet 2001;29:92–5.

    CAS  PubMed  Article  Google Scholar 

  22. 22.

    Meulmeester E, Frenk R, Stad R, de Graaf P, Marine J-C, Vousden KH, et al. Critical role for a central part of Mdm2 in the ubiquitylation of p53. Mol Cell Biol. 2003;23:4929–38.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. 23.

    Dobbelstein M, Sørensen CS. Exploiting replicative stress to treat cancer. Nat Rev Drug Discov. 2015;14:405–23.

    CAS  PubMed  Article  Google Scholar 

  24. 24.

    Blow JJ, Ge XQ, Jackson DA. How dormant origins promote complete genome replication. Trends Biochem Sci. 2011;36:405–14.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. 25.

    Quinet A, Carvajal-Maldonado D, Lemacon D, Vindigni A. DNA fiber analysis: mind the gap! In: Eichman BF (ed). DNA Repair Enzymes: Cell, Molecular, and Chemical Biology. Methods in Enzymology, vol 591. (Elsevier, Amsterdam, 2017) pp 55–82.

  26. 26.

    Plunkett W, Huang P, Searcy CE, Gandhi V. Gemcitabine: preclinical pharmacology and mechanisms of action. Semin Oncol 1996;23:3–15.

    CAS  PubMed  Google Scholar 

  27. 27.

    Wen W, Peng C, Kim MO, Ho Jeong C, Zhu F, Yao K, et al. Knockdown of RNF2 induces apoptosis by regulating MDM2 and p53 stability. Oncogene. 2014;33:421–8.

    CAS  PubMed  Article  Google Scholar 

  28. 28.

    Kuser-Abali G, Gong L, Yan J, Liu Q, Zeng W, Williamson A, et al. An EZH2-mediated epigenetic mechanism behind p53-dependent tissue sensitivity to DNA damage. Proc Natl Acad Sci. 2018;115:201719532.

    Article  CAS  Google Scholar 

  29. 29.

    Aguilera A, García-Muse T. R loops: from transcription byproducts to threats to genome stability. Mol Cell. 2012;46:115–24.

    CAS  PubMed  Article  Google Scholar 

  30. 30.

    Boguslawski SJ, Smith DE, Michalak MA, Mickelson KE, Yehle CO, Patterson WL, et al. Characterization of monoclonal antibody to DNA.RNA and its application to immunodetection of hybrids. J Immunol Methods. 1986;89:123–30.

    CAS  PubMed  Article  Google Scholar 

  31. 31.

    El Hage A, French SL, Beyer AL, Tollervey D. Loss of Topoisomerase I leads to R-loop-mediated transcriptional blocks during ribosomal RNA synthesis. Genes Dev. 2010;24:1546–58.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. 32.

    Fischer M, Steiner L, Engeland K. The transcription factor p53: not a repressor, solely an activator. Cell Cycle. 2014;13:3037–58.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. 33.

    Momand J, Villegas A, Belyi VA. The evolution of MDM2 family genes. Gene. 2011;486:23–30.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. 34.

    Fåhraeus R, Olivares-Illana V. MDM2’s social network. Oncogene. 2014;33:4365–76.

    PubMed  Article  CAS  Google Scholar 

  35. 35.

    Riley MF, Lozano G. The many faces of MDM2 binding partners. Genes Cancer. 2012;3:226–39.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. 36.

    Haupt S, Mejía-Hernández JO, Vijayakumaran R, Keam SP, Haupt Y. The long and the short of it: the MDM4 tail so far. J Mol Cell Biol. 2019;11:231–44.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. 37.

    Stad R, Little NA, Xirodimas DP, Frenk R, Eb AJ, van der, Lane DP, et al. Mdmx stabilizes p53 and Mdm2 via two distinct mechanisms. EMBO Rep. 2001;2:1029.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. 38.

    Lee J, Dunphy WG. The Mre11-Rad50-Nbs1 (MRN) complex has a specific role in the activation of Chk1 in response to stalled replication forks. Mol Biol Cell. 2013;24:1343–53.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. 39.

    Dewaele M, Tabaglio T, Willekens K, Bezzi M, Teo SX, Low DHP, et al. Antisense oligonucleotide–mediated MDM4 exon 6 skipping impairs tumor growth. J Clin Invest. 2015;126:68–84.

    PubMed  PubMed Central  Article  Google Scholar 

  40. 40.

    Matijasevic Z, Steinman HA, Hoover K, Jones SN. MdmX promotes bipolar mitosis to suppress transformation and tumorigenesis in p53-deficient cells and mice. Mol Cell Biol. 2008;28:1265–73.

    CAS  PubMed  Article  Google Scholar 

  41. 41.

    Marine J-C, Jochemsen AG. MDMX (MDM4), a Promising target for p53 reactivation therapy and beyond. Cold Spring Harb Perspect Med. 2016;6.

  42. 42.

    Valentin-Vega YA, Box N, Terzian T, Lozano G. Mdm4 loss in the intestinal epithelium leads to compartmentalized cell death but no tissue abnormalities. Differentiation. 2009;77:442–9.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  43. 43.

    Garcia D, Warr MR, Martins CP, Brown Swigart L, Passegué E, Evan GI. Validation of MdmX as a therapeutic target for reactivating p53 in tumors. Genes Dev. 2011;25:1746–57.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. 44.

    Heijkants RC, Nieveen M, Hart KC’t, Teunisse AFAS, Jochemsen AG. Targeting MDMX and PKCδ to improve current uveal melanoma therapeutic strategies. Oncogenesis. 2018;7:33.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. 45.

    Miranda PJ, Buckley D, Raghu D, Pang J-MB, Takano EA, Vijayakumaran R, et al. MDM4 is a rational target for treating breast cancers with mutant p53. J Pathol. 2017;241:661–70.

    CAS  PubMed  Article  Google Scholar 

  46. 46.

    Park DE, Cheng J, Berrios C, Montero J, Cortés-Cros M, Ferretti S, et al. Dual inhibition of MDM2 and MDM4 in virus-positive Merkel cell carcinoma enhances the p53 response. Proc Natl Acad Sci. 2019;116:1027–32.

    CAS  PubMed  Article  Google Scholar 

  47. 47.

    Carvajal LA, Neriah DBen, Senecal A, Benard L, Thiruthuvanathan V, Yatsenko T, et al. Dual inhibition of MDMX and MDM2 as a therapeutic strategy in leukemia. Sci Transl Med. 2018;10:eaao3003.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  48. 48.

    Pellegrino M, Mancini F, Lucà R, Coletti A, Giacchè N, Manni I, et al. Targeting the MDM2/MDM4 interaction interface as a promising approach for p53 reactivation therapy. Cancer Res. 2015;75:4560–72.

    CAS  PubMed  Article  Google Scholar 

  49. 49.

    Bernal F, Wade M, Godes M, Davis TN, Whitehead DG, Kung AL, et al. A stapled p53 helix overcomes HDMX-mediated suppression of p53. Cancer Cell. 2010;18:411–22.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. 50.

    Li Y, Yang J, Aguilar A, McEachern D, Przybranowski S, Liu L, et al. Discovery of MD-224 as a first-in-class, highly potent, and efficacious proteolysis targeting chimera murine double minute 2 degrader capable of achieving complete and durable tumor regression. J Med Chem. 2019;62:448–66.

    CAS  PubMed  Article  Google Scholar 

  51. 51.

    Técher H, Koundrioukoff S, Azar D, Wilhelm T, Carignon S, Brison O, et al. Replication dynamics: Biases and robustness of DNA fiber analysis. J Mol Biol. 2013;425:4845–55.

    PubMed  Article  CAS  Google Scholar 

Download references


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.

Author information




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.

Corresponding author

Correspondence to Matthias Dobbelstein.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Wohlberedt, K., Klusmann, I., Derevyanko, P.K. et al. Mdm4 supports DNA replication in a p53-independent fashion. Oncogene 39, 4828–4843 (2020).

Download citation

Further reading


Quick links