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Supporting Information Supporting Information 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] Supplementary Materials and Methods: Antibodies and reagents Mouse monoclonal anti-Flag (F3165, RRID: AB_259529) antibodies, anti-HA (H9658, RRID: AB_260092) antibodies, and etoposide (E1383) were purchased from Sigma-Aldrich. Mouse monoclonal anti-Myc (M047-3, RRID: AB_591112), anti-histidine (D291–3, RRID: AB_10597733), and rabbit polyclonal anti-β-actin (PM053, RRID: AB_10598196) antibodies were purchased from MBL. Mouse monoclonal anti-H3 (BE3015) antibodies were purchased from EASYBIO. Mouse monoclonal anti-γH2AX (05–636, RRID: AB_309864) antibodies and MG132 (2772605) were purchased from Millipore. Mouse monoclonal anti-HDAC1 (sc-81598, RRID: AB_2118083) antibodies were purchased from Santa Cruz Biotechnology. Anti-USP38 antibodies (17767-1-AP, RRID: AB_2213383) were purchased from Proteintech. Monoclonal anti-ubiquitin (P4D1, RRID: AB_10998070) antibodies were purchased from ENZO. Anti- K48-linkage specific polyubiquitin (D9D5, RRID: AB_10859893) and anti-K63-linkage specific polyubiquitin (D7A11, RRID: AB_10827985) antibodies were purchased from Cell Signaling Technology. Anti-H3K56Ac polyclonal antibodies (PTM118) were purchased form PTM Bio. Anti-SIN3A polyclonal antibodies (A1577, RRID: AB_2763188), anti-MTA1 polyclonal antibodies (A16085, RRID: AB_2763526), and anti-RCOR1 polyclonal antibodies (A12845, RRID: AB_2759686) were purchased from ABclonal. LPS (055: B5) were purchased from Sigma-Aldrich. TGF-β (240-B), IL-4(204-IL) and IL-5(205-IL) were purchased from R&D Systems. Anti-δ dextran (mouse) was purchased form Fina Biosolutions. FITC Rat Anti- Mouse IgG1 (553443, RRID: AB_394862), FITC Rat Anti-Mouse IgG3 (553403, RRID: AB_394840), FITC Rat Anti-Mouse IgA (559354, RRID: AB_397235), PE-Cy™5 Anti-IgM PE (551079, RRID: AB_394036), Rat Anti-Mouse B220 (553089, RRID: AB_394619), FITC Rat Anti-Mouse CD43 (561856, RRID: AB_10895376), and APC Rat Anti-Mouse CD19 (561738, RRID: AB_10893995) were purchased from BD Biosciences. Plasmids The cDNA of USP38 was kindly provided by Dr. Lingqiang Zhang at the Beijing Institute of Radiation Medicine. USP38 WT, USP38 C454A (USP38 CA) and USP38C454A/H857A (USP38 CAHA) were constructed and cloned into the pcDNA3 Myc vector. HDAC1 was cloned into the pcDNA3 Flag vector. His-HDAC1 was amplified by PCR and cloned into the pET-28a vector. GST-USP38 was cloned into the pGEX-4T-1 vector. All plasmids were verified by DNA sequencing. Cell culture and transfection HEK293T, HeLa, A498 and 786-O cells were cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco). USP38WT and USP38-/- MEF cells were cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1× MEM Non-Essential Amino Acids (Gibco). HEK293T cells were transfected with PEI (Polyscience) according to the manufacturer’s instructions. HeLa cells were transfected with X-tremeGENE™ HP DNA Transfection Reagent according to the manufacturer’s instructions (Sigma). Generation of USP38-/- cell lines by CRISPR-Cas9 A paired-guide sgRNA CRISPR-Cas9 system was used as described previously (1). Briefly, three pairs of single-guide RNAs (Supplementary table S1) targeting different regions in the first exon of the human USP38 gene were designed and cloned into a lentiviral sgRNA vector containing the mCherry selection marker using the Golden Gate method (2). Next, cells were co-transfected with the sgRNA vector and Cas9 vector. After 48 h of transfection, mCherry- positive cells were selected by FACS (MOFLO, Cytomation). Single clones were obtained after 10 days of selection. The knockout efficiency was confirmed by immunoblotting. USP38 gene mutations were verified by PCR and sequencing. Generation of USP38 or HDAC1 knockdown cells by shRNA lentivirus shRNA oligos (Supplementary table S1) were cloned into the pLKO.1 plasmid and verified by sequencing. The shRNA pLKO.1 plasmid was transfected into HEK293T cells with the pLP1 and pLP2 packaging plasmids and the pLP/VSVG envelope plasmid. After 48 h of transfection, the viruses were collected and stored at -80°C. During transfection, the lentiviral particle solution (MOI: 108 pfu) was added to the cells with polybrene at a ratio of 1:1000. The cell media were changed to fresh media 48 hours after infection. To select infected cells, 1 μg/mL puromycin was added to the media until the USP38 or HDAC1 protein level was stably knocked down. The knockdown efficiency was confirmed by immunoblotting. IR treatment IR treatment was performed following procedures described previously (2,3). After treatment with irradiation at the indicated dose, cells were incubated at 37 °C for 1 h or 24 h. Mass spectrometry (MS) HEK293T cells were lysed in modified RIPA buffer, sonicated and precleared with protein G beads. The supernatants were incubated with anti-FLAG affinity beads at 4°C for 4 h and eluted with FLAG peptide. The eluates were precipitated with TCA and subjected for mass spectrometric analysis. The MS data were aligned with Human Reviewed Swiss-Port database by Proteome Discoverer 2.2 software. Proteins were considered as major hits (positive) when matching the following criteria: 1) not found in negative control group; 2) the Protein FDR Confidence is high (FDR<0.01); 3) peptides≥5. Co-immunoprecipitation (Co-IP) Cell lysate preparation, immunoprecipitation and immunoblotting were performed as described previously (2,3) with slightly modifications. Briefly, cells were lysed in modified RIPA buffer and sonicated. After preclearing with protein G beads, the supernatants were incubated with the indicated antibody or IgG at 4°C for 4 h or overnight, followed by incubation with 30 μL protein G beads for 3 h. Finally, the beads were washed 3 times using modified RIPA buffer and denatured with 2× SDS loading buffer. His-ubiquitin pulldown assay His-ubiquitin pulldown assays were performed following a method described in a previous study (4). Briefly, HEK293T cells were transfected with his-ubiquitin and the indicated plasmids. After 48 h, the cells were harvested and lysed in His-pulldown buffer. The lysate was incubated with 60 µl of Ni2+ beads for 4 h and then washed 4 times with wash buffer. The beads were denatured with 2× SDS loading buffer and ubiquitin was assessed using indicated antibodies. Clonogenic survival assay First, 150–750 cells were seeded in 6-well plates in triplicate. After 24 h, cells were cultured in medium containing a different concentration of etoposide for 1 h and washed twice with DMEM or treated with the indicated doses of IR. After 12 days, cells were washed with PBS, fixed in precooled methanol for 10 min at -20°C and stained with crystal violet (0.1% wt/vol) for 15 min. The number of clones was counted. The survival fraction was normalized to the number of untreated cells. NHEJ assays and homologous recombination (HR) assays NHEJ assays were performed following a procedure described previously (2,5). USP38WT and USP38-/- HEK293T cells were cotransfected with a linear pEGFP-Pem1- Ad2 plasmid digested with HindIII and a dsRED plasmid. For the HR assay, USP38WT and USP38-/- HEK293T cells were cotransfected with DR-GFP, an I-SceI expression vector, and a dsRED plasmid. At the indicated time after transfection (36 h for NHEJ assays and 48 h for HR assays), cells were harvested and washed with PBS. Green (EGFP) and red (dsRED) fluorescence was measured by FACS on an LSRFortessa instrument (BD Biosciences). The repair efficiency is given by calculating the percentage of EGFP and dsRED double-positive cells in dsRED positive cells. The results are normalized to those of the USP38WT cells. Immunofluorescence microscopy HeLa cells were transfected with the indicated plasmids using PEI and treated with 10 Gy IR or 20 μM etoposide. At 24 h after transfection, cells were collected and fixed in precooled methanol for 10 min at -20°C following a procedure described previously (4). Images were obtained using a confocal microscope (Zeiss LSM-710 NLO & DuoScan, Germany) with a 40× objective lens or 63× oil objective. Quantification analysis was performed using Imaris 7.6 software (Bitplane, UK). Neutral comet assays Comet assays were performed with the Single Cell Gel Electrophoresis Assay-kit (Trevigen). Briefly, cells were treated as indicated, trypsinized and counted. Approximately 1 × 105 cells were mixed with low-melting agarose at a 1:10 ratio (v/v), after which 70 μL of the resulting mixture was transferred onto Gel Bond film. Samples were incubated at 4°C in the dark for 30 min to solidify. Cells were lysed by incubation with lysis solution for 60 min. The slides were immersed in neutral electrophoresis buffer (100 mM Tris Base, pH 9.0, 300 mM sodium acetate) for 30 min, after which they were subjected to 21 volts for 45 min on a horizontal electrophoresis apparatus in neutral electrophoresis buffer, fixed in 70% (v/v) ethanol for 30 min and allowed to dry overnight. Cells were visualized with propidium iodide. Images were collected using a standard Olympus epifluorescence microscope. Analysis of comets was performed with Comet Score software (TriTek).
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