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. 2009 Dec 17;462(7275):935-9.
doi: 10.1038/nature08657.

Mammalian SUMO E3-ligases PIAS1 and PIAS4 promote responses to DNA double-strand breaks

Yaron Galanty  1 , Rimma Belotserkovskaya, Julia Coates, Sophie Polo, Kyle M Miller, Stephen P Jackson
Affiliations

Mammalian SUMO E3-ligases PIAS1 and PIAS4 promote responses to DNA double-strand breaks

Yaron Galanty et al. Nature. .

Abstract

DNA double-strand breaks (DSBs) are highly cytotoxic lesions that are generated by ionizing radiation and various DNA-damaging chemicals. Following DSB formation, cells activate the DNA-damage response (DDR) protein kinases ATM, ATR and DNA-PK (also known as PRKDC). These then trigger histone H2AX (also known as H2AFX) phosphorylation and the accumulation of proteins such as MDC1, 53BP1 (also known as TP53BP1), BRCA1, CtIP (also known as RBBP8), RNF8 and RNF168/RIDDLIN into ionizing radiation-induced foci (IRIF) that amplify DSB signalling and promote DSB repair. Attachment of small ubiquitin-related modifier (SUMO) to target proteins controls diverse cellular functions. Here, we show that SUMO1, SUMO2 and SUMO3 accumulate at DSB sites in mammalian cells, with SUMO1 and SUMO2/3 accrual requiring the E3 ligase enzymes PIAS4 and PIAS1. We also establish that PIAS1 and PIAS4 are recruited to damage sites via mechanisms requiring their SAP domains, and are needed for the productive association of 53BP1, BRCA1 and RNF168 with such regions. Furthermore, we show that PIAS1 and PIAS4 promote DSB repair and confer ionizing radiation resistance. Finally, we establish that PIAS1 and PIAS4 are required for effective ubiquitin-adduct formation mediated by RNF8, RNF168 and BRCA1 at sites of DNA damage. These findings thus identify PIAS1 and PIAS4 as components of the DDR and reveal how protein recruitment to DSB sites is controlled by coordinated SUMOylation and ubiquitylation.

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Figures

Figure 1
Figure 1. SUMOs and Ubc9 accumulate at DNA-damage sites by mechanisms requiring MDC1, 53BP1 and BRCA1
a, U2OS cells or U2OS cells transfected with HA-SUMO1 or HA-SUMO3 were irradiated (5Gy; +IR) or mock-irradiated (−IR) and probed. b, As in (a) but with laser micro-irradiation. c, U2OS cells co-transfected with GFP-Ubc9 and RFP-SUMO1 or RFP-SUMO2 were micro-irradiated and live cells imaged after 20 minutes. d, U2OS cells transfected with siRNAs were laser micro-irradiated then probed. For siRNA depletions, see Fig. 3e and Supplementary Fig. 10a.
Figure 2
Figure 2. PIAS1 and PIAS4 are recruited to DNA-damage sites and mediate 53BP1 recruitment and sumoylation
a,b, U2OS cells stably expressing GFP-MDC1 were treated, micro-irradiated and probed (Supplementary Figs. 4c and 10a-c for quantifications and depletions, respectively). c, Cells expressing GFP-PIAS1 or RFP-PIAS4 were micro-irradiated and probed. d, Cells expressing GFP-PIAS1 or RFP-PIAS4 wild-type (WT), ligase dead (LD), delta SAP (data not shown) or ligase-dead delta SAP (LDΔSAP) were micro-irradiated and imaged. e, Cells stably expressing GFP-53BP1 subjected to FRAP (n=11 independent measurements; error bars = s.e.d.). f-h, U2OS cells stably expressing GFP-SUMO1 or GFP (f, g) or HEK293 cells co-transfected with full-length (1-1972) HA-53BP1 and GFP-SUMO1 or GFP (h) were treated with or without IR (10 Gy).
Figure 3
Figure 3. PIAS1 and PIAS4 promote BRCA1 accumulation and sumoylation, RPA phosphorylation, and DSB repair
a, U2OS cells treated, micro-irradiated and probed as indicated; representative images with % of γH2AX positive cells also positive for BRCA1, each image represents >200 γH2AX-positive cells in two independent experiments. b, U2OS cells stably expressing GFP-BRCA1 and Flag-BARD1 were subjected to FRAP; data from Luciferase (n=7 independent measurements), PIAS1 (n=8) and PIAS4 (n=11); error bars = s.e.d. c, Essentially as Fig. 2h, except cells were co-transfected with HA-BRCA1 and Flag- BARD1. d,e, Extracts were prepared and analyzed 2 h following mock (−) or 10 Gy IR treatment. f-h, Effects of PIAS1/4 depletion on HR-mediated gene-conversion (f), NHEJ (g) and IR sensitivity (h); error bars = s.e.d.; data accumulated over four independent experiments (in each of f-h).
Figure 4
Figure 4. Linkage between PIAS1/4 and RNF8/168
a, U2OS cells were treated and probed as indicated. b,c, As (a) except cells stably expressed GFP-RNF8 or GFP-BRCA1/Flag-BARD1; see Supplementary Figs. 9a and 9b for quantifications. d, U2OS cells were treated and probed as indicated; see Supplementary Figs. 9d 10c for quantifications and siRNA efficiencies, respectively. e,f, U2OS cells stably expressing GFP-PIAS1 (e) or RFP-PIAS4 (f) were treated and probed as indicated. g, Model; dashed arrows indicate protein requirements for accumulation, solid arrows indicate target-protein modifications.
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