Supplementary information

Transcription

1 Supplementary information The E3 ligase RNF8 regulates KU80 removal and NHEJ repair Lin Feng 1, Junjie Chen 1 1 Department of Experimental Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA. Correspondence should be addressed to J.C. (jchen8@mdanderson.org).

2 Supplementary Figures Supplementary Fig. 1. RNF8 and RNF168 promote respectively K48-linked and K63linked ubiquitin chain formation at DSB sites. (a) K48- and K63-foci shown in Fig.1a colocalized with γh2ax. All of the cells exhibited the same or similar staining pattern with a given antibody. (b) Expression of RINGdeletion mutant of RNF8 (ΔRING) does not change K48- or K63- staining in the cell. Experiments were performed as described in Fig.1a. Immunostaining was conducted using ubiquitin chain specific antibodies (K48 or K63) and anti-flag antibodies. All cells exhibited similar staining pattern. (c) 293T cells stably expressing SFB-K48 only (upper panel) or SFB-K63 only (lower panel) ubiquitin mutants were transfected with constructs

3 encoding GFP-tagged proteins as indicated. 24 hours post-transfection, cells were fixed and processed for GFP fluorescence and anti-flag immunofluorescent staining. (d) RNF8 does not catalyze mono-ubiquitin accumulation at DSBs. HeLa cells were cotransfected with constructs encoding SFB-tagged RNF8 and HA-ubiquitin mutant K0 (all 7-lysines were mutated to arginines), cells were treated as described in Fig.1a and immunostained with anti-flag and anti-ha antibodies. (e) Ubiquitin chain formation in the absence of IR. HeLa cells were transfected with constructs encoding SFB-tagged RNF8 (upper panel), RNF168 (middle panel), and MIU domains of RNF168 (lower panel). 36 hours post-transfection cells were fixed without pre-extraction and stained with ubiquitin chain specific antibodies (K48 or K63) and anti-flag antibody.

4 Supplementary Fig. 2. RNF8 facilitates its own degradation. (a) HeLa cells were transfected with constructs encoding SFB-RNF8 ΔRING along with empty vector or those encoding wild-type or RING domain deletion mutant of RNF8. 48 hours post-transfection cells were lysed and extracts were immunoblotted with antibodies as indicated. (b) Rnf8-/- MEF cells were reconstituted with HA-Flag-tagged wild-type or ΔRING domain mutant of RNF8. Cells were treated with 5 Gy IR. 4 hours later, cells were fixed and immunostained with anti-53bp1 and FK2 antibodies. (c) Rnf8-/MEFs reconstituted with wild-type or ΔRING domain mutant of RNF8 were irradiated in the presence or absence of 5 µm MG hours later, cells were fixed and

5 immunostained with anti-flag and γh2ax antibodies. (d) MEFs as shown in (c) were lysed at the indicated time points following the addition of 10 µm MG132 and extracts were immunoblotted with anti-flag and GAPDH antibodies. (e) MEF cells as shown in (c) were treated with cycloheximide (CHX; 50 µg ml -1 ) to block protein synthesis and collected at the indicated time points. Total cell lysates were examined by immunoblotting as described in (d). (f) 293T cells transfected with constructs encoding SFB-tagged RNF8 or RNF168, along with His Ub K48 only (K48-only ubiquitin mutant) or His Ub K63 only (K63-only ubiquitin mutant) were lysed in 6M Guanidine-HCl. The ubiquitinated proteins were recovered with Ni-NTA (nickel-nitrilotriacetic acid) resins, followed by immunoblotting with anti-flag antibodies. Arrowheads indicate unmodified proteins.

6 Supplementary Fig. 3. In vitro ubiquitination of RNF8. (a) RNF8 catalyzes the synthesis of poly-ubiquitin chains. Recombinant GST-RNF8 protein was incubated with E1, E2 (UbcH5a) and ATP in the absence or presence of wild-type ubiquitin (Ub) or lysine-free ubiquitin (K0). After 20 min at room temperature, the reactions were stopped by the addition of SDS-loading buffer and analyzed by SDS- PAGE. Immunoblotting was conducted using anti-ubiquitin antibody. (b) In vitro RNF8 ubiquitination assay was performed with RNF8ΔRING mutant as substrate. Recombinant GST-RNF8 was incubated with E1, E2 (UbcH5a), ATP and ubiquitin in the absence or presence of the recombinant MBP-RNF8ΔRING. After 2 hours at 30 C, the reactions were stopped by the addition of SDS-loading buffer, analyzed by SDS-PAGE and immunoblotted with anti-mbp antibody. Commassie Blue staining of GST-RNF8 and MBP-RNF8ΔRING recombinant proteins purified from Escherichia coli strain BL21 was also presented.

7 Supplementary Fig. 4. RNF8 ubiquitinates and degrades CHK2. (a) Degradation of CHK2 by RNF8 depends on its intact RING and FHA domain. HeLa cells were transfected with plasmids encoding HA-CHK2 along with empty vector or constructs encoding SFB-tagged wild-type (WT), ΔRING or ΔFHA mutants of RNF8. 48 hours post-transfection, cells were harvested and extracts were examined by immunoblotting as indicated. (b) RNF8 interacts with CHK2. 293T cells were cotransfected with plasmids encoding various proteins as indicated. 24 hours posttransfection, cells were treated with 20 µm MG132 for additional 2 hours and lysed in NETN buffer. Precipitation was conducted with S beads and blotted with anti-ha and anti-flag antibodies. (c) RNF8 ubiquitinates CHK2 in vivo. HeLa cells were transfected with expression constructs encoding His-tagged ubiquitin (Ub), HA-CHK2 and SFB-

8 RNF8 or ΔRING mutant of RNF8 as indicated. 36 hours post-transfection, cells were treated 20 µm MG132 for additional 10 hours and lysed in 6M Guanidine-HCl. The ubiquitinated proteins were recovered with Ni-NTA resin, followed by immunoblotting with anti-ha antibody. (d) Depletion of RNF8 enhances the stability and activation of CHK2. Control or RNF8 stable knockdown HeLa cells were exposed to 10 Gy of ionization radiation and harvested at the indicated times. Immunoblotting was conducted using indicated antibodies. Total and phosphorylated CHK2 were assessed by immunoblotting with antibodies against total CHK2, phosphorylated Thr68 of CHK2 (trans-phosphorylation site by ATM/DNA-PK), or phosphorylated Thr383 of CHK2 (autophosphorylation site of CHK2) (see review 1 ). The enhanced Thr68-phosphorylation is a simply refection of elevated total protein level of CHK2 but not due to hyperactivation of ATM as phosphorylation of Ser824 on KAP1 (an ATM phosphorylation site 2 ) did not change in RNF8-depleted cells.

9 Supplementary Methods Protein expression and purification Constructs encoding GST-RNF8 and MBP-RNF8ΔRING were transformed into Escherichia coli BL21. Bacterial cells were collected after 4 hours induction with 0.2 mm isopropyl-d-1-thiogalactopyranoside (IPTG), and resuspended in NETN buffer containing 100 µm PMSF. After lysis by sonication, MBP fusion proteins were affinitypurified by Amylose Resin (NEB) and eluted with 10 mm maltose, GST fusion proteins were affinity-purified by Glutathione Sepharose 4B beads (GE Healthcare) and eluted with 10 mm Reduced Glutathione. Eluted proteins were dialyzed in E3 buffer (10 mm Tris-HCl ph 7.4, 50 mm NaCl, 2.5 mm MgCl 2, 0.1 mm DTT) with 20% glycerol and stored at 80 C. In vitro ubiquitination assay In vitro ubiquitination assays were carried out in E3 buffer containing 2 mm ATP, 1.25 µg ml -1 of E1, 1.25 µg ml -1 of E2 (UbcH5a), 50 µg ml -1 ubiquitin (E1, E2, ubiquitin and mutant were obtained from Boston Biochem) and 10 µg ml -1 of E3 (20 µl total volume). All ubiquitination reactions were initiated by the addition of ubiquitin and stopped by the addition of 5X SDS-PAGE loading buffer. References 1. Antoni, L., Sodha, N., Collins, I. & Garrett, M.D. CHK2 kinase: cancer susceptibility and cancer therapy - two sides of the same coin? Nat Rev Cancer 7, (2007).

10 2. Ziv, Y. et al. Chromatin relaxation in response to DNA double-strand breaks is modulated by a novel ATM- and KAP-1 dependent pathway. Nat Cell Biol 8, (2006).