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Arginine methylation and ubiquitylation crosstalk controls DNA end-resection and homologous recombination repair - PubMed

  • ️Fri Jan 01 2021

Arginine methylation and ubiquitylation crosstalk controls DNA end-resection and homologous recombination repair

Maria Pilar Sanchez-Bailon et al. Nat Commun. 2021.

Abstract

Cross-talk between distinct protein post-translational modifications is critical for an effective DNA damage response. Arginine methylation plays an important role in maintaining genome stability, but how this modification integrates with other enzymatic activities is largely unknown. Here, we identify the deubiquitylating enzyme USP11 as a previously uncharacterised PRMT1 substrate, and demonstrate that the methylation of USP11 promotes DNA end-resection and the repair of DNA double strand breaks (DSB) by homologous recombination (HR), an event that is independent from another USP11-HR activity, the deubiquitylation of PALB2. We also show that PRMT1 is a ubiquitylated protein that it is targeted for deubiquitylation by USP11, which regulates the ability of PRMT1 to bind to and methylate MRE11. Taken together, our findings reveal a specific role for USP11 during the early stages of DSB repair, which is mediated through its ability to regulate the activity of the PRMT1-MRE11 pathway.

© 2021. The Author(s).

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. PRMT1 interacts with and methylates USP11.

a Flag-tagged USP11 co-immunoprecipitates endogenous PRMT1 in MCF7 cells. Representative image from three independent repeats. b Recombinant USP11 is methylated by recombinant PRMT1 as determined by in vitro methylation assay. Methylation of histone H4 serves as a positive control. Representative image from three independent repeats. c Recombinant PRMT1 methylates endogenous USP11 isolated from 293T cells as determined by in vitro methylation assay. Incubation with 20 µM AdOx for 24 h generates hypomethylated substrate appropriate for enzymatic modification (compare lanes 4 and 5). Representative image from three independent repeats. d USP11 is methylated in vivo. 293T cells were transfected with the indicated plasmids and de novo protein synthesis inhibited by CAM/CHX treatment. Following [3H]-methyl-methionine labelling, Flag-USP11 was immunoprecipitated and incorporated methyl groups detected by SDS-PAGE and autoradiography. Flag-GFP serves as a control for protein synthesis inhibition. The graph represents quantification of methyl incorporation adjusted for the amount of protein immunoprecipitation (mean ± SD; n = 3 biologically independent experiments). e In vivo endogenous USP11 methylation is PRMT1-dependent. MCF7 cells stably expressing shPRMT1 were treated with CAM/CHX, labelled with [3H]-methyl-methionine, followed by endogenous USP11 immunoprecipitation. The incorporation of methyl groups was determined by SDS-PAGE and autoradiography. Asterisk represents non-specific protein immunoprecipitating with IgG control. Graph represents quantification of methyl-incorporation adjusted for USP11 immunoprecipitation and normalised to MCF7-shCTRL (mean ± SD; n = 3 biological independent experiments; **p = 0.00108 (Student’s t test, two-sided, equal variance). Uncropped blots and processed graphical data are provided as a Source Data file.

Fig. 2
Fig. 2. PRMT1 methylates USP11 at R433.

a Higher energy Collisional Dissociation (HCD) MS/MS spectrum and the associated peptide sequence of the arginine methylated peptide EYVELCDAAGRPDQEVAQNHK, residues 423–443 derived from USP11. b R433 is a methyl-acceptor site in cells. Autoradiograph and immunoblots of 293T cells transfected with Flag-USP11, Flag-USP11-R433K, Flag-USP11-R693K and Flag-USP11-R433K/R693K, treated with CAM/CHX and labelled with [3H]-methyl-methionine. The graph represents the quantification of n = 2 independent experiments. Data are depicted as methyl incorporation adjusted for USP11 immunoprecipitation and normalised to wild-type USP11. c USP11-R433K is the major PRMT1-mediated methylation site as determined by in vitro methylation assay. Histone H4 serves as a positive control. The graph to the right represents the quantification of n = 3 independent experiments. Data are depicted as methyl incorporation adjusted for substrate levels (USP11) and normalised to wild-type USP11 (mean ± SD). NV no value. d Protein sequence alignment by MUSCLE (multiple sequence comparison by log-expectation) using USP11 as a reference, showing conservation of R433 in several USP11 orthologues and in USP11 paralogues, USP4 and USP15. Uncropped blots and processed graphical data provided as a Source Data file.

Fig. 3
Fig. 3. Methylation of USP11 at R433 regulates DUB activity.

a Predicted structure of the catalytic core of USP11 using USP4 and USP15 structures as templates (PDB IDs: 2Y6E, 6GHA) showing the location of the distal (S1) ubiquitin-binding site, the catalytic residues (orange) and arginine 433 (red). b In vitro DUB assay using K63-linked di-ubiquitin (K63-diUb) as substrate. 293T cells were depleted of endogenous USP11 and then transfected with the indicated constructs and Flag-tagged USP11 immunoprecipitated. Following Flag peptide elution, immunoprecipitated USP11 was incubated with K63-diUb for time-course analysis of di-ubiquitin chain processing (representative image of n = 3 independent biological repeats). Lanes 1–14 were run on a single gel, dotted line is to aid interpretation. c Depletion of PRMT1 reduces USP11 activity as determined by in vitro DUB assay using K63-linked di-ubiquitin as substrate. Flag-USP11 was immunoprecipitated from 293T Flp-In-Flag-USP11 cells stably depleted for PRMT1 and peptide eluted. Flag-USP11 was incubated with K63-diUb for time-course analysis of di-ubiquitin chain processing (representative image of n = 3 independent biological repeats). All lanes were run on a single gel, the dotted line is to aid interpretation. d Methylation of USP11 alters its DUB activity. Recombinant wild-type or R433K His-tagged USP11 was incubated with GST-PRMT1, SAM (as indicated), and increasing concentrations of Ub-AMC. DUB activity was measured through fluorescence detection of cleaved AMC. Data were fitted according to Michaelis–Menten model using GraphPad Prism. Enzymatic parameters Km, Vmax and catalytic efficiency (kcat/Km) are shown in the table below (mean ± SD; n = 3 independent biological repeats: one-way ANOVA and Tukey post hoc test: Vmax: His-USP11/GST-PRMT1/SAM versus His-USP11-R433K/GST-PRMT1/SAM **p = 0.0035; His-USP11/GST-PRMT1/SAM versus His-USP11/GST-PRMT1 *p = 0.0438; kcat/Km: His-USP11/GST-PRMT1/SAM versus His-USP11-R433K/GST-PRMT1/SAM **p = 0.0034; His-USP11/GST-PRMT1/SAM versus His-USP11/GST-PRMT1 *p = 0.0104. NS not significant. Uncropped blots and processed graphical data provided as a Source Data file.

Fig. 4
Fig. 4. Methylation of USP11 at R433 is required for homologous recombination-mediated repair of DSBs.

a MCF7 cells stably expressing USP11 or USP11-R433K were transfected with siUSP11, exposed to increasing doses of olaparib and cell viability determined by colony survival assay (mean ± SD; n = 3 independent biological repeats; Student’s t test (two-sided, equal variance) *p = 0.010, **p = 0.00257 comparing USP11 with USP11-R433K). b HeLa-USP11 and HeLa-USP11-R433K cells were transfected with siUSP11, exposed to increasing doses of olaparib and cell viability determined by colony survival assay (mean ± SD; n = 3 independent biological repeats; Student’s t test (two-sided, equal variance) **p = 0.006 (1 μM); *p = 0.0412 (2 μM); *p = 0.0539 (3 μM)). c–e MCF7 cells stably expressing doxycycline-inducible shUSP11 and ectopic USP11 or USP11-R433K were treated with doxycycline for 72 hrs, exposed to 3 Gy IR, and harvested at the time points indicated (mean ± SD; n = 3 independent biological repeats). Mitosin or CENPF-positive cells were scored for: c 53BP1 (Student’s t-test (two-sided, equal variance): *p = 0.04, ***p = 0.0005); d RAD51 (Student’s t test (two-sided, equal variance): *p = 0.02 NT versus shUSP11; *p = 0.04 shUSP11/USP11 versus shUSP11/USP11-R433K); e RPA (Student’s t test (two-sided, equal variance): *p = 0.03 NT versus shUSP11 (2 h post IR); *p = 0.02 shUSP11/USP11 versus shUSP11/USP11-R433K (2 h post IR); p = 0.05 NT versus shUSP11 (6 h post IR); *p = 0.03 shUSP11/USP11 versus shUSP11/USP11-R433K (6 h post IR)). Scale bar represents 5 μm. f HeLa cells stably expressing USP11, USP11-R433K or USP11-C318S were transfected with siUSP11, exposed to 3 Gy IR, and harvested 6 hrs later. CENPF-positive cells were scored for RPA foci formation (mean ± SD; n = 3 independent biological repeats, **p = 0.0057 (Student’s t test (two-sided, equal variance); ****p < 0.0001; (ordinary one-way ANOVA and Tukey post hoc test)). g PRMT1 and USP11 are epistatic in the regulation of RPA foci formation after IR (3 Gy) in HeLa late S/G2 cells (mean ± SD; n = 3 independent biological repeats; ****p < 0.0001; one-way ANOVA and Tukey post hoc test). Uncropped blots, raw and processed graphical data provided as a Source Data file.

Fig. 5
Fig. 5. Methyl-USP11 regulates end-resection, ATR signalling and G2/M checkpoint.

a USP11 and MRE11 are epistatic in the regulation of RPA foci formation after IR (3 Gy) in late S/G2 HeLa cells. CENPF-positive cells were scored for RPA foci formation (mean ± SD; n = 3 independent biological repeats; ****p < 0.0001; one-way ANOVA and Tukey post hoc test). b USP11 is not epistatic with CtIP in the regulation of RPA foci formation after IR (3 Gy) in late S/G2 HeLa cells (mean ± SD; n = 3 independent biological repeats; Student’s t test (two-sided, equal variance): ***p = 0.0096). c Methylated USP11 is epistatic with MRE11 in the regulation of RPA foci formation after IR in late S/G2 MCF7 cells. CENPF-positive cells were scored for RPA foci formation (mean ± SD; n = 3 independent biological repeats; one-way ANOVA and Tukey post hoc test: groups A and B *p = 0.031; groups A–C **p = 0.0046; groups A–D **p = 0.0022; groups A–E *p = 0.0162; groups A–F ***p = 0.0006). d Methylated USP11 is epistatic with MRE11 in the regulation of RPA foci formation after IR in late S/G2 HeLa cells. CENPF-positive cells were scored for RPA foci formation (mean ± SD; n = 3 independent biological repeats; one-way ANOVA and Tukey post hoc test: ****p < 0.0001). e HeLa-USP11-R433K cells display defective ATR signalling. HeLa-USP11 or HeLa-USP11-R433K cells were transfected with siUSP11, exposed to 10 Gy IR and then harvested at the time-points indicated for immunoblot analysis. Representative image of n = 3 independent biological repeats. f HeLa-USP11-R433K cells exhibit a G2/M checkpoint deficiency. Cells were treated with 5 Gy IR and harvested at the time points indicated. Proportion of mitotic cells was determined by phospho-Histone H3 (S10) staining (mean ± SEM; n = 3 biological repeats; Student’s t test (two-sided, equal variance): **p = 0.0023). Data on bar graph underneath represents the 90 min time point (mean ± SEM; n = 3 biological repeats; Student’s t test (two-sided, equal variance): **p = 0.0023). Uncropped blots, raw and processed graphical data provided as a Source Data file.

Fig. 6
Fig. 6. Methylated USP11 regulates PRMT1 activity towards MRE11.

a USP11 regulates MRE11 methylation. Knockdown of USP11 was induced by doxycycline treatment of HeLa-pTRIPZ-shCTRL and shUSP11 cells, and asymmetric dimethylation of MRE11 was detected by PLA (antibodies directed towards myc-tagged MRE11 and ADMA). Data are from three independent biological experiments (median PLA foci for shUSP11 (+dox) and shUSP11 (−dox) were 19 and 34, respectively; Mann–Whitney n1 = 203; n2 = 207; p < 0.0001; two-tailed). The middle and right panels display representative PLA images and immunoblots. Scale bar represents 5 μm. b Methyl-USP11 regulates MRE11 methylation. HeLa cells stably expressing myc-MRE11 and USP11 wildtype or R433K were transfected with siUSP11 to deplete endogenous protein, and levels of MRE11 methylation were detected by PLA. Data are from three independent experiments (median PLA foci for USP11 WT and USP11-R433K were 36 and 16, respectively; Mann–Whitney n1 = 209; n2 = 207; p < 0.0001; two-tailed). The middle and right panels display representative PLA images and immunoblots. Scale bar represents 5 μm. c Depletion of USP11 reduced PRMT1 and MRE11 interactions as determined by co-immunoprecipitation. HeLa-myc-MRE11/pTRIPZ-shCTRL or HeLa-myc-MRE11/pTRIPZ-shUSP11 cells were treated with doxycycline and MRE11 immunoprecipitated with anti-myc antibodies. Associated PRMT1 was detected by immunoblotting (representative image of n = 3 independent biological experiments). d Expression of USP11-R433K decreases PRMT1 and MRE11 interactions in HeLa cells as determined by co-immunoprecipitation. HeLa-myc-MRE11 cells were generated to stably express USP11 or USP11-R433K, and MRE11 was immunoprecipitated with anti-myc antibodies. Associated PRMT1 was detected by immunoblotting (representative image of n = 3 independent biological experiments). e PRMT1-mediated methylation of USP11 and MRE11 is epistatic for RPA foci formation after ionising radiation in late S/G2 cells. HeLa cells stably expressing USP11, USP11-R433K, MRE11 and MRE11-R/K (MRE11 mutated in all PRMT1-methyl acceptor sites) were transfected with siUSP11, exposed to 3 Gy IR, and harvested 6 h later. CENPF-positive cells were scored for RPA foci formation (mean ± SD; n = 3 independent biological experiments; one-way ANOVA and Tukey post hoc test: ****p < 0.0001). Uncropped blots, raw and processed graphical data provided as a Source Data file.

Fig. 7
Fig. 7. USP11 deubiquitylates PRMT1, increasing MRE11 methylation.

a PRMT1 is ubiquitylated in 293T cells as determined by MultiDSK pulldown. DSK mut = MultiDSK that cannot bind ubiquitin (representative image of n = 3 independent biological experiments). b PRMT1 ubiquitylation status is USP11-dependent. 293T-pTRIPZ-shCTRL or shUSP11 cells were transfected with the indicated constructs and treated with doxycycline for 48 hrs. PRMT1 ubiquitylation was determined by MultiDSK pulldown (representative image of n = 3 independent experiments). c PRMT1 ubiquitylation is decreased in G2/M arrested cells in a USP11-dependent manner. HeLa-pTRIPZ-shUSP11 cells were transfected with the indicated constructs and doxycycline was added for 72 hrs. Ro-3306 (9 μM) was added 20 hrs prior to lysis and ubiquitylation status was determined by MultiDSK pulldown (representative image of n = 3 independent experiments). d MRE11 methylation is increased in G2/M arrested cells in a USP11 dependent manner. HeLa myc-MRE11/pTRIPZ-shCTRL or shUSP11 cells were treated with doxycycline for 48 h and then Ro-3306 (9 μM) for a further 20 hrs. Levels of MRE11 methylation were determined by myc-ADMA PLA. Data are from three independent biological experiments, with >75 nuclei scored per repeat (medium PLA foci for shCTRL (+Ro-3306) and shUSP11 (+Ro-3306) were 87 and 54, respectively; Mann–Whitney n1 = 243; n2 = 234; p < 0.0001; two-tailed). e Stable knockdown or overexpression of USP11 does not change PRMT1 protein levels in MCF7 and MDA-MB-231 cells. Representative image of n = 3 independent experiments. f USP11 but not PRMT1 expression levels are upregulated in S/G2 cells. 293T cells were synchronised by double thymidine block, released into complete media and harvested at the time points indicated. Cyclin A levels indicate cells in the S/G2 phase (representative image of n = 2 independent biological experiments). g Proposed model of cross-talk between arginine methylation, deubiquitylation and the control of DNA end-resection. USP11 levels increase in the S/G2 phase of the cell cycle providing substrate for PRMT1 to methylate USP11 at R433. In turn, methyl-USP11 catalyses the deubiquitylation of PRMT1, enhancing PRMT1-MRE11 interactions and promoting MRE11 methylation in S/G2 cells. This stimulates DNA end-resection that commits a cell to HR-mediated repair of DSBs. Unmethylated USP11 deubiquitylates PALB2. Uncropped blots and raw graphical data provided as a Source Data file.

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