RNF4 sustains Myc-driven tumorigenesis by facilitating DNA replication
Joonyoung Her, … , Haiyan Zheng, Samuel F. Bunting
Joonyoung Her, … , Haiyan Zheng, Samuel F. Bunting
Published March 26, 2024
Citation Information: J Clin Invest. 2024;134(10):e167419. https://doi.org/10.1172/JCI167419.
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Research Article Genetics Oncology

RNF4 sustains Myc-driven tumorigenesis by facilitating DNA replication

Abstract

The mammalian SUMO-targeted E3 ubiquitin ligase Rnf4 has been reported to act as a regulator of DNA repair, but the importance of RNF4 as a tumor suppressor has not been tested. Using a conditional-knockout mouse model, we deleted Rnf4 in the B cell lineage to test the importance of RNF4 for growth of somatic cells. Although Rnf4–conditional-knockout B cells exhibited substantial genomic instability, Rnf4 deletion caused no increase in tumor susceptibility. In contrast, Rnf4 deletion extended the healthy lifespan of mice expressing an oncogenic c-myc transgene. Rnf4 activity is essential for normal DNA replication, and in its absence, there was a failure in ATR-CHK1 signaling of replication stress. Factors that normally mediate replication fork stability, including members of the Fanconi anemia gene family and the helicases PIF1 and RECQL5, showed reduced accumulation at replication forks in the absence of RNF4. RNF4 deficiency also resulted in an accumulation of hyper-SUMOylated proteins in chromatin, including members of the SMC5/6 complex, which contributes to replication failure by a mechanism dependent on RAD51. These findings indicate that RNF4, which shows increased expression in multiple human tumor types, is a potential target for anticancer therapy, especially in tumors expressing c-myc.

Authors

Joonyoung Her, Haiyan Zheng, Samuel F. Bunting

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Figure 3

Phenotypes of Rnf4Δ/Δ cells are caused by accumulation of SUMOylated proteins.

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Phenotypes of Rnf4Δ/Δ cells are caused by accumulation of SUMOylated pro...
(A) Analysis of viability of Rnf4Δ/Δ cells transduced with constructs expressing GFP, WT RNF4, a catalytic mutant of RNF4 (RNF4-CS), or RNF4 defective for SUMO binding (RNF4-ΔSIM). (B) Quantification of EdU uptake by Rnf4Δ/Δ cells transduced with GFP or RNF4 cDNAs. (C) Western blot of cells grown in vitro for 2 days with or without continual treatment with USP7 inhibitor P22077 (2 μM) or P5091 (1 μM). (D) Analysis of nascent DNA tract length in WT and Rnf4Δ/Δ B cells. Cells were cultured in vitro for 48 hours with or without P22077 (2 μM) or P5091 (1 μM), then incubated with CldU for 20 minutes, followed by IdU for 20 minutes. CldU and IdU tract lengths were analyzed after DNA combing. Mean ± SD of n = 3 experiments shown. (E) Cell viability after 72 hours of culture with or without P22077 (1 μM, 2 μM). Viable cells were identified based on ability to exclude DAPI. (F) As in E, using P5091 (0.5 μM, 1 μM). (G) Analysis of chromosome aberrations in B cells cultured 48 hours with or without P22077 treatment (2 μM). (H) As in G, using P5091 (1 μM). (I) Analysis of chromosome aberrations in B cells cultured 48 hours with or without continuous treatment with SUMO inhibitor 2-D08 (40 μM) or ML-792 (20 nM). (J) Cell viability measured by quantification of DAPI-negative cells after 72 hours of culture with or without 2-D08 (40 μM) or ML-792 (20 nM). Error bars in A, B, and E–J show SD of the mean. P values were calculated with unpaired 2-tailed t test (A, B, and J), 1-way ANOVA with Dunnett’s multiple-comparison test (D and I), 2-way ANOVA with Dunnett’s multiple-comparison test (E and F), and 1-way ANOVA with Tukey’s multiple-comparison test (G and H). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.