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1 USP51 Deubiquitylates H2AK13, 15Ub and Regulates DNA Damage USP51 deubiquitylates H2AK13, 15ub and regulates DNA damage response Zhiquan Wang1, 5, Honglian Zhang1, 5 Ji Liu1, Abigael Cheruiyot2, Jeong-Heon Lee1, 3, Tamas Ordog3, Zhenkun Lou4, Zhongsheng You2, and Zhiguo Zhang1, 3* 1Department of Biochemistry and Molecular Biology, 3Center for Individualized Medicine 4Division of Oncology Research Mayo Clinic College of Medicine, Rochester, MN, 55905, USA 2Department of Cell Biology and Physiology Washington University School of Medicine 660 S. Euclid Avenue, MO 63110 5 These authors contributed equally to this work *Corresponding author: [email protected] (p): 507-538-6074, (f): 507-284-9759 1 Supplemental Figures. Supplemental Fig. 1. USP51 depletion resulted in formation of spontaneous RNF168-dependent DNA damage foci and G1 phase arrest. (A) USP51 depletion does not affect the protein levels of BRCA1 and 53BP1. (B-D) Formation of spontaneous 53BP1 and BRCA1 foci after USP51 depletion is dependent on RNF168. U2OS cells were infected with USP51 shRNA or co-infected with RNF168 shRNA 2 for 72 hours. Cells were fixed and stained with antibodies against 53BP1 and BRCA1. USP51 and RNF168 knockdown efficiency is shown in C and percentage of cells showing more than 3 53BP1 or 5 BRCA1 foci is shown in D. (E-F) USP51 depletion leads to G1 phase arrest. USP51 depleted or control U2OS cells were pulsed with BrdU and stained with antibodies against BrdU as well as Propidium iodide (PI). Quantification of percentage of cells at each phase of the cell cycle is shown in F. 3 Supplemental Fig. 2. USP51 functions downstream of the recruitment of RNF168 and RNF169 in response to IR. (A) IR-induced 53BP1, BRCA1 and FK2 foci in Figure 2 co-localize with γH2Ax and are dependent on RNF168. U2OS cells were 4 infected with RNF168 or non-target control shRNA and exposed to IR (10Gy) 72 hours after infection. Cells were fixed and stained with antibodies against γH2AX, 53BP1, BRCA1 and FK2. (B-C) Overexpression of USP51 but not its catalytically inactive mutant suppresses IR-induced 53BP1 foci but not γH2Ax foci. U2OS cells were transiently transfected with EGFP-USP51 or EGFP-USP51/CI and exposed to IR (10Gy). Immunofluorescence using antibodies against the 53BP1, γH2Ax was performed. Percentage of cells with more than 10 53BP1 or γH2Ax foci were counted shown in C. (D) Transfected EGFP-RNF169 forms IR induced DNA damage foci. U2OS cells were transiently transfected with EGFP-RNF169 and exposed to IR (10Gy). Immunofluorescence using antibodies against γH2Ax was performed. (E-F) Overexpression of USP51 but not its catalytically inactive mutant suppresses IR-induced RNF169 foci. U2OS cells were transiently co-transfected with EGFP-RNF169 and FLAG-USP51 and exposed to IR (10Gy). Immunofluorescence using antibodies against FLAG epitope was performed. Representative images (E) and quantification of IR-induced RNF169 foci (F) is shown. 5 Supplemental Fig. 3. USP51 regulates H2AK13-K15ub but not H2AK118-119ub in 293T cells. (A) Depletion USP51 does not affect H2AK118-119ub in 293T cells. 293T cells expressing FLAG-tagged H2A (K13, 15 R) were infected with two independent USP51 shRNAs (shUSP51/1 and shUSP51/2). After 72 hours, FLAG-H2A-containing mononucleosomes were purified using M2 beads. H2A and its ubiquitylated species were detected by Western blot using antibodies against FLAG epitope. WCL: whole cell lysate. (B) Overexpression USP51 affects H2AK15ub. FLAG-H2A (K118,119R) stable line was transfected with EGFP-tagged USP3, USP16 or USP51. FLAG-H2A were immunoprecipitated under denaturing conditions . FLAG-H2A and its ubiquitylated species were detected by Western blot using antibodies against FLAG epitope. WCL: whole cell lysate.. 6 Supplemental Fig. 4. Characterization of antibodies against H2AK15ub under a variety of conditions (A) The H2AK15ub antibodies recognizes ubiquitylated species produced by RNF168. Recombinant mononucleosomes were ubiquitylated by recombinant RNF168 in vitro in the presence or absence of E1 enzyme. Reaction 7 mixtures were analyzed by Western blot using antibodies against H2A (lower) and against H2AK15ub (upper). Molecular weight markers (kDa) as well as ubiquitylated H2A species were indicated. (B) Antibodies against H2AK15ub do not recognize H2K119ub. FLAG-H2A (K13,15R)-containing mononucleosomes were purified from 293T cells and analyzed by Western blot using H2AK15ub antibodies. Recombinant mononucleosomes ubiquitylated by RNF168 in vitro in the presence of FLAG-ubiquitin were used as controls. (C) Antibodies against H2AK15ub do not recognize di-ubiquitin. K63 and K27 linked di-ubiquitin species were analyzed by Western blot using H2AK15ub antibodies. FK2 antibodies were utilized to compare the ubiquitylation levels and RNF168-ubiquitylated recombinant mononucleosomes were used as controls. (D-E) Depletion of USP51, but not USP3 and USP16, results in an increase in H2AK15ub. 293T cells were infected with shRNAs targeting USP3, USP16 or USP51. 72 hours after infections, RNA was isolated and RT-PCR was performed to analyze depletion (D) and histones were isolated by acid extractions for Western blot analysis using antibodies against H2AK15ub. (F) The increase in H2AK15ub after USP51 depletion is dependent on RNF168. 293T cells stably expressing FLAG-H2A (K118, 119R) mutant were infected with lentivirus targeting USP51, RNF168 alone or in combination. FLAG-H2A (K118, 119R) was purified under denaturing conditions and analyzed by Western blotting with indicated antibodies. (G) Mouse ES cells with USP51 knock out show increased H2AK15ub levels. Histones were purified using acid extraction from USP51 knock out mouse ES cells and analyzed by western blot using H2AK15ub and H2AK119ub antibodies. (H) 8 Antibodies against H2AK15ub recognize IR-induced ubiquitin species. Histones were isolated from 293T cells with or without RNF168 depletion and in the presence or absence of 25Gy irradiation treatment. The samples were analyzed by Western blot using antibodies against H2AK15ub. Asterisk indicated nonspecific band. (I) USP51 overexpression suppresses IR-induced H2AK15ub. FLAG-USP51 or FLAG-USP51/CI was transfected to 293T cells, which were then irradiated at 25Gy. Histones were purified by acid extraction and analyzed by Western blot. The membrane below 45 kDa was probed with antibodies against H2AK15ub. 9 Supplemental Fig. 5. USP51 deubiquitylates both histone H2AK15ub and H2AK13ub and also exhibit activity for K27 or K63 linked di-ubiquitin. (A) USP51 catalyzes the removal of H2AK13ub and H2AK15ub in vitro. FLAG-H2A (K13,118,119R)- or FLAG-H2A(K15,118,119R)-containing mononucleosomes were purified from 293T cells and were then ubiquitylated by RNF168 and FLAG-ubiquitin. Ubiquitylated mononucleosomes were used as substrates for de-ubiquitylation reactions in the presence of recombinant USP51 or catalytic inactive USP51 (CI). Ubiquitylated species were detected using antibodies against FLAG epitope. (B) USP51 exhibits activities against K27 and K63 linked di-ubiquitin. K27 or K63 linked di-ubiquitin and recombinant USP51 or catalytic inactive USP51 (CI) 10 were assembled for de-ubiquitylation reactions. Ubiquitin was detected by Coomassie blue staining, and USP51 and mutant by Western blot. 11 Supplemental Fig. 6. USP51 regulates dynamic assembly and disassembly of H2AK15ub foci at DNA breaks. (A-B) USP51 depletion results in accelerated formation of H2AK15ub foci shortly after irradiation. USP51 depleted or control U2OS cells were irradiated with 2.5 Gy. Immunofluorescence was performed after different time of IR treatment using antibodies against H2AK15ub. Representative 12 images at indicated time point after irradiation were shown. The mean and SD of three independent experiments was shown (*p < 0.05). (C) USP51 depletion results in persistent H2AK15ub foci after irradiation. The experiments were performed as described above except that cells at later time points after IR treatment were used for analysis. Representative images at indicated time point after irradiation were shown. The quantification was shown in Fig. 6C. 13 Supplemental Fig. 7. USP51 regulates dynamic assembly and disassembly of 53BP1 foci at DNA breaks. (A) USP51 depletion results in increased rate of forming 53BP1 foci. USP51 depleted or control U2OS cells stable expressing FLAG-RNF168 were irradiated with 2.5 Gy. Immunofluorescence was performed at different time after IR treatment using antibodies against FLAG and 53BP1. Representative images 14 at indicated time point after irradiation were shown in A. Quantification results from three independent experiments were shown at left panel (* p < 0.05). (B) USP51 depletion results in persistent 53BP1 foci after irradiation. The experiments were performed as described above using cells at different time after IR treatment. Representative images at indicated time point after irradiation were shown. The quantification results were shown in Fig. 6E. (C) IR-induced 53BP1 foci were also persistent in USP51 knockout mouse ES cells. Representative images at indicated time point after irradiation were shown and quantification results were shown in Fig. 6G. 15 Supplemental Fig. 8. USP51 impairs DNA damage response. (A) USP51 depletion results in increased γH2Ax foci in the presence and absence of IR. USP51 depleted or control U2OS cells were irradiated with 2.5 Gy. Immunofluorescence was performed 24 hours after IR treatment using antibodies against γH2Ax. Representative images at indicated time point after irradiation were shown. Quantification results were shown in lower
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