The Deubiquitinase USP38 Promotes NHEJ Repair Through Regulation Of

Author Manuscript Published OnlineFirst on December 24, 2019; DOI: 10.1158/0008-5472.CAN-19-2149 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

The deubiquitinase USP38 promotes NHEJ repair through regulation of HDAC1 activity and regulates cancer cell response to genotoxic insults Yongfeng Yang1,2, Chuanzhen Yang1，2, Tingting Li3, Shuyu Yu1,2,Tingting Gan4, Jiazhi Hu4, Jun Cui5,6, and Xiaofeng Zheng1,2,* 1 State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China. 2Department of Biochemistry and Molecular Biology, School of Life Sciences, Peking University, Beijing, China. 3State Key Laboratory of Proteomics, National Center of Biomedical Analysis, Institute of Basic Medical Sciences, Beijing, China. 4Department of Cell Biology, School of Life Sciences, Peking University, Beijing, China. 5Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China. 6Collaborative Innovation Center of Cancer Medicine, Sun Yat-sen University, Guangzhou, China.

*To whom correspondence should be addressed. Xiaofeng Zheng, School of Life Sciences, Peking University, Beijing 100871, China. Tel: +86 10-6275-5712; Email: [email protected]

Running title

USP38 promotes NHEJ through regulation of HDAC1 activity

Key words

USP38, NHEJ, HDAC1 ubiquitination and activity, genome stability, resistance to DNA- damage therapy

Funding This work was supported by National Natural Science Foundation of China (81730080 and 31670786) and the National Key Research and Development Program of China (2016YFC1302401).

Conflict of interest No potential conflicts of interest were disclosed.

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 24, 2019; DOI: 10.1158/0008-5472.CAN-19-2149 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

The DNA damage response (DDR) is essential for maintaining genome integrity. Mounting evidence reveals that protein modifications play vital roles in the DDR. Here, we show that USP38 is involved in the DDR by regulating the activity of HDAC1. In response to DNA damage, USP38 interacted with HDAC1 and specifically removed the K63-linked ubiquitin chain promoting the deacetylase activity of HDAC1. As a result, HDAC1 was able to deacetylate H3K56. USP38 deletion resulted in persistent focal accumulation of NHEJ factors at DNA damage sites and impaired NHEJ efficiency, causing genome instability and sensitizing cancer cells to genotoxic insults. Knockout of USP38 rendered mice hypersensitive to IR and shortened survival. In addition, USP38 was expressed at low levels in certain types of cancers including renal cell carcinoma, indicating dysregulation of USP38 expression contributes to genomic instability and may lead to tumorigenesis. In summary, this study identifies a critical role of USP38 in modulating genome integrity and cancer cell resistance to genotoxic insults by deubiquitinating HDAC1 and regulating its deacetylation activity.

Significance: This study demonstrates that USP38 regulates genome stability and mediates cancer cell resistance to DNA-damaging therapy, providing insight into tumorigenesis and implicating USP38 as a potential target for cancer diagnosis.

Introduction Many intrinsic and extrinsic factors have been shown to generate different types of DNA damage (1). Among such factors, DNA double-strand breaks (DSBs) are the most cytotoxic DNA lesions. To maintain chromatin stability, mammalian cells have developed a complicated protective system called DNA damage response (DDR) (2-4). Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are two major pathways responsible for DSB repair (4). Proper functioning of the DDR is vital for cellular homeostasis because defective DNA damage repair causes chromosomal aberrations or mutations and leads to various diseases, including cancers (5). The DDR is strictly modulated by various post-translational protein modifications (PTMs) (6-9). For example, ubiquitination and deubiquitination play central roles in the DDR. In addition to E3 ubiquitin ligases that catalyze ubiquitin signaling to promote recruitment of repair proteins (10-13), deubiquitinating enzymes (DUBs) also function in multiple DDR pathways by either acting directly at DNA damage sites or regulating the activities of key proteins involved in the DDR (14). Among them, USP1 contributes to DNA replication (15), the auto deubiquitination of USP4 and USP15 promotes HR (16,17), USP7 targets MDC1, USP21 targets BRCA2, USP34 targets RNF168, and USP51 targets H2A to regulate the DDR (5,18-20). It is of great importance to identify novel DUBs and elucidate the underlying mechanisms by which they fine-tune the DDR process. Acetylation also functions in the DDR by regulating transcription of DDR-related proteins, modulating chromatin structure and equilibrating dynamic acetylation level of DDR-related proteins (21-23). Histone acetylation is tightly controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs) (24). HDACs are a class of enzymes that remove acetyl groups from N-acetyllysine amino acids on histones, allowing tighter wrapping of DNA on histones. This epigenetic modulation results in the formation of an inactive chromatin structure that represses DNA transcription (25-27). By targeting histone or non-histone proteins, HDACs play critical roles in cellular growth, apoptosis, DNA damage response, and tumorigenesis (28). A recent study demonstrated that HDAC1 and HADC2 regulate the DDR by promoting NHEJ repair through regulation of H3K56 acetylation (29). In addition, HDAC3, HDAC4, HDAC9, HDAC10, SIRT1, SIRT6 and SIRT7 are also involved in the DDR (30). Although so many HDACs are implicated in the DDR, the precise mechanisms underlying HDAC regulation during DNA damage repair remain poorly understood.

Here, by screening DUBs that translocate to DNA damage sites, we found that USP38 is recruited to DNA damage lesions, where it regulates K63-linked ubiquitination of HDAC1. Furthermore, USP38 depletion enhances H3K56 acetylation (H3K56Ac) and reduces NHEJ efficiency. We also found that USP38 is generally expressed at low levels in kidney renal clear cell carcinoma (KIRC), and mice with depleted USP38 show increased genome instability. In this study, we identified USP38 as a regulator of the DDR and elucidated the mechanism underlying HDAC1 regulation by USP38 during the DDR.

Materials and Methods Cell lines HeLa cells were purchased from ATCC and HEK293T was from the National Infrastructure of Cell Line Resource. A489 and 786-O cells were kindly provided by Weimin CI at the Beijing Institute of Genomics. The identities of cell lines were authenticated by short tandem repeat analysis. All cell lines were confirmed to have no mycoplasma contamination by PCR analysis. All cells were passaged for less than 1 month after resuscitation and were used at the third through fourteenth passage in culture for this study.

In vitro deacetylation assay Flag-HDAC1 protein was immunoprecipitated from HEK293T cells and eluted using Flag peptide. For the deacetylation assay, HDAC1 and histone protein were incubated in

deacetylation buffer (25 mM Tris, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2) at 30°C for 30 min. The resulting products were subjected to western blot analysis with anti- H3K56Ac antibodies.

In vitro deubiquitination assay HEK293T cells were transfected with Flag-HDAC1/His-ub, Flag-ev, Flag-USP38 WT, or Flag-USP38 CAHA. After 48 h, cells were harvested and precipitated using Flag beads. Ubiquitinated HDAC1 proteins, Flag-USP38 WT, and Flag-USP38 CAHA were eluted using Flag peptide and incubated together in deubiquitination buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA, 10 mM DTT, 5% glycerol) at 37°C for 2 h. HDAC1 ubiquitination was detected by western blotting with anti-His antibodies.

Laser microirradiation Laser microirradiation was carried out following procedures described previously (31). HeLa cells were grown on thin glass-bottom plates and irradiated with an ultraviolet laser (16 Hz pulse, 60% laser output). Images were taken using a Dragonfly (Andor) confocal imaging system for the indicated period of time.

Radio sensitivity of MEFs and mice USP38WT and USP38-/- MEF cells were plated into six well plates in triplicate. After 10 h, the plates were exposed to IR (0, 2, 4, or 8 Gy). Survival rates were calculated 12 days after IR.

USP38-/- C57BL/6 mice were kindly provided by Dr. Cui Jun at Sun Yat-sen University. Animals were housed in specific pathogen-free barrier facilities. All experiments were approved by the Peking University Laboratory Animal Center. All mice were handled following the ‘Guide for the Care and Use of Laboratory Animals’. Production license for laboratory animals (SCXK) number is SCXK-2016-0010. Twenty USP38WT and USP38-/- littermates were irradiated with 10 Gy of whole body IR, and survival rates were calculated every day after irradiation. Observation of the mice was continued for 7 weeks.

Statistical analysis Statistical analysis was performed using a Student’s t-test. All of the results are presented as mean ± SEM (* p<0.05, ** p <0.01, *** p <0.001).

Bioinformatics analysis Bioinformatics analysis of the expression of USP38 in different types of cancer and adjacent normal tissues was carried out using the TCGA database. The data were downloaded through the FireBrowse RESTful API with R package "FirebrowseR" (http://firebrowse.org/api- docs/).

Additional materials and methods are described in the Supplementary Information.

Results

USP38 is recruited to DNA damage sites and is important for maintaining genome stability Since DUBs play vital roles in maintaining genome stability, we performed laser microirradiation to identify DUBs involved in the DDR. As shown in Fig. 1A and S1A, we found that in response to DNA damage, ubiquitin-specific protease 38 (USP38) was recruited to DNA damage sites obviously among the DUBs examined. At 3 min after micro-irradiation, USP38 was recruited to DNA damage sites in the nucleus. These observations suggest that USP38 may function in the DDR pathway. To further investigate the role of USP38 in the DDR, USP38 was stably knocked down in HeLa cells with two different USP38 shRNA lentiviruses (shUSP38 #1 and shUSP38 #2, Supplementary Table S1), after which γH2AX foci, a biomarker for the DDR, were monitored by immunofluorescence assays. As the knockdown efficiency of the shRNA lentiviruses was similar, we used shUSP38 #1 transfected HeLa cells as representative cells (Supplementary Fig. S1B). In comparison with control cells transfected with the scrambled shRNA lentivirus (shctrl), cells with depleted USP38 showed an increased number of γH2AX foci (Fig. 1B). Moreover, at different time points after IR or etoposide treatment, USP38- depleted cells contained more γH2AX foci than did wild-type cells (Fig. 1B; Supplementary Fig. S1C). These results indicate that depletion of USP38 results in a notable decrease in genome stability. Next, to explore the role of USP38 in genome stability, we generated USP38-/- HEK293T cells using the CRISPR-Cas9 system (Supplementary Figs. S1D and S1E). We performed neutral comet assays using USP38WT and USP38-/- HEK293T cells treated with or without IR. Under normal conditions, the comet tails of USP38-/- cells were slightly longer than those of USP38WT cells. When IR- or etoposide-induced DSBs occurred, USP38 deletion significantly increased the length of the comet tails (Fig. 1C; Supplementary Fig. S1F), suggesting that depletion of USP38 resulted in a notable decrease in genome stability. As USP38 could promote the DSB repair, we tested whether USP38 influences the NHEJ and HR pathways. Deletion of USP38 significantly reduced the efficiency of NHEJ (Fig. 1D), but inhibited HR only slightly (Fig. 1E), suggesting that USP38 is involved in the DDR through regulating NHEJ pathway. We also investigated how USP38 is regulated. Interestingly, the inhibition of both ATM and ATR impaired the recruitment of USP38 to DNA damage sites

(Supplementary Fig. S1G), indicating that USP38 functions downstream of ATM and ATR. Taken together, these data indicate that USP38 regulates genome stability during the DDR.

USP38 is physically associated with HDAC1 To illustrate the regulatory mechanism of USP38 in the DDR, we performed immunoprecipitation (IP) assays followed by mass spectrometry to identify USP38 interacting proteins (Fig. 2A and supplementary Tables S2, S3). HDAC1, a deacetylase that functions in the NHEJ pathway, was shown to be a potential partner of USP38 (Fig. 2A). Co- immunoprecipitation (co-IP) assays showed that USP38 interacted with HDAC1 (Fig. 2B and Supplementary Fig. S2A). Although HDAC2 has ~85% similarity to HDAC1, HDAC2 showed much weaker interaction with USP38 in comparison with HDAC1 (Supplementary Fig. S2B). Furthermore, the direct interaction between USP38 and HDAC1 was demonstrated by in vitro GST pulldown assay (Fig. 2C). We also mapped the critical domain of USP38 that interacts with HDAC1 by co-IP assays. As expected, the N terminus of USP38, but not the C terminal catalytic domain, is responsible for its interaction with HDAC1 (Fig. 2D). Surprisingly, all tested HDAC1 domains interacted with USP38 (Supplementary Fig. S2C). These data demonstrate that USP38 is a novel partner of HDAC1.

USP38 deubiquitinates HDAC1 in vivo and in vitro As a new partner of HDAC1, we speculated that deubiquitinating enzyme USP38 may function in the DDR by regulating ubiquitination of HDAC1. To verify this speculation, two USP38 mutants, USP38 C454A (USP38 CA) and USP38C454A/H857A (USP38 CAHA), were constructed (Supplementary Fig. S3A). HEK293T cells transfected with Flag-HDAC1 together with wild-type or mutant USP38 were subjected to IP to assess the role of USP38 in HDAC1 ubiquitination. Interestingly, the deubiquitinase activity of the USP38 was fully abolished by CAHA mutant but not CA mutant (Fig. 3A). In addition, by His-pulldown assay, we also confirmed the effect of USP38 on the ubiquitination of endogenous HDAC1 under denatured condition (Fig. 3B). In contrast, knockout (Fig. 3C) and knockdown (Supplementary Fig. S3B) of USP38 significantly increased HDAC1 ubiquitination. Consistently, a higher level of HDAC1 ubiquitination was observed in USP38-/- cells, and re- expression of USP38 recovered its inhibitory effect on HDAC1 ubiquitination (Fig. 3D). Moreover, we performed in vitro deubiquitination assays using purified USP38 and ubiquitinated HDAC1. We found that USP38 WT effectively removed ubiquitin chains from ubiquitinated HDAC1, while the inactive USP38 CAHA mutant did not alter HDAC1 ubiquitination (Fig. 3E). These results demonstrate that USP38 is a DUB of HDAC1. 8

As recent reports have shown that HDAC1 can be degraded by K48-linked ubiquitination (32,33), we next investigated whether USP38 could regulate the stability of HDAC1. No influence of USP38 on HDAC1 stability was observed in cells transfected with different amounts of USP38 WT (Fig. 3F), which was confirmed in USP38-/- and knockdown cells (Fig. 3G and 3H). These data indicate that USP38-mediated deubiquitination of HDAC1 does not affect the stability of HDAC1. Next, we explored which type of HDAC1 ubiquitination was affected by USP38. Immunoprecipitation assays showed that USP38 efficiently removed the K63-linked polyubiquitin chain of HDAC1 (Fig. 3I). Consistently, IP assays revealed that knockout of USP38 promoted K63-linked ubiquitination, but had no effect on K48-linked ubiquitination of HDAC1 (Fig. 3J). These data indicate that USP38 regulates HDAC1 by specifically removing the K63-linked ubiquitin chain of HDAC1.

USP38 is involved in the DDR by regulating the deacetylase activity of HDAC1 Previous studies showed that HDAC1 is involved in the NHEJ pathway through modulation of H3K56 deacetylation (29). Given the regulatory effect of USP38 on HDAC1 ubiquitination, we wondered whether the effect of USP38 on HDAC1 ubiquitination affected the deacetylase activity of HDAC1. As the specificity of some commercial H3K56Ac antibodies are not ideal (34), we first demonstrated the specificity of H3K56Ac antibody used in this study by dot blot analysis (Supplementary Fig. S4A). Then we used this antibody to examine the effect of USP38 on the deacetylase activity of HDAC1. The level of H3K56Ac decreased in cells with USP38 WT overexpression but not changed in cells with overexpressed USP38 CAHA (Fig. 4A). Consistently, MEF cells isolated from USP38-/- mouse embryos exhibited a higher level of H3K56Ac in comparison with that of USP38WT mice (Fig. 4B). These data indicate that deletion of USP38 reduces HDAC1 activity. As DSB-induced damage decreased H3K56Ac mediated by HDAC1 (35), we next determined whether USP38 dynamically affected H3K56Ac in response to DNA damage. USP38WT and USP38-/- HEK293T cells were treated with IR or etoposide, after which the abundance of H3K56Ac was determined. The level of H3K56Ac decreased in USP38WT cells 1 h and recovered 12 h after treatment. However, in USP38-/- cells, the level of H3K56Ac increased slightly after IR or etoposide treatment (Fig. 4C; Supplementary Fig. S4B). These observations suggest that USP38 deletion abrogates HDAC1-mediated H3K56 deacetylation in response to DNA damage.

To explore how USP38 functions in the DDR through regulation of HDAC1, the effect of DNA damage on the interaction between USP38 and HDAC1 was assessed. As expected, IR- or etoposide-induced DNA damage enhanced USP38-HDAC1 interaction (Fig. 4D; Supplementary Fig. S4C), which subsequently reduced HDAC1 ubiquitination, while in USP38-/- cells, DNA damage induced by IR or etoposide did not decrease HDAC1 ubiquitination (Fig. 4E; Supplementary Fig. S4D). Based on these findings, we hypothesized that the deacetylase activity of HDAC1 is affected by USP38-modulated deubiquitination in the presence of DNA damage. Therefore, we designed an in vitro deacetylation assay to test this hypothesis. As shown in Fig. 4F, we expressed Flag-tagged wild-type HDAC1 and deacetylase activity-dead HDAC1 mutant H141Y together with His-ubiquitin in HEK293T cells and purified ubiquitinated HDAC1 proteins by IP using an anti-Flag antibody. Meanwhile, we obtained histone 3 (H3) via acid extraction. To better understand the effect of USP38 on HDAC1 deacetylase activity, we also purified ubiquitinated HDAC1 from USP38- /- HEK293T cells. Next, in vitro deacetylation assays were performed using these purified proteins and the abundance of H3K56Ac was examined by immunoblot assay. As expected, HDAC1 prepared from USP38WT HEK293T cells, but not USP38-/- HEK293T cells, reduced H3K56Ac efficiently, and a similar result was observed using inactive HDAC1 H141 (Fig. 4G). The HDAC1 ubiquitination level of USP38-/- cells was higher than that of USP38WT cells (Fig. 4G). Taken together, these results suggest that ubiquitination of HDAC1 dictates its deacetylase activity; a higher level of HDAC1 ubiquitination is associated with a lower level of deacetylase activity. As USP38 removed only the K63-linked ubiquitin chain of HDAC1 (Figs. 3I and 3J), we separately assessed the effects of K48-linked and K63-linked ubiquitination on HDAC1 deacetylase activity by performing in vitro deacetylation assay. As expected, K63-linked ubiquitination, but not K48-linked ubiquitination, affected HDAC1 activity (Fig. 4H). These results demonstrate that DNA damage promotes physical interaction between USP38 and HDAC1, and USP38 regulates DNA damage repair by deubiquitinating HDAC1 and thus affecting its deacetylase activity. Elia et al. showed that HDAC1 ubiquitination was altered at seven lysine sites in response to DNA damage: K66, K74, K123, K126, K242, K361, and K412 (36). To verify the effect of ubiquitination on HDAC1 deacetylase activity in the DDR, we mutated all the seven lysines to arginines to generate HDAC1 K7R. Mutations in the seven selected ubiquitination sites reduced the ubiquitination level of HDAC1 under normal conditions (Fig.

4I lane 4 vs. lane 1). Treatment with IR or etoposide reduced HDAC1 ubiquitination in cells transfected with Flag-tagged HDAC1 WT, but not in cells transfected with Flag-tagged HDAC1 K7R (Fig. 4I), suggesting that ubiquitination at some or all of the seven selected sites is altered in response to DNA damage. Overexpression of USP38 did not decrease the ubiquitination level of HDAC1 K7R (Fig. 4J), indicating that USP38 removed ubiquitin from the selected lysines. Furthermore, ubiquitination of HDAC1 K7R did not inhibit its deacetylase activity in comparison with that of HDAC1 WT (Fig. 4K). These results indicate that USP38 regulates HDAC1 ubiquitination and its deacetylase activity in response to the DDR.

USP38 promotes NHEJ-mediated DNA repair As deletion of USP38 impairs NHEJ (Fig. 1D), we wanted to know whether USP38 regulates the NHEJ pathway by modulating HDAC1 activity, we examined the effect of USP38 on NHEJ efficiency in USP38WT and USP38-/- HEK293T cells treated with or without HDAC1 inhibitor TSA. In the absence of TSA, cells with knockout USP38 showed reduced NHEJ efficiency in comparison with that of wild-type cells; however, when HDAC1 activity was abolished by TSA, deletion of USP38 had no effect on NHEJ efficiency (Fig. 5A). In accordance with these results, when the HDAC1 was knocked down, USP38 deficiency did not further reduce NHEJ efficiency (Fig. 5B). Furthermore, we found that in USP38-/- cells, rescued with USP38 WT but not USP38 CAHA could promote NHEJ efficiency to a level similar to that of wild-type cells (Fig. 5C). As HDAC1 ubiquitination regulated its deacetylase activity, we overexpressed HDAC1 WT or HDAC1 K7R in USP38-/- HEK293T cells and accessed the effect of HDAC1 ubiquitination on NHEJ rate. In USP38-/- cells, reintroduce of HDAC1 WT slightly increased NHEJ efficiency, while reoverexpression of HDAC1 K7R could promote NHEJ efficiency significantly (Fig. 5D). These results indicate that USP38 promotes NHEJ repair by regulating HDAC1 ubiquitination and deacetylase activity. Next, to investigate whether the involvement of HDAC1 in DSB-induced DDR is dependent on USP38, recruitments of HDAC1 to damage sites was monitored in HeLa cells with depleted or wild-type USP38 by microscopy. As shown in Fig. 5E, USP38 depletion impaired recruitment of GFP-HDAC1 to damage sites. As HDAC1 participates in NHEJ by affecting the persistence of NHEJ factors (37,38), we examined the effect of USP38 on recruitment of NHEJ factors Ku70 and Artemis at DSB sites. GFP-Ku70 and Artemis were rapidly recruited to laser-induced DNA damage sites in USP38 control and USP38

knockdown cells (Fig. 5F and 5G). The initial kinetics of Ku70 and Artemis accumulation at DSB sites in both cells were comparable. As NHEJ factors persist at DSBs when the NHEJ pathway is defective (39), we examined the persistence of Ku70 and Artemis at DSB sites by performing time-lapse imaging for a period of 2 h following microirradiation. Interestingly, the intensity of the Ku70 and Artemis accumulation at 2 h was approximately ~20% of the maximal level in USP38 control cells, while it was approximately ~50% of the maximal level in USP38 knockdown cells. These results suggest that USP38 deficiency prolonged the presence of NHEJ factors at DSBs sites, which was indicative of defective NHEJ. Taken together with the results showing that cells with depleted USP38 had more γH2AX foci (Fig. 1B) and reduced NHEJ efficiency (Figs. 1D and 5A-D), these results show that USP38 participates in maintaining genome stability by promoting NHEJ.

USP38 is implicated in cell survival and carcinogenesis As USP38 regulates HDAC1 ubiquitination and H3K56 acetylation in response to DNA damage, it is likely that USP38 affects cell survival under DNA damage states. Indeed, clonogenic survival analysis revealed that the knockdown of USP38 reduced cell survival under DNA damage (Supplementary Fig. S5A) and that the reintroduction of wild-type USP38 but not USP38 CAHA mutant to USP38-/- cells rendered the cells more resistant to etoposide and IR stimulation (Fig. 6A). These data suggest that the deubiquitinase activity of USP38 plays an important role in its regulation of the sensitivity of cells to DNA damage stimuli. Because DNA damaging reagents, including etoposide and IR, are potent inducers of apoptosis, we examined the effect of USP38 on apoptosis under DNA damage stimulation. As shown in Fig. 6B, expression of wild-type USP38, but not deubiquitinase activity- deﬁcient mutant USP38, in USP38-/- HEK293T cells rescued cell viability and rendered cells resistant to etoposide and IR treatment. These data indicate that the regulatory effect of USP38 on DNA damage-induced cell apoptosis is dependent on the deubiquitinase activity of USP38. As USP38 plays important roles in DNA repair and maintaining genome integrity, loss of USP38 might lead to genome instability and tumorigenesis. Therefore, we investigated the relationship between dysregulation of USP38 and human cancers. Immunochemical assays comparing the expression level of USP38 in KIRC and adjacent normal tissue revealed that USP38 expression was significantly decreased in KIRC (Fig. 6C, 6D and Supplementary Fig. S5B). Consistently, analyses of the TCGA database also showed decreased USP38

expression level in KIRC in comparison with that of normal tissue (Fig. 6E). Furthermore, analysis of 33 types of human tumor data from the TCGA revealed low USP38 expression in most tumors (Supplementary Fig. S5C). These findings suggest that USP38 depletion and reduced USP38 expression are associated with tumorigenesis. To elucidate the specific mechanism underlying the function of USP38 in kidney carcinoma, we performed the following experiments using A498 kidney carcinoma cells and 786-O renal cell adenocarcinoma cells. Co-IP assays confirmed the endogenous interaction between HDAC1 and USP38 in both A498 cells and 786-O cells (Fig. 6F). Depletion of USP38 increased the ubiquitination of HDAC1 (Fig. 6G), suggesting that USP38 is a DUB of HDAC1 in kidney cancer cells. Next, we examined the regulatory effect of USP38 on the deacetylase activity of HDAC1 in these two cell lines. As expected, the knockdown of USP38 enhanced the H3K56Ac level (Fig. 6H), which is consistent with the results from 293T and MEF cells (Fig. 4A and 4B). These results demonstrate that in kidney cancer cells, USP38 can also modulate HDAC1 deacetylase activity by deubiquitinating HDAC1. Moreover, by neutral comet assay, we assessed the effect of USP38 knockdown on genome stability in kidney cancer cells treated with or without IR. Under normal conditions, the comet tails of USP38 knockdown A498 and 786-O cells were slightly longer than those of control cells. When IR-induced DSBs occurred, USP38 depletion significantly increased the length of the comet tails compared with those of control cells (Fig. 6I and 6J). Furthermore, control and USP38 knockdown A498 and 786-O cells were exposed to different doses of IR and etoposide, and their sensitivity to DNA damage was assessed. Both A498 and 786-O cells in which USP38 was depleted were more sensitive to IR or etoposide treatment than the control cells (Fig. 6K and 6L). Taken together, these results indicate that USP38 plays important roles in maintaining genome stability and modulating sensitivity to genotoxic insults in kidney cancer cells by regulating HDAC1 ubiquitination and deacetylase activity.

Deletion of USP38 renders mice hypersensitive to IR We further confirmed the regulatory role of USP38 in maintaining genome stability using USP38-/- mice (Supplementary Fig. S6A). Increased radiation sensitivity is a hallmark of a defective DDR. Therefore, we assessed whether knockout of USP38 rendered mice hypersensitive to IR. USP38-/- and USP38WT littermates (n=20) were irradiated with a dose of 10 Gy, after which survival was monitored. As expected, all USP38-/- mice died within 20 days after irradiation, while 85% of USP38WT mice were alive 5 weeks after irradiation (Fig. 7A). A blood routine test showed that neither USP38WT nor USP38-/- mice exhibited a

significant decrease in red blood cells (RBCs), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) after IR irradiation compared to untreated mice (Supplementary Fig. S6B), suggesting that IR treatment does not induce anemia in mice. Moreover, the results of immunochemical assays showed that cells from the livers and kidneys of USP38-/- mice exhibited more γH2AX staining than those from USP38WT mice (Fig. 7B). We next isolated MEF cells from USP38WT and USP38-/- mice embryos and examined chromosome aberrations by performing chromosome separation assays. Metaphase analysis showed that USP38-/- MEF cells had more chromosomal aberrations than did USP38WT cells (Fig. 7C). Next, neutral comet assays were performed to test the effect of USP38 on genome stability. Similar to the results obtained in HEK293T cells, USP38 deletion resulted in genome instability in MEF cells (Fig. 7D). Furthermore, second-passage primary USP38-/- and USP38WT MEFs were exposed to IR or treated with different doses of etoposide to assess their sensitivity to these treatments. The surviving cells were counted on the 10th day after treatment with IR or etoposide. USP38-/- MEFs were more sensitive to IR and etoposide than were USP38WT MEFs (Fig. 7E). Deletion of USP38 in mice and MEFs increases sensitivity to irradiation, suggesting that USP38 plays important roles in regulation of the DDR and genome integrity.

Discussion Ubiquitination of many key factors in the DDR pathway is crucial for damage monitoring, signal transduction and DNA repair. A variety of DUBs function in the DDR through different mechanisms due to the complexity of the DDR process. Therefore, the identification of novel DUBs and elucidation of their underlying mechanisms in the DDR would provide a more detailed and comprehensive understanding of the roles of DUBs in DNA damage. In this study, we showed that USP38 plays a role in the NHEJ pathway and genome stability. Mechanically, in response to DNA damage stimulation, USP38 physically interacts with HDAC1 and specifically removes K63-linked HDAC1 ubiquitin, which promotes the deacetylase activity of HDAC1 and decreases the abundance of H3K56Ac. Consequently, USP38 promotes NHEJ repair. Moreover, we found that USP38 is expressed at a low level in various types of cancers. When USP38 is deficient, increased ubiquitination of HDAC1 inhibits its deacetylase activity, which enhances H3K56Ac and affects the persistence of key NHEJ factors at DNA damage sites, causing genome instability (Fig. 7F). This finding was verified in vivo using a mouse model. In addition, as H3K56Ac is one of the substrates of HDAC1 in the DDR, it is possible that HDAC1 influences DSB repair by directly targeting NHEJ factors themselves or other regulators, in addition to regulating H3K56Ac, which should be studied in the future. Mounting evidence indicates that histone modifications such as acetylation, phosphorylation and ubiquitination are key regulators of chromatin structure that affect DNA-based processes, including the DDR (40,41). Among them, HDAC1 plays a critical role in NHEJ by modulating H3K56 acetylation in a deacetylase activity-dependent manner (29). Studies exploring the mechanisms underlying regulation of HDAC1 deacetylase activity have focused primarily on HDAC inhibitors and modification such as ubiquitination and acetylation. HDAC1 acetylation at K432 blocks its deacetylase activity (42). Some studies of HDAC1 ubiquitination have investigated K48-linked ubiquitination and subsequent degradation (32,33,43), but little is known about the manner in which K63-linked HDAC1 ubiquitination is regulated. Recently, USP19 was found to remove K63-linked ubiquitination of HDAC1 in the DDR (44); however, the underlying mechanism is not well understood. In this study, we found that USP38 regulates K63- but not K48-linked ubiquitination of HDAC1, thus increase HDAC1 deacetylase activity. Since HDAC1 is usually functioned in three major multiprotein co-repressor complexes, Sin3, NuRD (nucleosome remodelling and

deacetylation), and CoREST (co-repressor for element-1-silencing transcription factor), to deacetylase histones (45), we examined the influence of USP38 on the interaction between HDAC1 and the three major components of the complexes. The deubiquitination of HDAC1 mediated by USP38 increased the interaction between HDAC1 and Sin3 (Supplementary Figs. S7A and S7B), suggesting that the K63- linked ubiquitin chain might block the interaction surface of HDAC1 and Sin3A and suppress its deacetylase activity. However, further studies are necessary to reveal the underlying mechanism. We also demonstrated that USP38 plays an important role in NHEJ repair. Knockout of USP38 significantly reduced NHEJ efficiency. Cells with depleted USP38 showed elevated accumulation of DNA damage foci in comparison with control cells, indicating that USP38 is required for a normal DDR. Moreover, when the HDAC1 activity is blocked, knockout of USP38 no longer affects NHEJ, suggesting that USP38 functions in a HDAC1-dependent manner. The prolonged residence of NHEJ factors at damage sites in USP38 depleted cells reflects defective NHEJ repair. Taken together, our findings show that USP38 promotes NHEJ repair via regulation of HDAC1 ubiquitination and its deacetylation activity. Considering that classical non-homologous end-joining (C-NHEJ) plays an essential role in the V(D)J rearrangement and also involves in IgH class switch recombination (CSR), we next examined whether defects in V(D)J rearrangement and CSR could be detected in USP38-/- mice. However, no significant change was detected for V(D)J rearrangement in USP38-/- mice versus their wild-type littermates (Supplementary Fig. S8A), which might due to the accumulative effect or functional redundancy. With regard to the accumulative effect, the USP38-/- mice have sufficient time to develop B lymphocytes though in the presence of some (minor or modest) defect in V(D)J recombination. Similarly, depletions of some C-NHEJ factors such as ATM in mice show no significant V(D)J recombination defect, although ATM deficiency causes lots of abnormal translocation during V(D)J recombination (46). Moreover, the most recently identified C-NHEJ factors XLF and PAXX are dispensable for V(D)J recombination in pro-B cell lines due to functional redundancy (47,48), which might also be true for USP38. Therefore, further study is required to identify possible factors function together with USP38 in regulation of NHEJ in the future. Our result also showed no significant CSR defect in both wild-type and USP38-/- mice (Supplementary Fig. S8B). This may be due to that the alternative end-joining pathway is also involved in CSR (49). Recent research on USP38 has focused primarily on its role in immunity (50,51). However, less is known about the role of USP38 in cancer. A study of gene expression in

patients with HER2 subtype breast cancer revealed a significant association of this subtype with USP38 expression (52). A recent study revealed that USP38 enhances the drug resistance of human colon cancer cells by regulating LSD1 ubiquitination and degradation (5). Although these studies revealed a relationship between USP38 and certain types of cancer, the specific mechanisms underlying this connection remain unknown. Given that genome instability may contribute to tumor development, and considering the roles of HDAC1 in DNA damage repair and genome integrity revealed by our study, we investigated the mechanism of USP38 in tumorigenesis. It is well known that the DDR functions like a double-edged sword. The DDR renders cancer cells resistant to chemotherapy and radiotherapy, but loss of proper DDR functioning in normal cells leads to genome instability and can promote tumorigenesis. In our study, USP38-depleted cancer cells were more sensitive to IR and etoposide treatment than were USP38WT cells. And USP38-/- mice showed more genome aberration and enhanced sensitivity to IR in comparison with USP38WT mice. Bioinformatics and immunohistochemical analyses also revealed low USP38 expression in cancer. Taken together, our results show that USP38 plays an important role in the maintenance of genome integrity in cells and mice, suggesting that reduced USP38 expression may lead to tumorigenesis. Interestingly, we did not observe tumor development in vivo in USP38-/- mice although MEF cells from the mice showed chromosomal aberration. It is likely due to the elimination of cells with persistent DSBs or oncogenic translocations via p53-dependent G1/S checkpoint in the mice. Consistent with this observation, the core NHEJ factors like XRCC4-, Lig4- or KU80-deficient mice do not spontaneously develop cancer, unless the p53-dependent checkpoint pathway is also deficient (53-55). Overall, our data show that USP38 plays a role in the DDR in response to DNA DSBs by regulating HDAC1 ubiquitination and its deacetylase activity, indicating a role of PTM crosstalk in fine-tuning the DDR signaling. Our findings provide mechanistic insight into the function of USP38 in maintaining genome stability and its role in carcinoma cells resistance to DNA damage therapy.

Acknowledgement We sincerely thank Prof. Wensheng Wei for providing CRISPR/Cas9-related plasmids, Prof. Weimin CI for providing A489 and 786-O cells and Prof. Huadong Pei for providing the HR and NHEJ systems. We appreciate the assistance from Xiaochen Li, Guilan Li, Liying Du, Dong Liu and Hongxia Lv from the Core Facilities of Life Sciences at Peking University. We

also appreciate the help of PTM Bio Company with the test of the specificity for H3K56Ac antibody.

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Figure legends

Figure 1. USP38 is involved in the DDR and is vital for genome stability. (A) HeLa cells transfected with GFP-USP38 were subjected to laser micro-irradiation to generate DSBs in a line pattern. The dynamic relocation of GFP-USP38 to DSB sites was monitored in a time course as indicated. Scale bar, 5 μm. (B) Irradiated wild-type and USP38 knocked down HeLa cells were immunostained with a γ-H2AX antibody (green) and DAPI (blue) to examine the effect of USP38 depletion on γ-H2AX foci formation; representative images of γ-H2AX foci from three independent experiments are shown. Scale bar, 10 μm. The total numbers of γ-H2AX foci per cell were quantified; data are shown as mean ± SEM; ***p<0.001. (C) Neutral comet assays were performed using USP38WT and USP38-/- 293T cells that were untreated (-) or treated with IR (+); representative images are shown. Quantification of tail moments for cells treated with (+) or without (-) IR. The graphs show means ± SEM, n = >100 cells from each sample. USP38 knockout efficiency and etoposide treatment were confirmed by immunoblotting. (D and E) The efficiency of NHEJ and HR was determined in USP38WT and USP38-/- HEK293T cells. NHEJ (D) or HR (E) efficiency of USP38-/- HEK293T cells is presented as a relative value in comparison with that of WT HEK293T cells. Statistical analysis was performed using a Student’s t-test. Data are shown as mean ± SEM. ***p<0.001. The experiments were performed three times.

Figure 2. USP38 interacts with HDAC1. (A) Tandem affinity purification was performed using HEK-293T cells transfected with Flag-USP38. Flag-USP38 was immunopurified with anti-FLAG affinity beads and eluted with FLAG peptide. The eluates were resolved on SDS/PAGE and silver stained, followed by mass spectrometric analysis. Major hits from the mass spectrometry analysis are shown in the table. (B) The interaction between USP38 and HDAC1 was confirmed by co-IP in HEK293T cells. (C) The in vitro interaction between USP38 and HDAC1 was confirmed by GST pulldown assay using purified GST-USP38 and His-HDAC1. (D) HEK293T cells transfected with the indicated Myc-USP38 truncations were subjected to Co-IP assays. The interactions between endogenous HDAC1 and USP38 truncations were confirmed by immunoblotting

Figure 3. USP38 deubiquitinates HDAC1 in vivo and in vitro. (A and B) HEK293T cells transfected with different plasmids were subjected to IP ubiquitination analysis (A) or His- ubiquitination pulldown analysis (B) using the indicated antibodies. (C) USP38WT and 22

USP38-/- HEK293T cells transfected with Flag-HDAC1 were subjected to His-ubiquitination pulldown analysis. (D) USP38WT HEK293T cells and USP38-/- HEK293T cells transfected with Myc-empty vector or Myc-USP38 were subjected to IP ubiquitination analysis. (E) HEK293T cells were transfected with Flag-USP38 WT or Flag-USP38 CAHA and Flag- HDAC1 together with his-ubi, respectively. Ubiquitinated HDAC1 and USP38 proteins were immunoprecipitated with Flag beads and purified with Flag peptide, respectively, and incubated for 3 h at 37°C. And then HDAC1 ubiquitination was assessed by immunoblotting. (F) HEK293T cells were transfected with different amounts of USP38, and the effect of USP38 on HDAC1 expression were analyzed by immunoblotting using the indicated antibodies. (G and H) The HDAC1 abundance was examined in USP38 knockdown HEK293T cells (G) or USP38-/- HEK293T cells (H) by immunoblotting using the indicated antibodies. (I) HEK293T cells transfected with the indicated plasmids were treated with DMSO or 10 μM MG132 for 8 h and then subjected to His-ubiquitin pulldown analysis using the indicated antibodies. (J) USP38WT and USP38-/- HEK293T cells transfected with the indicated plasmids were treated with DMSO or 10 μM MG132 for 8 h before etoposide treatment, and then subjected to IP ubiquitination analysis using the indicated antibodies.

Figure 4. USP38 participates in the DDR by regulating HDAC1 ubiquitination. (A) HEK293T cells were transfected with Flag-USP38, and the level of H3K56Ac was analyzed by immunoblotting using the indicated antibodies. (B) The abundance of H3K56Ac was compared between USP38WT and USP38-/- MEF cells by immunoblotting using the indicated antibodies. (C) USP38WT and USP38-/- HEK293T cells were treated with IR (10 Gy) for 1 h or treated with IR (10 Gy) and recovered for 12 h, after which the cell lysates were analyzed by immunoblotting using the indicated antibodies. (D) HEK293T cells were transfected with the indicated plasmid and treated with IR (10 Gy), and the interaction between USP38 and HDAC1 was examined by co-IP assay. (E) USP38WT and USP38-/- HEK293T cells were transfected with the indicated plasmid and treated with IR (10 Gy), and the effect of USP38 on HDAC1 ubiquitination was analyzed by His-ubiquitin pulldown using the indicated antibodies. (F) Diagram of the in vitro deacetylation assay used for G, H and K. (G) The effect of USP38 on HDAC1 deacetylase activity was examined by in vitro deacetylation assay. USP38WT and USP38-/- HEK293T cells were transfected with Flag-HDAC1 WT or Flag-HDAC1 deacetylase inactive mutant H141Y. HDAC1 was immunoprecipitated with Flag beads, eluted by Flag peptide and then incubated with acid-extracted H3 at 37°C for 0.5

h. H3K56Ac was assessed by immunoblotting. (H) HEK293T cells were transfected with Flag-HDAC1 and only K48- or K63-linked ubiquitin. Ubiquitinated HDAC1 was immunoprecipitated with Flag beads, eluted by Flag peptide and then incubated with acid- extracted H3 at 37°C for 0.5 h. The effect of HDAC1 ubiquitination on its deacetylase activity was analyzed by examination of H3K56Ac level. (I) HEK293T cells transfected with the indicated plasmid were treated with IR (10 Gy) or etoposide (40 μM) for 1 h, and the effect of DNA damage on WT or mutant HDAC1 ubiquitination was assessed by IP ubiquitination analysis using the indicated antibodies. (J) HEK293T cells were transfected with the indicated plasmid and the effect of USP38 on HDAC1 K7R ubiquitination was analyzed by IP analysis using the indicated antibodies. (K) The deacetylase activity of HDAC1 WT or K7R mutant was examined by in vitro deacetylation assay.

Figure 5. USP38 promotes NHEJ efficiency in a HDAC1-dependent manner. (A-D) The effect of USP38 on NHEJ efficiency. USP38WT and USP38-/- HEK293T cells were treated with DMSO or 1.3 μM HDAC inhibitor TSA for 12 h (A), or stably knocked down with shctrl or shHDAC1 (B), or transfected with Myc-ev or USP38 WT or USP38 CAHA (C), or transfected with Flag–ev or HDAC1 WT or K7R (D). And then the NHEJ efficiency was determined and presented as a relative value in comparison with that of WT HEK293T cells. The experiments were performed three times. Statistical analysis was performed using a Student’s t-test. Data are shown as mean ± SEM. **p<0.01, ****p<0.0001. N.S., not significant. (E-G) HeLa cells stably expressing the control vector or USP38 shRNA were transfected with GFP-HDAC1 (E), GFP-Ku70 (F) or GFP-Artemis (G). And then cells were subjected to UV laser micro-IR and live-cell imaging at the indicated time points. Representative images are shown. Statistical analysis was performed using a Student’s t-test. The graphs show mean ± SEM, n=10 for each group. **p<0.01, ***p<0.001. The USP38 knockdown efficiency was confirmed by immunoblotting.

Figure 6. USP38 knockdown cells exhibit reduced survival. (A) USP38-/- HEK293T cells expressing empty vector, USP38 WT or USP38 CAHA were treated with 0–8 Gy IR or 0–15 μM etoposide, after which clonogenic survival assays were performed. (B) USP38-/- HEK293T cells transfected with the control vector, USP38 WT or the USP38 CAHA mutant were treated with 40 μM etoposide and 10 Gy IR. The proportion of apoptotic cells was determined by flow cytometry. The experiments were performed three times. Statistical

analysis was performed using a Student’s t-test, *** p <0.001. (C) Immunohistochemistry of KIRC samples was performed with anti-USP38 antibodies. Representative images of immunohistochemical staining of USP38 in adjacent normal tissue and KIRC are shown; scale bar, 100 μm. (D) The staining shown in panel E was quantified by ImageJ software. The results are presented in box plots. ****P < 0.0001, Student’s t-test. The USP38 expression scoring method is described in the ‘Materials and Methods’ section. (E) Bioinformatics analysis of the TCGA data set for the expression of USP38 in kidney paracancerous tissue and KIRC samples. The data were downloaded through the FireBrowse RESTful API with R package "FirebrowseR" (http://firebrowse.org/api-docs/). Data are presented with box plots. Statistical analysis was performed using a Student’s t-test, *** p <0.001. (F) The interaction between USP38 and HDAC1 in A498 and 786-O cells was confirmed by co-IP assays using anti-HDAC1 antibodies. (G and H) USP38 knockdown and control A498 and 786-O cells were subjected to IP ubiquitination analysis (G) or immunoblotting (H) using the indicated antibodies. (I and J) Neutral comet assays were performed using USP38 knockdown and control A498 (I) and 786-O (J) cells treated with or without IR; representative images are shown. The quantified tail moments are shown for each group. The graphs show the mean ± SEM, with n >100 cells from each sample. The USP38 knockdown efficiency and IR treatment were confirmed by immunoblotting. (K and L) The radio sensitivity and etoposide sensitivity of USP38 knockdown and control A498 (K) and 786-O (L) cells are plotted as the fraction of surviving cells relative to the number of untreated cells. The USP38 protein level was confirmed by immunoblotting.

Figure 7. USP38 is required for mouse genome integrity. (A) USP38WT and USP38-/- littermates (n=20 for each genotype) were exposed to 10 Gy of whole-body IR and monitored for 35 days. (B) Mice were dissected on the 15th day after they were treated as in (A), and liver and kidney tissues were subjected to immunochemistry (IHC) with anti-γH2AX antibody. Representative γH2AX staining is shown. (C) USP38WT and USP38-/- MEFs were treated with demecolcine (0.1 μg/mL) for 2 h and subjected to the metaphase spread assay. At least 30–35 metaphase spreads were analyzed for chromosome aberrations for each genotype. The main types of chromosome aberrations are chromatid breaks and chromosome fusion. Representative aberrations in USP38WT and USP38-/- MEFs are shown. The arrows indicate chromosome aberrations. The experiments were performed three times. Statistical analysis was performed using a Student’s t-test. Data are shown as mean ± SEM. **p<0.01. (D)

Neutral comet assays were performed using USP38WT and USP38-/- MEFs that were untreated, treated with IR, or treated with IR after 24 h; representative images are shown. The quantified tail moments are shown for each group. The graphs show mean ± SEM, n >100 cells from each sample. The USP38 knockout efficiency and IR treatment were confirmed by immunoblotting. (E) Radio sensitivity (left) and etoposide sensitivity (right) of USP38WT and USP38-/- MEFs were plotted as the fraction of surviving cells relative to the number of untreated cells of the same genotype. (F) Model for the role of USP38 in regulation of the NHEJ pathway. Schematic diagram of USP38 in the NHEJ pathway. When DSBs occur, USP38 interacts with HDAC1 and specifically removes K63-linked ubiquitination of HDAC1, which increases the deacetylase activity of HDAC1. Consequently, HDAC1 deacetylates H3 on K56, and reduced H3K56Ac promotes NHEJ repair. When USP38 is depleted, HDAC1 polyubiquitination inhibits its deacetylase activity, which results in the persistence of NHEJ factors at DNA damage sites, leading to genome instability.

Figure 1 shctrl shUSP38 A B Time after microirradation Hours after IR γH2AX Merge γH2AX Merge Laser - 3 min 15 min 30 min

0 h

GFP-USP38

1 h USP38WT USP38-/- 1 USP38-/- 2 C D

NO IR B 4 h 150 ****** fWT) *** IR 100 8 h

50 10 *** EJ efficiency (% o EJ efficiency *** 8 N H 16 h *** 0

6 *** o ment

4 Tail m Tail 24 h 2 E 150 N.S. 0 N.S. f WT)f *** 60 100 WT No IR IR *** shUSP38 *** 40 50 *** AX per cell per γ H2 AX R efficiency (% o efficiency H R f o *** IR - -- + + + 0 20 USP38 *** Number β-actin

γH2AX 0 Downloaded from cancerres.aacrjournals.orgHo onurs afterSeptember IR 0 28,1 2021. 4 © 2019 8 American16 Association24 for Cancer Research. Author Manuscript Published OnlineFirst on December 24, 2019; DOI: 10.1158/0008-5472.CAN-19-2149 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 2

KDa protein peptide Coverage(%) A 170 130 USP38 USP38 85 72 96 72 55 HDAC1 LDHA 11 45 43 RPS27A 11 43 34 6 19 26 HDAC1 KRT6B 10 19

PRL17 9 53 17 CKB 9 44

QARS 11 17 11 MCM6 8 13

C GST-USP38 - + B His-HDAC1 + + KDa His-HDAC1 GST KDa 55 pulldown HDAC1 GST-USP38 130 55 130 His-HDAC1 USP38 55 USP38 130 Input HDAC1 55 Input β-actin 43 GST 26

Flag-HDAC1 + + + Myc-USP38 N - + - - - + KDa D Myc-USP38 C Flag-HDAC1 55 1 445 949 1042 Myc-USP38 C 72 IP: Myc USP38 USP Myc-USP38 N 43 1 400 USP38 N Myc-USP38 C 72 401 1042 Flag-HDAC1 Input USP38 C DownloadedUSP from cancerres.aacrjournals.org55 on September 28, 2021. © 2019 American Association for Cancer Myc-USP38 N 43 Research. Author Manuscript Published OnlineFirst on December 24, 2019; DOI: 10.1158/0008-5472.CAN-19-2149 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 3 USP38WT USP38-/- A B C #1 #2 Myc-USP38 - - + CAHA CA Myc-USP38 - - + CAHA CA His-ubi - + + + + + + - + + + + KDa Flag-HDAC1 + + His-ubi KDa His-ubi - + + + + KDa

HDAC1 HDAC1 72 72 His IP:Flag

72 His-pulldown

Flag-HDAC1 His-pulldown 55 His His Myc-USP38 130 72 Flag-HDAC1 55 Input 72 β-actin 43 Myc-USP38 130 WCL USP38 130 HDAC1 55 WCL HDAC1 55 USP38WT USP38-/- D E Flag-USP38 - WT CAHA Myc-USP38 - - + KDa Flag-HDAC1/His-ubi + + + KDa

ubi F Flag-USP38 - IP: HDAC1 KDa His HDAC1 55

72 deu biquitination Flag-USP38 130 β-actin HDAC1 55 43

72 vitroIn USP38 130 Flag-USP38 130 HDAC1 Input Input -/- 55 Flag-HDAC1 WT USP38 55 USP38 #1 #2 β-actin 43 + Flag-HDAC1 + + + KDa G I J shUSP38 Myc-USP38 - - + - + - + K63-ubi shRNA shctrl #1 #2 KDa His-ubi - + + k48 k48 k63 k63 KDa HDAC1 55 130 HDAC1 72 IP: Flag USP38 72 β-actin 43

USP38-/- K48-ubi

H USP38WT #1 #2 KDa His His-pulldown 72 HDAC1 55 Flag 55 130 72 USP38 130 WCL Myc-USP38 USP38 Downloaded from cancerres.aacrjournals.org130 WCL on September 28, 2021. © 2019 American Association for Cancer β-actin 43 Flag-HDAC1 HDAC1 55 Research.55 Author Manuscript Published OnlineFirst on December 24, 2019; DOI: 10.1158/0008-5472.CAN-19-2149 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 4 Myc-USP38 - + + A B C USP38WT USP38-/- D MEF IR - - + KDa FLAG-USP38 - WT CAHA IR 0 1h post IR - 1h post IR mUSP38 +/+ -/- KDa HDAC1 55 1 0.7 0.95 KDa 1 0.57 0.94 1.7 1.8 1.7 KDa IP: Myc H3K56Ac 17 H3K56Ac 17 H3K56Ac 17 Myc-USP38 130 FLAG-USP38 130 H3 17 Myc-USP38 130 H3 17 H3 17 130 USP38 130 mUSP38 HDAC1 55 Input

USP38WT USP38-/- γH2AX 17 γH2AX 17 E His-ubi + + + + Hours after IR - 1h - 1h KDa WT -/- F G USP38 USP38 H His-ubi EV K63 K48 IP: ubi-HDAC1 His-ubi + + + IP: ubi-HDAC1 Flag-HDAC1 + + + Flag-HDAC1 and His- Flag-HDAC1 + H141Y + + + + acid extraction ubiquitin co-transfection H3 KDa HDAC1 H3 acid extraction + + + KDa H3K56Ac IP from cells H3K56Ac 17 72 17 ubiquitinated HDAC1 Histone 3 H3 17 (Acid extraction ) H3 17 His pulldown His His de acetylation His His de acetylation in vitro deacetylation 72 72 72 in vitro in

Flag-HDAC1 vitro in Flag-HDAC1 55 USP38 130 55 130 Flag-HDAC1 WCL USP38 55 Input HDAC1 55 WB analysis of H3K56Ac Input Flag-HDAC1 55 H3 17 γH2AX 17 H3 17 His-ubi - + - + Flag-HDAC1 WT K7R I J K Flag-HDAC1 WT WT K7RK7R IP: ubi-HDAC1 His-ubi + + + + + + Myc-USP38 - + H3 + + + + acid extraction Treatment - IR Eto - IR Eto KDa Flag-HDAC1 K7R + + KDa KDa H3K56Ac 17 17 H3 Ubi IP: Flag IP: Flag Ubi Ubi

72 72 72

Flag-HDAC1 55 Flag-HDAC1 K7R 55 deacetylation vitro in Flag-HDAC1 55 130 Flag-HDAC1 Myc-USP38 55 Input Flag-HDAC1 Input 55 Input γH2AX 17 Flag-HDAC1 K7R 55 Downloaded from cancerres.aacrjournals.org on September 28, 2021. ©H3 2019 American17 Association for Cancer K7R: K66R, K74R, K123R, K126R, K242R, K361R, and K412R Research. Author Manuscript Published OnlineFirst on December 24, 2019; DOI: 10.1158/0008-5472.CAN-19-2149 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 5

A B C D 150 *** 150 **** 150 N.S. 150 **** *** N.S. **** *** **** **** **** **** 100 100 100 100 N.S. N.S. **

50 50 50 50 NHEJ efficiency (% ofWT) efficiency NHEJ NHEJ efficiency (% ofWT) efficiency NHEJ NHEJ efficiency (% ofWT) efficiency NHEJ 0 (% ofWT) efficiency NHEJ 0 0 0

untreated TSA shctrl shHDAC1 -/- USP38-/- USP38

USP38 WT WT-/- -/- TSA - - + + KDa USP38 WT WT-/- -/- USP38 130 shHDAC1 - - + + KDa Flag-HDAC1 - - WT K7R KDa 130 β-actin 43 USP38 Myc-USP38 - - WTCAHA KDa USP38 130 USP38 130 H3K56Ac 17 HDAC1 55 Flag-HDAC1 55 β-actin 43 β-actin 43 β-actin 43 H3 17

Time after microirradation E shctrl Laser - 1 min 3 min 6 min 9 min 12 min 15 min 160 shUSP38 *** 140 *** shctrl *** 120 *** *** *** 100 GPF-HDAC1

shUSP38 (%) intensity Relative 80 15129631 Time after irradiation (min) Time after microirradation 150 1 min 30 min 60 min 90 min 120 min F Laser - shctrl shUSP38 100 * shctrl *** *** *** 50 GPF-Ku70

shUSP38 (%) intensity Relative 0 0 1 30 60 90 120 Time after irradiation(min) Time after microirradation 150 Laser - 1 min 30 min 60 min 90 min 120 min shcrtl G shUSP38 100 * ** shctrl *** *** 50 USP38 shUSP38 GPF-Artemis

Relative intensity (%) intensity Relative β-actin 0 0 1 30 60 90 120 Downloaded from cancerres.aacrjournals.orgTime after irradiation(min) on September 28, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 24, 2019; DOI: 10.1158/0008-5472.CAN-19-2149 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 6 A B IR Etoposide USP38-/- USP38-/- 40 40 N.S. -/- N.S. USP38 + WT 100 USP38-/-+ WT *** 100 )%(tnemgarf lavivruS )%(tnemgarf -/- *** ** USP38 + CAHA ** USP38-/-+ CAHA 30 30 Myc-USP38 - WT CAHA *** * N.S. N.S. 20 20 Myc-USP38 10 10 N.S. ** β-actin *** 10 (%) cells po ptotic 10 ptotic cells (%) cells po ptotic

N.S. A

N.S. A N.S.

Survival fragment(%) Survival 0 1 1 0 0 2 4 8 0 5 10 15 IR (Gy) Etoposide (μM)

USP38 C KIRC adjacent tissue KIRC tissue KIRC D E

P=7.57e-5 **** USP38 score USP38 log2(RSEM+0.001)

Adjacent tissue KIRC 0 1 2 3 4 5 (n=75) (n=75) tumor normal p value: 1.09172293897073e−30 G A498 786-O USP38 shRNA USP38 shRNA F A498 786-O H shRNA Ctrl #1 #2 Ctrl #1 #2 KDa A498 786-O USP38 shRNA USP38 shRNA KDa ubi IP: HDAC1 shRNA Ctrl #1 #2 Ctrl #1 #2 KDa USP38 130 72 H3K56Ac 17 HDAC1 55 HDAC1 55 USP38 USP38 130 130 USP38 130 Input Input HDAC1 55 HDAC1 55 H3 17

I J K L ctrl USP38WT USP38-/- 1 USP38-/- 2 USP38WT USP38-/- 1 USP38-/- 2 ctrl shUSP38 #1 100 shUSP38 #1 100 shUSP38 #2 shUSP38 #2 NO IR NO IR ** ** ** 10 *** 10 ** IR IR *** Survival fragment(%) Survival Survival fragment(%) Survival 1 1 0 2 4 8 0 2 4 8 IR (Gy) IR (Gy) *** 10 10 *** ctrl *** *** ctrl 8 8 100 shUSP38 #1 100 shUSP38 #1 *** *** shUSP38 #2 shUSP38 #2 6 6 * *** *** *** ** 4 4 10 10 Tail moment Tail

Tail moment Tail ** **** 2 2 ** Survival fragment(%) Survival 0 0 fragment(%) Survival 1 1 0 5 10 15 0 5 10 15 Etoposide (μM) Etoposide (μM)

No IR IR No IR IR

IR - -- + + + IR - -- + + + USP38 USP38 USP38 USP38 β-actin β-actin β-actin β-actin γH2AX γH2AX Downloaded from cancerres.aacrjournals.org on SeptemberA 4928,8 cell 2021. © 2019 American786-O cell Association for Cancer A498 cell 786-O cell Research. Author Manuscript Published OnlineFirst on December 24, 2019; DOI: 10.1158/0008-5472.CAN-19-2149 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 7 liver kidney D No IR IR Post IR A B 100 )%( l )%(

WT USP38WT a vivrus eg vivrus USP38 50

a WT

tnecreP USP38 γH2 AX -/-

-/- USP38 USP38-/-

0 USP38 0 10 20 30 40 Days elapsed 8 ***

USP38 WT MEF USP38 -/- MEF 6

USP38WT en t *** C *** 5 USP38-/- *** 4 il mom il a T ge (%) 4 2 *** 3

pe rcent a 0 *** 2 USP38 WTWTWT-/- -/- -/- ssions

a 1 No IR IR Post IR

Abe rr 0 break fusion total No IR IR Post IR mUSP38 WTWTWT-/- -/- -/-

mUSP38 β-actin mUSP38 γH2AX β-actin

DNA damage stimuli F E DSB K63 linked ubiquitin USP38 low or deficient USP38 normal

WT WT 100 USP38 100 ** USP38 USP38 )%(tnemg * USP38-/- * USP38-/- ** HDAC1 HDAC1 ? ** USP38 * ** ** regulators a * ra gment(%) rf l rf 10 10 HDAC1 activity HDAC1 activity

* l f a a

vivruS NEHJ NEHJ factors factors Su rviv NEHJ 1 1 factors H3K56Ac H3K56Ac 0 5 10 15 0 2 4 8 NEHJ Etoposide (μM) IR (Gy) factors

NHEJ defect NHEJ and Downloaded from cancerres.aacrjournals.org on September 28,Genome 2021. instability © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 24, 2019; DOI: 10.1158/0008-5472.CAN-19-2149 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

The deubiquitinase USP38 promotes NHEJ repair through regulation of HDAC1 activity and regulates cancer cell response to genotoxic insults

Yongfeng Yang, Chuanzhen Yang, Tingting Li, et al.

Cancer Res Published OnlineFirst December 24, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-19-2149

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