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 1

Genomic instability and apoptosis in RNF4-conditional-knockout B cells.

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Genomic instability and apoptosis in RNF4-conditional-knockout B cells. ...
(A) Schematic representation of Cre-induced deletion of Rnf4 exon 6. (B) Flow cytometry analysis of cell populations in the spleens of WT and Rnf4-conditional-knockout (Rnf4Δ/Δ) mice. (C) Western blot analysis of protein expression in Rnf4Δ/Δ B cells after 2 days of activation in vitro. (D) Western blot to measure induction of markers of DNA damage signaling in activated WT and Rnf4Δ/Δ B cells after 2 days and 3 days of in vitro culture. (E) Analysis of chromosome aberrations in metaphase spreads prepared from WT and Rnf4Δ/Δ B cells after 1 or 2 days of in vitro culture. Arrows show examples of chromosome rearrangements, and arrowheads show examples of chromosome breaks. (F) Cell cycle analysis of fixed B cells after 48 hours of activation in vitro. (G) Flow cytometry analysis of cell death in splenic B cells cultured for 2 or 3 days in vitro. Left: Figures indicate the percentage of cells that remained viable, based on propidium iodide exclusion. (H) Chromosome aberrations in metaphase spreads from WT, Rnf4Δ/+, and Rnf4Δ/Δ B cells. (I) Viability of B cells after 2 and 3 days of in vitro culture, measured by DAPI exclusion. Error bars indicate SD of the mean. P values were calculated with unpaired 2-tailed t test (E–G), 1-way ANOVA with Dunnett’s multiple-comparison test (H), and 2-way ANOVA with Dunnett’s multiple-comparison test (I). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.