Zhou and Xu Cell Death and Disease (2019) 10:69 https://doi.org/10.1038/s41419-019-1317-7 Cell Death & Disease ARTICLE Open Access Cereblon attenuates DNA damage-induced apoptosis by regulating the transcription- independent function of p53 Liang Zhou1 and Guoqiang Xu1 Abstract Cereblon (CRBN) is the substrate receptor of the cullin 4-RING E3 ligase complex and has been employed for targeted protein degradation in the treatment of cancers. However, its normal physiological functions and molecular mechanism in the regulation of DNA damage response are largely unknown. Here we find that CRBN plays a protective role against DNA damage-induced apoptosis in cell lines and primary cells. Mechanistic studies demonstrate that although CRBN does not affect the ubiquitination and degradation of the tumor suppressor p53, it directly interacts with p53 and therefore, suppresses the interaction between p53 and anti-apoptotic regulators Bcl-2 and Bcl-XL. CRBN depletion enhances the interaction between p53 and Bcl-2/Bcl-XL, reduces mitochondrial membrane potential, increases the cleavage of caspase-3 and poly(ADP-ribose) polymerase 1, and thus promotes DNA damage- induced apoptosis in cell lines and primary cells upon etoposide treatment. Moreover, Crbn knockout mice exhibit increased mortality upon etoposide challenge. Taken together, our data elucidate a novel molecular mechanism by which CRBN inhibits DNA damage response in vitro and in vivo. This work extends our understanding of the broad spectrum of physiological roles for CRBN and may suggest its potential application in the treatment of DNA damage- associated diseases. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Introduction regulation of apoptosis8. The transcription-independent p53 is one of the most studied tumor suppressors, which pro-apoptotic functions of p53 are closely associated with 9 10 11 is critical for the regulation of DNA damage-induced Bcl-2 family members , such as Bcl-2 , Bcl-XL , and – apoptosis1 4. As a transcription factor, nuclear p53 pro- BAX5,11,12. The stable interaction between p53 and anti- motes the expression of pro-apoptotic genes, such as apoptotic regulators Bcl-2 and Bcl-XL is the important apoptosis regulator BAX5 and Bcl-2-binding component 3 molecular mechanism underlying DNA damage-induced 6 13,14 PUMA . Their increased protein products then initiate apoptosis . p53 stably binds to Bcl-2 and Bcl-XL, affects mitochondrial damage, reduce mitochondrial membrane mitochondrial outer membrane permeabilization, and potential (MMP), release cytochrome c, activate caspases, counteracts the protective role of Bcl-2 and Bcl-XL in the and lead to the cleavage of poly(ADP-ribose) polymerase 1 regulation of mitochondrion-mediated apoptosis10,11. (PARP1) and apoptosis4,7. However, the nuclear localiza- Therefore, both the transcription-dependent and -inde- tion signal deleted p53 can still promote apoptosis, pendent functions of p53 are involved in the regulation of indicating the important role of cytoplasm p53 in the DNA damage-induced cell death. Cullin 4-RING E3 ligases (CRL4s) are a group of protein complexes consisting of cullin-4A/B, damage-specific Correspondence: Guoqiang Xu ([email protected]) DNA-binding protein 1, RING finger protein, and mul- 1Jiangsu Key Laboratory of Neuropsychiatric Diseases and College of tiple substrate receptors, which recognize a specific set of Pharmaceutical Sciences, Jiangsu Key Laboratory of Preventive and substrates for ubiquitination15. CRL4s monoubiquitinate Translational Medicine for Geriatric Diseases, Soochow University, Suzhou, Jiangsu 215123, China histone H2A and regulate DNA damage repair, which is Edited by D. 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Official journal of the Cell Death Differentiation Association Zhou and Xu Cell Death and Disease (2019) 10:69 Page 2 of 13 mediated by the substrate receptor DDB216. The substrate experiments showed that Crbn KO mice exhibit increased receptor Cdt2 promotes CRL4-mediated mono- mortality rate upon etoposide challenge. This work indi- ubiquitination of proliferating cell nuclear antigen in cates that CRBN could protect cells from DNA damage- response to DNA damage17. Cereblon (CRBN), acting as a induced apoptosis, which provides a novel understanding substrate receptor of the CRL4 E3 ligase, regulates a of physiological roles of CRBN in p53-mediated apoptosis. variety of cellular processes through the enzymatic func- – tion of its associated E3 ligase18 20. CRBN promotes the Results ubiquitination of large-conductance calcium-activated CRBN reduces DNA damage-induced apoptosis 20 potassium (BKCa) channel , chloride channel protein 1 Although CRL4 E3 ligases regulate DNA damage – (CLC-1)21, and presynaptic proteins22, and thus poten- response36 40, the role of one of its substrate receptors tially regulates learning and memory23,24. The pharma- CRBN in DNA damage response is largely unknown. To cological intervention in animal models demonstrates that explore this function, we first determine if CRBN affects the learning and memory deficits in Crbn knockout (KO) apoptosis induced by DNA damage. We cultured the mouse are affected by exaggerated AMPK activity, inhi- primary fibroblasts dissected from wild-type (WT) and bition of mTORC1 signaling pathway24, and increased Crbn KO littermate mice (Supplementary Figure S1) and 23 activity of BKCa channel . CRBN also enhances the ubi- then treated them with the DNA damage inducers eto- quitination and degradation of c-Jun, reduces the activity poside and cisplatin. The immunofluorescence images of of c-Jun associated transcription factors, and thus propidium iodide (PI) staining showed that Crbn KO suppresses lipopolysaccharide-induced inflammatory fibroblasts are more susceptible to apoptosis induced by response25. CRBN mediates immunomodulatory drug etoposide and cisplatin (Fig. 1a, b). Given the important (IMiD)-induced death of multiple myeloma cells by pro- role of mitochondria in DNA damage-induced apoptosis, moting the ubiquitination-mediated degradation of two we then examined the MMP in WT and Crbn KO transcription factors IKZF1 and IKZF326,27. This principle fibroblasts after the induction of DNA damage. Crbn KO has been utilized for the development of proteolysis tar- fibroblasts exhibited more severe MMP reduction than geting chimera in drug discovery by targeting previously the WT fibroblasts, as indicated by tetramethylrhodamine “undruggable” proteins28,29. Furthermore, CRBN exhibits methyl ester (TMRM) staining (Fig. 1c, d), which is in non-enzymatic functions independent to its associated E3 concert with the result obtained from PI staining. Fur- ligase in multiple processes, such as on epigenetic reg- thermore, flow cytometry analyses showed that expression ulation of potassium voltage-gated channel subfamily A of CRBN protects cells from etoposide-induced apoptosis member 3 (Kcna3)30, formation of sequestosome-1/p62 (Supplementary Figure S2). Taken together, our data bodies31, and inhibition of ubiquitination of tumor indicate that CRBN exhibits protective roles in DNA necrosis factor receptor-associated factor 6 and TAK1- damage-induced apoptosis. binding protein 232 through its interaction with DNA or proteins. In addition, two CRBN mutants C391R (Cys to p53 is required for the regulation of CRBN on DNA Arg mutation at position 391) and R419X (Arg to stop damage-induced apoptosis codon mutation at position 419) have been discovered to Considering the critical role of p53 in DNA damage- be the genetic causes for different degree of intellectual induced apoptosis1, we examined whether p53 is involved disability, indicating the important roles of CRBN in the in the CRBN-mediated protection of DNA damage- – regulation of neuronal functions33 35. Moreover, it has induced apoptosis. As etoposide could activate the also been discovered that CRBN protects neuronal cells apoptotic mitochondrial pathway followed by activation from death induced by pathogenic protein aggregates in of executioner caspase-3 (Fig. 1c)41,42, we then investi- neurodegenerative diseases31. However, it is unknown gated the effects of knockdown of CRBN and p53 by small whether CRBN could regulate DNA damage in neuronal interfering RNA (siRNA) on the MMP. Immunoblotting and non-neuronal cells. experiments confirmed the knockdown efficiency of In this work, through pharmacological intervention and CRBN and p53 in human embryonic kidney (HEK) 293 genetic manipulation, we discovered that CRBN defi- cells (Fig. 2a). CRBN knockdown results in more severe ciency in cell lines, primary fibroblasts, and cortical neu- reduction of MMP than the control knockdown, whereas rons leads to their increased sensitivity