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. 2012 Apr 16;197(2):179-87.
doi: 10.1083/jcb.201106152.

DNA damage-inducible SUMOylation of HERC2 promotes RNF8 binding via a novel SUMO-binding Zinc finger

Jannie Rendtlew Danielsen  1 , Lou Klitgaard Povlsen, Bine Hare Villumsen, Werner Streicher, Jakob Nilsson, Mats Wikström, Simon Bekker-Jensen, Niels Mailand
Affiliations

DNA damage-inducible SUMOylation of HERC2 promotes RNF8 binding via a novel SUMO-binding Zinc finger

Jannie Rendtlew Danielsen et al. J Cell Biol. .

Abstract

Nonproteolytic ubiquitylation of chromatin surrounding deoxyribonucleic acid (DNA) double-strand breaks (DSBs) by the RNF8/RNF168/HERC2 ubiquitin ligases facilitates restoration of genome integrity by licensing chromatin to concentrate genome caretaker proteins near the lesions. In parallel, SUMOylation of so-far elusive upstream DSB regulators is also required for execution of this ubiquitin-dependent chromatin response. We show that HERC2 and RNF168 are novel DNA damage-dependent SUMOylation targets in human cells. In response to DSBs, both HERC2 and RNF168 were specifically modified with SUMO1 at DSB sites in a manner dependent on the SUMO E3 ligase PIAS4. SUMOylation of HERC2 was required for its DSB-induced association with RNF8 and for stabilizing the RNF8-Ubc13 complex. We also demonstrate that the ZZ Zinc finger in HERC2 defined a novel SUMO-specific binding module, which together with its concomitant SUMOylation and T4827 phosphorylation promoted binding to RNF8. Our findings provide novel insight into the regulatory complexity of how ubiquitylation and SUMOylation cooperate to orchestrate protein interactions with DSB repair foci.

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Figures

Figure 1.
Figure 1.
PIAS4- and RNF8-dependent SUMOylation of HERC2 and RNF168 in response to DSBs. (A) HeLa/FLAG-SUMO cell lines left untreated or induced to express SUMO1 or SUMO2 by addition of doxycycline (DOX) for 24 h were subjected to IR or not and harvested 1 h later. Cells were lysed under denaturing conditions, and protein SUMOylation was analyzed by immunoblotting of FLAG IPs with the indicated antibodies. MM, molecular mass; WCE, whole-cell extract. (B) HeLa/FLAG-SUMO1 cells exposed to IR or UV were processed as in A. (C) HeLa/FLAG-SUMO1 cells transfected with control (CTRL), PIAS1, or PIAS4 siRNAs 24 h before addition of doxycycline were processed as in A. Knockdown efficiency of PIAS1 and PIAS4 siRNAs is shown in Fig. S1 E. (D) U2OS cells transfected or not with HA-tagged PIAS4 plasmid for 24 h were exposed to IR and harvested 1 h later. Cell extracts were subjected to HA IP followed by immunoblotting. (E) HeLa/FLAG-SUMO1 cells transfected with control or RNF8 siRNAs for 24 h were processed as in A.
Figure 2.
Figure 2.
SUMOylation of HERC2 promotes its interaction with RNF8. (A) SUMOylation of the C-terminal region of HERC2. U2OS cells were cotransfected with combinations of SUMO1- and HERC2-C (amino acids 4421–4834) expression constructs for 24 h, lysed, and processed for Strep-Tactin pulldowns. Bound complexes were analyzed by immunoblotting. Migration of molecular mass (MM) markers are indicated. WB, Western blot; WCE, whole-cell extract. (B) U2OS cells transfected with the indicated constructs were processed as in A. (C) U2OS cells transfected with siRNAs for 48 h were exposed to IR 1 h before lysis, separated into soluble and chromatin-enriched fractions, and immunoblotted with HERC2 and H2AX antibodies. CTRL, control. (D) U2OS cells transfected with indicated siRNAs for 48 h were exposed to IR and harvested 1 h later. Association between RNF8 and HERC2 was analyzed by immunoblotting of RNF8 IPs with RNF8 and HERC2 antibodies. R8, RNF8. (E) U2OS/Strep-HA-PIAS4 siR cell lines were transfected with siRNAs and induced or not with doxycycline (DOX) for 48 h, treated with IR, and harvested 1 h later. RNF8 IPs were analyzed by immunoblotting. (F) HEK293T/Strep-HA-Ubc13 cells transfected with the indicated siRNAs for 48 h were treated or not with doxycycline for an additional 24 h to induce expression of ectopic Strep-HA-Ubc13. Strep-Tactin pulldowns were immunoblotted with the indicated antibodies.
Figure 3.
Figure 3.
The ZZ Zinc finger motif in HERC2 is a novel SUMO-binding domain. (A) Cell extracts from U2OS cells harvested 1 h after treatment or not with IR were incubated with SUMO1-immobilized agarose or unconjugated agarose beads. Bound complexes were immunoblotted for HERC2 and RNF8. MM, molecular mass. (B) U2OS cells were transfected with plasmids encoding FLAG-tagged HERC2 fragments for 24 h, and SUMO binding was analyzed as in A. WB, Western blot. (C) Schematic depiction of conserved motifs in the SUMO-binding HERC2-F4 fragment. Residues mutated to disrupt the Zinc-coordinating ability of the ZZ finger are indicated. (D) Extracts of HEK293T cells transfected with the indicated Strep-HA-HERC2(2600–2900) constructs or empty vector for 24 h were incubated with SUMO1 or ubiquitin agarose. Bound complexes and inputs were analyzed by anti-HA immunoblotting. (E) ITC showing direct binding between the HERC2 ZZ domain and SUMO1/SUMO2. Purified SUMO1 or SUMO2 was titrated into the ITC sample cell at 10°C containing folded, recombinant HERC2 ZZ domain (see Fig. S3, B and C) until saturation was achieved. Heat effects (top) and cumulative heat effects (bottom) of the SUMO1/2-ZZ domain interaction are shown. Solid lines represent the single binding site model fit to the experimental data. ITC thermodynamic values are indicated.
Figure 4.
Figure 4.
Roles of PIAS4-mediated SUMOylation of HERC2 and RNF168 in cellular DSB responses. (A) Schematic depiction of HERC2 C-terminal fragments (HERC2-C3000) used in B. (B) HEK293T cells transfected with the indicated versions of GFP-HERC2-C3000 for 24 h were exposed to IR and lysed 1 h later. GFP IPs were immunoblotted with the indicated antibodies. MM, molecular mass; WCE, whole-cell extract. (C) U2OS cells were left untreated or transfected with Ubc9-1 siRNA for 48 h, transfected with HERC2 ZZ (residues 2690–2770) constructs for an additional 24 h, and processed as in Fig. 2 A. (D) U2OS cells transfected with siRNAs for 48 h were transfected with the indicated HA-tagged constructs for an additional 24 h, exposed to 4 Gy IR, and fixed 1 h later. Cells were immunostained with HA and 53BP1 antibodies. Arrows indicate cells proficient for 53BP1 accumulation in IR-induced foci. siCTRL, control siRNA. Bar, 10 µm. (E) Quantification of data in D. At least 200 cells were counted for each treatment. Results depict the mean (±SD) of three independent experiments. (F) Cells were treated as in D and coimmunostained with HA and FK2 antibodies. Bar, 10 µm. (G) HeLa/FLAG-SUMO1 cells were transfected with the indicated siRNAs for 72 h and processed as in Fig. 1 C. DOX, doxycycline.
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