Volume 66, Issue 4, Pages 458-472.e5 (May 2017) RNF8- and Ube2S-Dependent Ubiquitin Lysine 11-Linkage Modification in Response to DNA Damage  Atanu Paul, Bin Wang  Molecular Cell  Volume 66, Issue 4, Pages 458-472.e5 (May 2017) DOI: 10.1016/j.molcel.2017.04.013 Copyright © 2017 Elsevier Inc. Terms and Conditions

Molecular Cell 2017 66, 458-472.e5DOI: (10.1016/j.molcel.2017.04.013) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Lys11-Linkage Ub Conjugation in Response to DNA Damage (A) A live-cell imaging system to monitor linkage-specific Ub mutant conjugation at DNA damage sites. U2OS cells stably expressing either GFP or GFP-tagged Ub WT or mutant were subjected to laser microirradiation (marked by a dotted line) with a UV laser. Images at the indicated time points are shown. (B) Ube2S and Ube2C are associated with chromatin. Cell fraction analysis was carried out with U2OS cells treated or not treated with 10-Gy IR followed by a 30-min incubation at 37°C. (C) GFP-Ube2S accumulates to DNA damage sites. Images show U2OS cells stably expressing GFP-Ube2S at the indicated times after laser microirradiation. (D) Ube2S colocalizes with γH2AX at DNA damage sites. U2OS cells 5–10 min after laser microirradiation were analyzed. See also Figure S1. Molecular Cell 2017 66, 458-472.e5DOI: (10.1016/j.molcel.2017.04.013) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 IR-Induced Lys11-Linkage Chromatin Ubiquitination Depends on Ube2S/Ube2C Enzyme, but Not APC/C E3 Ligase (A) IR-induced Lys11-linkage chromatin ubiquitination. U2OS cells expressing HA-K11 Ub were either treated or not treated with 10-Gy IR followed by a 1-hr incubation at 37°C. MG132 (10 μM) was added 5 hr before IR. (B) Linkage-specific ubiquitination on chromatin in response to IR. U2OS cells expressing HA-Ub WT or mutant were treated with IR similarly as in (A). (C) Ube2S/Ube2C are required for Lys11-linkage chromatin ubiquitination. U2OS cells were transfected with the indicated siRNAs, followed by transient transfection of HA-K11 Ub. (D) Ube2S catalyzes Lys11-linkage chromatin ubiquitination. HEK293T cells expressing control or GFP-tagged siRNA-resistant Ube2S construct were transfected with the indicated siRNAs, followed by transient transfection of HA-K11 Ub. (E) APC/C-independent Lys11-linkage chromatin ubiquitination. (F) APC/C E3 ligase activity-independent Lys11-linkage chromatin ubiquitination. U2OS expressing HA-K11 Ub were treated with APC/C inhibitor (proTAME 25 μM) for 18 hr before treatment with IR. See also Figure S2. Molecular Cell 2017 66, 458-472.e5DOI: (10.1016/j.molcel.2017.04.013) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Lys11-Linkage Chromatin Ubiquitination Is Regulated by ATM, MDC1, and RNF8 (A) ATM and MDC1, but not ATR, are required for Lys11-linkage ubiquitination on chromatin in response to IR. U2OS cells were transfected with the indicated siRNAs, followed by transient transfection of HA-K11 Ub. (B) H2AX is required for Lys11-linkage ubiquitination on damaged chromatin. (C) RNF8 regulates Lys11-linkage ubiquitination on chromatin. See also Figure S3. Molecular Cell 2017 66, 458-472.e5DOI: (10.1016/j.molcel.2017.04.013) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Lys11-Linkage Ubiquitination Is Dispensable for Recruitment of DNA Damage Repair Proteins 53BP1 and BRCA1 (A–E) U2OS cells were transfected with control siRNA or siRNAs targeting Ube2S/Ube2C. Then 48 hr post-transfection, cells were subject to 10-Gy IR, followed by a 2-hr incubation at 37°C and immunostaining with antibodies to conjugated Ub (FK2 antibody) (A), 53BP1 (B), BRCA1 (C), Abraxas (D), or Rap80 (E). The percentage of foci-positive cells (cells containing more than ten foci) was quantified (mean ± SD with p value indicated). See also Figure S4. Molecular Cell 2017 66, 458-472.e5DOI: (10.1016/j.molcel.2017.04.013) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Ube2S- and RNF8-Dependent Lys11-Linkage Ubiquitination of Histone H2A/H2AX in Response to DNA Damage (A) Linkage-specific ubiquitination of acid-extracted histone faction. U2OS cells expressing HA-WT or mutant Ub were treated similarly as in Figure 2. Western blot with HA antibody and Ponceau staining of acid-extracted histones are shown. (B) Histone H2A as a substrate of Lys11-linkage ubiquitination. Western blot was carried out with antibodies against ubiquitinated H2A (ubH2A). (C) DNA damage-induced Lys11-linkage ubiquitination of histone H2A/H2AX. HEK293T cells co-transfected with FLAG-H2A or H2AX and HA-K11 Ub were irradiated at 12 Gy and incubated at 37°C for 1 hr. FLAG immunoprecipitation was carried out under denaturing condition. (D) Ube2S- and RNF8-dependent histone H2A Lys11-linkage ubiquitination. HEK293T cells depleted of Ube2S or RNF8 were co-transfected with FLAG-H2A and HA-K11 Ub. See also Figure S5. Molecular Cell 2017 66, 458-472.e5DOI: (10.1016/j.molcel.2017.04.013) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 RNF8 Interacts with Ube2S, Catalyzing Lys11-Linkage Ub Conjugation, and Is Antagonized by a Lys11-Linkage Deubiquitinating Enzyme, Cezanne (A) RNF8-dependent Lys11-linkage ubiquitination is antagonized by Cezanne. U2OS cells were transfected with the indicated siRNAs individually or in combination, followed by transient transfection of HA-K11 Ub. (B) RNF8 autoubiquitination by Lys11-linkage modification. HEK293T cells were co-transfected with FLAG-RNF8 and HA-Ub mutant. Cells were subject to 10-Gy IR (or left untreated) followed by a 30-min incubation at 37°C. FLAG immunoprecipitation was carried out under denaturing condition. (C) Lys11-linkage autoubiquitination of RNF8 in vitro. Purified FLAG-RNF8 was eluted from FLAG beads after immunoprecipitation from lysates of HEK293T cells expressing HA-FLAG-RNF8. An equal amount of eluted FLAG-RNF8 was incubated in reaction mixtures as indicated. (D) Lys11-linkage autoubiquitination of RNF8 in vitro using recombinant GST-RNF8. (E) RNF8 interacts with Ube2S. HEK293T cells were co-transfected with FLAG-RNF8 and GFP-Ube2S before treatment with IR. (F) RNF8 and Ube2S/Ube2C catalyze histone H2A ubiquitination in vitro. Recombinant histone H2A was incubated with a reaction mixture containing purified Ub, E1, and increasing amounts of Ube2S/Ube2C (lanes 3 and 4 and 5 and 6), in the presence (lanes 5 and 6) or absence (lanes 1–4) of purified FLAG-RNF8. Reaction mixtures were analyzed by western blot with FK2 antibody. (G) H2A ubiquitination in vitro. H2A/H2B dimer was used in the reaction. The western blot was carried out with ubH2A antibody. See also Figure S6. Molecular Cell 2017 66, 458-472.e5DOI: (10.1016/j.molcel.2017.04.013) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 Lys11-Linkage Ubiquitination Regulates DNA Damage-Induced Transcription Silencing (A) A scheme for examining DNA damage-induced transcription inhibition. (B) Detection of 5-EU labeling and γH2AX staining 1 hr post-laser microirradiation. (C) Ube2S/Ube2C-deficient cells are defective in DNA damage-induced transcription inhibition. Quantification of 5-EU intensity normalized by γH2AX intensity in the laser-damaged region is presented as mean ± SEM. (D) Increased RNAPII hyperphosphorylation in Ube2S/Ube2C-deficient cells. U2OS cells stably expressing the indicated small hairpin RNAs (shRNAs) were subject to 10-Gy IR, harvested at the indicated time points, and nuclear fractions were isolated for western blot analysis. IIo and IIa designate hyperphosphorylated and hypophosphorylated forms of the large subunit Rpb1 of RNAPII, respectively. (E) Quantification of Rpb1 hyperphosphorylation in Ube2S/Ube2C-deficient cells. Hyperphosphorylated Rpb1 (IIo) and hypophosphorylated Rpb1 (IIa) band intensities were measured by ImageJ and normalized to untreated samples. (F) Increased cellular sensitivity of Ube2/Ube2C-deficient cells to IR. Percentage of survival in the clonogenic survival assay is quantified and presented as mean ± SD. (G) A proposed model for the Lys11-linkage ubiquitination at sites of DNA damage. See also Figure S7. Molecular Cell 2017 66, 458-472.e5DOI: (10.1016/j.molcel.2017.04.013) Copyright © 2017 Elsevier Inc. Terms and Conditions