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

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.

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.

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..

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.

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.

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.

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.

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 panel. (B) Overexpression of USP51, but not catalytically inactive mutant

USP51 (C/I), results in increased sensitivity to IR. U2OS cells overexpressing USP51 or USP51 (CI) mutant were irradiated with the indicated IR doses. Cell viability was measured 3 days after irradiation. Right panel shows the expression of FLAG-tagged

USP51 or USP51 (CI) mutant.

Supplemental Fig. 9. USP51 impairs DNA repair in a RNF168 dependent manner. (A) Depletion of RNF168 suppresses the increase in NHEJ efficiency

17 caused by USP51 depletion. HeLa cells were infected with USP51 shRNA or co-infected with RNF168 shRNA. NHEJ efficiency was measured as Figure 7D.

Right panel showed USP51 and RNF168 knocking down efficiency. (B) RNF168 co-depletion abolished increased HR efficiency caused by USP51 depletion. DR-GFP cells were infected with USP51 shRNA or co-infected with RNF168 shRNA. HR efficiency was measured as described in experimental procedures. Right panel showed USP51 and RNF168 knocking down efficiency.

Supplemental Fig. 10: Full images for H2AK16ub Western blot of Fig. 5C, 5D and 6A.

18 Materials and Methods

Cell culture and transfection

Culture media were supplemented with 10% fetal bovine serum (Sigma) and

o maintained at 37 C and 5% CO2 atmosphere. U2OS cells (ATCC) were grown in

McCoy's 5A (Cellgro). HEK293T cells (ATCC) were grown in DMEM (Cellgro).

HeLa DR-GFP cells were cultured in DMEM supplemented with 2 µg/ml puromycin

(Invivogen). To generate stable cell lines, HEK293T cells were infected by lentiviral vectors encoding FLAG-H2A, FLAG-H2A (K118,119R), or FLAG-H2A (K13,15R) and selected with 3 µg/ml puromycin. Clones with low expression levels were used in experiments and were maintained in medium with 1 µg/ml puromycin. FLAG-USP51

U2OS stable cell lines were selected with 2µg/ml and maintained in 0.5µg/ml puromycin. HEK293T and U2OS cells were transfected with PEI (Polyethylenimine) and X-tremeGENE HP (Roche), respectively.

Generation and characterization of histone H2AK15ub monoclonal antibody

Monoclonal antibodies against histone H2A ubiquitylated at Lys15 (H2AK15ub)

(Clone EDL H2AK15-1, IgG1k; Clone EDL H2AK15-4, IgG2bk) were generated in the Mayo Clinic Antibody Hybridoma Core (Rochester, MN). The branched peptide

(Ac-ARAKAK[GGRL]TRSSC) corresponding to the ubiquitination site on human

H2AK15 was synthesized from EZBioLab. The peptide conjugated with keyhole limpet hemocyanin (KLH) was injected into mice for immunization. After

19 hybridoma fusion, the supernatants from hybridoma clones were initially screened by

ELISA using the branched peptide and the corresponding unmodified peptide. The positive clones were further screened by Western blotting analysis using in vitro ubiquitinated H2A. In vitro ubiquitinated H2A was generated by incubating 2.5 µg mononucleosomes purified from human cell with 30nM yeast E1, 1.5 µM UbcH5a, 4

µM RNF168 (1-113), 10 µM Flag-ubiquitin and 3 mM ATP in ubiquitylation buffer

(50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM Zncl2 and 1 mM

DTT) at 30 °C for 2h.. The reaction products in the absence of recombinant E1 were used as negative control for in vitro ubiquitination. Clone EDL H2AK15-4 was used in the immunofluorescence and Western blot assays.

Protein purification

The RING domain of human RNF168 (1-113) was expressed in E. coli and purified according to established protocols (Liu et al. 2013). Briefly, protein extracts were incubated with Nickel Sepharose beads (Qiagen) in buffer (50 mM HEPES pH 8,

500 mM NaCl, 10% glycerol, 10 mM imidazol, 1 µM ZnCl2, 1 mM TCEP). After extensive washing, proteins were eluted with 400 mM imidazol and dialyzed against buffer (50 mM HEPES pH 8, 250 mM NaCl, 10% glycerol, 10 mM imidazol,

1 µM ZnCl2, 1 mM DTT). Full-length USP51 and USP51/CI mutant were cloned to pBac-Fast with 6xHis tag and expressed in sf9 insect cells. Proteins were purified using nickel agarose beads, eluted with 400 mM imidazol and dialyzed against buffer

(50 mM HEPES pH 8, 250 mM NaCl, 10% glycerol, 10 mM imidazol,

20 1 µM ZnCl2, 1 mM DTT).

Plasmid construction

USP51 cDNA was purchased from GeneCopoeia. USP51 and H2A cDNAs were cloned into the pcDNA3 with an N-terminal FLAG tag. The cDNAs encoding the

USP51 catalytic inactive mutant (C372S, H665R), H2A (K13,15R), or H2A

(K118,119R) were generated by site-directed mutagenesis and were subcloned into pEGFP-C2 or pFast-Bac. The cDNAs encoding USP51 or H2A were subcloned into pTsin vector for lentivirus production and infection. FLAG-USP3 and FLAG-USP16 were purchased from Addgene and subcloned into pEGFP-C1. EGFP-RNF169 plasmid was kindly provided by Dr. Daniel Durocher.

Short hairpin RNA interference and lentivirus package

The shRNAs against USP51 and RNF168 were purchased from Sigma-Aldrich and validated for knockdown efficiency. The following shRNAs were selected for experiments: shUSP51: TRCN0000038849, TRCN0000038850; shRNF168:

TRCN0000034136, TRCN0000034137; shUSP3: TRCN0000320763,

TRCN0000320690; shUSP16: TRCN0000436256, TRCN0000435081. Lentiviral particles were produced in HEK293T cells.

Immunofluorescence

Immunofluorescence was performed as previously described (Chan et al. 2011; Chan

21 et al. 2013). Cells grown on chamber slides (Nunc) or coverslips were washed briefly with PBS followed by fixation with 3% paraformaldehyde at room temperature for

15min. Cells were permeabilized with 0.5% Triton X-100 solution for 5 min at room temperature and then blocked with 5% normal goat serum for 1 hour. Primary antibodies with appropriate dilutions (Flag (1:1000), 53BP1 (1:500), BRCA1 (1:200), ubiquitin FK2 (1:2000), MDC1 (1:500), γH2Ax (1: 2000) and H2AK15ub (1:500)) were added and incubated at 4oC overnight. Cells were then washed with PBS 3 times and probed with FITC/rhodamine-conjugated secondary antibodies at room temperature for 1 hour. DNA was counterstained with DAPI and samples were mounted with ProLong Gold Antifade reagents (Invitrogen) and examined using a

Zeiss Axioplan Fluorescence microscope.

Mononucleosome immunoprecipitation

Mononucleosomes were purified as previously described (Chan et al. 2013).

Chromatin from the stable cell lines expressing FLAG-H2A or FLAG-H2A mutants were digested by micrococcal nuclease (NEB, M0247S，1u/1000 cells) at 37 °C for 20 min. After clarification by centrifugation, the digested nucleosomes were incubated with 30 µl of Anti-FLAG M2 agarose beads (Sigma) at 4°C overnight. The beads were then washed four times using washing buffer (50 mM HEPES-KOH, pH 7.4,

200 mM NaCl, 0.5% Triton X-100, 10% glycerol, 100 mM EDTA and proteinase inhibitors) for 5 min each time. Proteins were eluted by 1 mg/ml FLAG peptide

(Sigma) at 16 °C for 30 min and precipitated by TCA.

22 Denatured immunoprecipitation

For denaturing immunoprecipitation, cells expressing FLAG-H2A or FLAG-H2A mutants were lysed in 100 µl of lysis buffer (50 mM HEPES-KOH, pH 7.4, 200 mM

NaCl, 0.5% NP40, 10% glycerol, 100 mM EDTA and proteinase inhibitors) and 1%

SDS, sonicated, and mixed with 9 volumes of lysis buffer. The lysate was mixed by pipetting, and centrifuged at 16,000 × g for 20 min at 4 °C. The supernatant were incubated with 30 µl of Anti-FLAG M2 agarose beads (Sigma) at 4°C overnight. The beads were then washed four times using washing buffer lysis buffer for 5 min each time. Proteins were eluted by 1 mg/ml FLAG peptide (Sigma) at 16 °C for 30 min and precipitated by TCA.

References

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