Journal: Molecular Cell

Article Title: PRMT5-Dependent Methylation of the TIP60 Coactivator RUVBL1 Is a Key Regulator of Homologous Recombination

doi: 10.1016/j.molcel.2017.01.019

Figure Lengend Snippet: PRMT5 Is Required for Effective Double-Strand Break Repair (A) HeLa-shCTRL, shPRMT5 hairpin 1 (1), and shPRMT5 hairpin 2 (2) cells were stimulated with 3Gy IR and co-stained for γH2AX and 53BP1 foci at the time points indicated. (B and C) Quantification of (A) is shown (mean ± SEM; n = 3). (D) Reconstitution of PRMT5-depleted HeLa cells with wild-type or catalytically inactive (MD) PRMT5. Quantification of 53BP1 foci 24 hr after IR (3 Gy) is shown (mean ± SEM; n = 3). (E) Primary, non-immortalized prmt5 f/f and prmt5 f/f ;CreERT2 mouse embryonic fibroblasts (MEFs) were cultured in the absence or presence of 4-hydroxy-tamoxifen (4-OHT; 500 nM for 24 hr), followed by 4 days in 4-OHT-deficient media, and then immunoblotted for PRMT5. (F–H) Cells were stimulated with 5Gy IR and co-stained for γH2AX and 53BP1 foci at the time points indicated (F). Quantification of γH2AX (G) and 53BP1 (H) foci is shown (mean ± SEM). (I) HeLa-shPRMT5 cells were exposed to increasing doses of IR and cell viability was determined by colony survival assay (mean ± SD; n = 3). (J) Analysis of chromatid gaps and breaks per metaphase is shown (mean ± SEM; ∗∗ p < 0.0098; n = 3). (K) Quantification of 53BP1 foci in mitosin-positive (late S/G2) cells 24 hr after 3Gy IR is shown (mean ± SEM; n = 3). Scale bar, 10 μm. See also Figure S1 .

Article Snippet: shPRMT5 (2) (5′-AGGGACTGGAAT ACGCTAATT-3′) , Broad Institute GPP Web Portal , http://portals.broadinstitute.org/gpp/public.

Techniques: Staining, Cell Culture, Clonogenic Cell Survival Assay

Journal: Molecular Cell

Article Title: PRMT5-Dependent Methylation of the TIP60 Coactivator RUVBL1 Is a Key Regulator of Homologous Recombination

doi: 10.1016/j.molcel.2017.01.019

Figure Lengend Snippet: PRMT5 Promotes Homologous Recombination-Mediated DSB Repair Independently of ATM Activation (A) HeLa-shCTRL and shPRMT5 cells were stimulated with 3Gy IR and RAD51 foci analyzed 6 hr later. Quantification of number of cells with >10 RAD51 foci is shown below (mean ± SEM; ∗∗ p = 0.006; n = 3). (B) Quantification of RPA foci in CENPF-positive (late S/G2) cells 8 hr after 3Gy IR is shown (mean ± SEM; ∗∗ p = 0.006; n = 3). (C) Time course of phosphorylated and total protein levels is shown. (D) U2OS-DR3 cells were treated with siRNA as indicated before transfection of I - Sce1 and myc-PRMT5. The number of GFP-positive cells (HR proficient) was determined by flow cytometry (mean ± SD; n = 3). (E) PRMT5-depleted cells were treated with increasing doses of camptothecin (CPT; left panel) or Olaparib (right panel), and survival was determined by colony assay (mean ± SD; n = 3). (F) Cells were treated with CPT (1 μM) for the time points indicated and immunoblotted for phosphorylated and total proteins. Scale bar, 10 μm. See also Figure S2 .

Article Snippet: shPRMT5 (2) (5′-AGGGACTGGAAT ACGCTAATT-3′) , Broad Institute GPP Web Portal , http://portals.broadinstitute.org/gpp/public.

Techniques: Homologous Recombination, Activation Assay, Transfection, Flow Cytometry, Colony Assay

Journal: Molecular Cell

Article Title: PRMT5-Dependent Methylation of the TIP60 Coactivator RUVBL1 Is a Key Regulator of Homologous Recombination

doi: 10.1016/j.molcel.2017.01.019

Figure Lengend Snippet: PRMT5 Binds to and Methylates RUVBL1 at R205 (A) SYPRO Ruby stain of PRMT5 protein complexes isolated from 293T cells stably expressing tetracycline-regulated TAP-tagged catalytically inactive PRMT5 (PRMT5 MD) is shown. (B) Co-immunoprecipitation of myc-PRMT5 MD with FLAG-tagged RUVBL1/RUVBL2 is shown. (C) Endogenous immunoprecipitation of RUVBL1 from HeLa cells co-immunoprecipitates endogenous PRMT5. (D) Transfected HeLa-shCTRL or shPRMT5 cell lines were treated with CHX/CAM and labeled with [ 3 H]-methyl methionine. FLAG-RUVBL1 was immunoprecipitated, and incorporated methyl groups were detected by SDS-PAGE followed by autoradiograph. Quantification of [ 3 H]-methyl signal intensity adjusted for immunoprecipitation (IP) and normalized to shCTRL is shown (mean ± SD; ∗ p = 0.03 and ∗∗∗ p = 0.0009). (E) Autoradiograph and immunoblots of 293T cells transfected with FLAG-RUVBL1 or FLAG-RUVBL1-R205K, treated with CHX/CAM and labeled with [ 3 H]-methyl methionine. Quantification of [ 3 H]-methyl signal intensity adjusted for IP and normalized to wild-type is shown (mean ± SD; n = 3). (F) Immunoblot analysis of endogenous RUVBL1-R205me2s in HeLa-shCTRL or shPRMT5 cell lines is shown. (G) Immunoblot analysis of endogenous RUVBL1-R205me2s in HeLa cells treated with Adox (100 μM; 24 hr) or GSK591 (5 μM; 24 hr) is shown. (H) Transfected 293T cells were damaged with 10Gy IR and myc-PRMT5 MD immunoprecipitated. Associated RUVBL1 was detected by FLAG immunoblotting. (I) Autoradiograph and immunoblots of 293T cells transfected with FLAG-RUVBL1, treated with CHX/CAM, and labeled with [ 3 H]-methyl methionine for 4 hr before treating with 10Gy IR are shown. (J) Immunoblot analysis of whole-cell lysates transfected with FLAG-RUVBL1 and damaged with 10Gy IR. See also .

Article Snippet: shPRMT5 (2) (5′-AGGGACTGGAAT ACGCTAATT-3′) , Broad Institute GPP Web Portal , http://portals.broadinstitute.org/gpp/public.

Techniques: Staining, Isolation, Stable Transfection, Expressing, Immunoprecipitation, Transfection, Labeling, SDS Page, Autoradiography, Western Blot

Journal: Molecular Cell

Article Title: PRMT5-Dependent Methylation of the TIP60 Coactivator RUVBL1 Is a Key Regulator of Homologous Recombination

doi: 10.1016/j.molcel.2017.01.019

Figure Lengend Snippet: PRMT5-Dependent Methylation of RUVBL1 at R205 Regulates 53BP1 Removal and Is Epistatic with TIP60 (A) HeLa-shCTRL or shPRMT5 cells were siRNA transfected, exposed to 3Gy IR, and cell viability was assessed by colony survival. Results are expressed normalized to the respective non-treatment control (mean ± SD; n = 3). (B and C) HeLa-shCTRL or shPRMT5 cells were siRNA transfected, exposed to 3Gy IR, and stained for 53BP1 (B) or CENPF/RPA (C) foci 24 and 8 hr after damage, respectively (mean ± SEM; n = 3). (D and E) HeLa-shCTRL or RUVBL1-R205K cells were siRNA transfected, exposed to 3Gy IR, and stained for 53BP1 (D) or CENPF/RPA (E) foci 24 and 8 hr after damage, respectively (mean ± SEM; n = 3). (F) Validation of siTIP60 knockdown is shown. (G) Cells were transfected with si53BP1 and the number of mitosin-positive cells with >10 RAD51 foci was quantified (mean ± SEM; n = 3). (H) Cells were transfected with si53BP1 and the number of CENPF-positive cells with >10 RPA foci was quantified (mean ± SEM; n = 3). (I) Validation of 53BP1 siRNA SMARTpool. See also Figure S6 .

Article Snippet: shPRMT5 (2) (5′-AGGGACTGGAAT ACGCTAATT-3′) , Broad Institute GPP Web Portal , http://portals.broadinstitute.org/gpp/public.

Techniques: Methylation, Transfection, Control, Staining, Biomarker Discovery, Knockdown

Journal: Molecular Cell

Article Title: PRMT5-Dependent Methylation of the TIP60 Coactivator RUVBL1 Is a Key Regulator of Homologous Recombination

doi: 10.1016/j.molcel.2017.01.019

Figure Lengend Snippet:

Article Snippet: shPRMT5 (2) (5′-AGGGACTGGAAT ACGCTAATT-3′) , Broad Institute GPP Web Portal , http://portals.broadinstitute.org/gpp/public.

Techniques: Recombinant, Staining, Mutagenesis, Plasmid Preparation, Sequencing, Negative Control, Software

Journal: Molecular Cell

Article Title: PRMT5-Dependent Methylation of the TIP60 Coactivator RUVBL1 Is a Key Regulator of Homologous Recombination

doi: 10.1016/j.molcel.2017.01.019

Figure Lengend Snippet: PRMT5 Is Required for Effective Double-Strand Break Repair (A) HeLa-shCTRL, shPRMT5 hairpin 1 (1), and shPRMT5 hairpin 2 (2) cells were stimulated with 3Gy IR and co-stained for γH2AX and 53BP1 foci at the time points indicated. (B and C) Quantification of (A) is shown (mean ± SEM; n = 3). (D) Reconstitution of PRMT5-depleted HeLa cells with wild-type or catalytically inactive (MD) PRMT5. Quantification of 53BP1 foci 24 hr after IR (3 Gy) is shown (mean ± SEM; n = 3). (E) Primary, non-immortalized prmt5 f/f and prmt5 f/f ;CreERT2 mouse embryonic fibroblasts (MEFs) were cultured in the absence or presence of 4-hydroxy-tamoxifen (4-OHT; 500 nM for 24 hr), followed by 4 days in 4-OHT-deficient media, and then immunoblotted for PRMT5. (F–H) Cells were stimulated with 5Gy IR and co-stained for γH2AX and 53BP1 foci at the time points indicated (F). Quantification of γH2AX (G) and 53BP1 (H) foci is shown (mean ± SEM). (I) HeLa-shPRMT5 cells were exposed to increasing doses of IR and cell viability was determined by colony survival assay (mean ± SD; n = 3). (J) Analysis of chromatid gaps and breaks per metaphase is shown (mean ± SEM; ∗∗ p < 0.0098; n = 3). (K) Quantification of 53BP1 foci in mitosin-positive (late S/G2) cells 24 hr after 3Gy IR is shown (mean ± SEM; n = 3). Scale bar, 10 μm. See also Figure S1 .

Article Snippet: shPRMT5 (1) (5′-GCGTTTCAAGAGGGAGTTCAT-3) , Broad Institute GPP Web Portal , http://portals.broadinstitute.org/gpp/public.

Techniques: Staining, Cell Culture, Clonogenic Cell Survival Assay

Journal: Molecular Cell

Article Title: PRMT5-Dependent Methylation of the TIP60 Coactivator RUVBL1 Is a Key Regulator of Homologous Recombination

doi: 10.1016/j.molcel.2017.01.019

Figure Lengend Snippet: PRMT5 Promotes Homologous Recombination-Mediated DSB Repair Independently of ATM Activation (A) HeLa-shCTRL and shPRMT5 cells were stimulated with 3Gy IR and RAD51 foci analyzed 6 hr later. Quantification of number of cells with >10 RAD51 foci is shown below (mean ± SEM; ∗∗ p = 0.006; n = 3). (B) Quantification of RPA foci in CENPF-positive (late S/G2) cells 8 hr after 3Gy IR is shown (mean ± SEM; ∗∗ p = 0.006; n = 3). (C) Time course of phosphorylated and total protein levels is shown. (D) U2OS-DR3 cells were treated with siRNA as indicated before transfection of I - Sce1 and myc-PRMT5. The number of GFP-positive cells (HR proficient) was determined by flow cytometry (mean ± SD; n = 3). (E) PRMT5-depleted cells were treated with increasing doses of camptothecin (CPT; left panel) or Olaparib (right panel), and survival was determined by colony assay (mean ± SD; n = 3). (F) Cells were treated with CPT (1 μM) for the time points indicated and immunoblotted for phosphorylated and total proteins. Scale bar, 10 μm. See also Figure S2 .

Article Snippet: shPRMT5 (1) (5′-GCGTTTCAAGAGGGAGTTCAT-3) , Broad Institute GPP Web Portal , http://portals.broadinstitute.org/gpp/public.

Techniques: Homologous Recombination, Activation Assay, Transfection, Flow Cytometry, Colony Assay

Journal: Molecular Cell

Article Title: PRMT5-Dependent Methylation of the TIP60 Coactivator RUVBL1 Is a Key Regulator of Homologous Recombination

doi: 10.1016/j.molcel.2017.01.019

Figure Lengend Snippet: PRMT5 Binds to and Methylates RUVBL1 at R205 (A) SYPRO Ruby stain of PRMT5 protein complexes isolated from 293T cells stably expressing tetracycline-regulated TAP-tagged catalytically inactive PRMT5 (PRMT5 MD) is shown. (B) Co-immunoprecipitation of myc-PRMT5 MD with FLAG-tagged RUVBL1/RUVBL2 is shown. (C) Endogenous immunoprecipitation of RUVBL1 from HeLa cells co-immunoprecipitates endogenous PRMT5. (D) Transfected HeLa-shCTRL or shPRMT5 cell lines were treated with CHX/CAM and labeled with [ 3 H]-methyl methionine. FLAG-RUVBL1 was immunoprecipitated, and incorporated methyl groups were detected by SDS-PAGE followed by autoradiograph. Quantification of [ 3 H]-methyl signal intensity adjusted for immunoprecipitation (IP) and normalized to shCTRL is shown (mean ± SD; ∗ p = 0.03 and ∗∗∗ p = 0.0009). (E) Autoradiograph and immunoblots of 293T cells transfected with FLAG-RUVBL1 or FLAG-RUVBL1-R205K, treated with CHX/CAM and labeled with [ 3 H]-methyl methionine. Quantification of [ 3 H]-methyl signal intensity adjusted for IP and normalized to wild-type is shown (mean ± SD; n = 3). (F) Immunoblot analysis of endogenous RUVBL1-R205me2s in HeLa-shCTRL or shPRMT5 cell lines is shown. (G) Immunoblot analysis of endogenous RUVBL1-R205me2s in HeLa cells treated with Adox (100 μM; 24 hr) or GSK591 (5 μM; 24 hr) is shown. (H) Transfected 293T cells were damaged with 10Gy IR and myc-PRMT5 MD immunoprecipitated. Associated RUVBL1 was detected by FLAG immunoblotting. (I) Autoradiograph and immunoblots of 293T cells transfected with FLAG-RUVBL1, treated with CHX/CAM, and labeled with [ 3 H]-methyl methionine for 4 hr before treating with 10Gy IR are shown. (J) Immunoblot analysis of whole-cell lysates transfected with FLAG-RUVBL1 and damaged with 10Gy IR. See also .

Article Snippet: shPRMT5 (1) (5′-GCGTTTCAAGAGGGAGTTCAT-3) , Broad Institute GPP Web Portal , http://portals.broadinstitute.org/gpp/public.

Techniques: Staining, Isolation, Stable Transfection, Expressing, Immunoprecipitation, Transfection, Labeling, SDS Page, Autoradiography, Western Blot

Journal: Molecular Cell

Article Title: PRMT5-Dependent Methylation of the TIP60 Coactivator RUVBL1 Is a Key Regulator of Homologous Recombination

doi: 10.1016/j.molcel.2017.01.019

Figure Lengend Snippet: PRMT5-Dependent Methylation of RUVBL1 at R205 Regulates 53BP1 Removal and Is Epistatic with TIP60 (A) HeLa-shCTRL or shPRMT5 cells were siRNA transfected, exposed to 3Gy IR, and cell viability was assessed by colony survival. Results are expressed normalized to the respective non-treatment control (mean ± SD; n = 3). (B and C) HeLa-shCTRL or shPRMT5 cells were siRNA transfected, exposed to 3Gy IR, and stained for 53BP1 (B) or CENPF/RPA (C) foci 24 and 8 hr after damage, respectively (mean ± SEM; n = 3). (D and E) HeLa-shCTRL or RUVBL1-R205K cells were siRNA transfected, exposed to 3Gy IR, and stained for 53BP1 (D) or CENPF/RPA (E) foci 24 and 8 hr after damage, respectively (mean ± SEM; n = 3). (F) Validation of siTIP60 knockdown is shown. (G) Cells were transfected with si53BP1 and the number of mitosin-positive cells with >10 RAD51 foci was quantified (mean ± SEM; n = 3). (H) Cells were transfected with si53BP1 and the number of CENPF-positive cells with >10 RPA foci was quantified (mean ± SEM; n = 3). (I) Validation of 53BP1 siRNA SMARTpool. See also Figure S6 .

Article Snippet: shPRMT5 (1) (5′-GCGTTTCAAGAGGGAGTTCAT-3) , Broad Institute GPP Web Portal , http://portals.broadinstitute.org/gpp/public.

Techniques: Methylation, Transfection, Control, Staining, Biomarker Discovery, Knockdown

Journal: Molecular Cell

Article Title: PRMT5-Dependent Methylation of the TIP60 Coactivator RUVBL1 Is a Key Regulator of Homologous Recombination

doi: 10.1016/j.molcel.2017.01.019

Figure Lengend Snippet:

Article Snippet: shPRMT5 (1) (5′-GCGTTTCAAGAGGGAGTTCAT-3) , Broad Institute GPP Web Portal , http://portals.broadinstitute.org/gpp/public.

Techniques: Recombinant, Staining, Mutagenesis, Plasmid Preparation, Sequencing, Negative Control, Software