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RNF168 Ubiquitylates 53BP1 and Controls Its Response to DNA Double-Strand Breaks

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RNF168 Ubiquitylates 53BP1 and Controls Its Response to DNA Double-Strand Breaks RNF168 ubiquitylates 53BP1 and controls its response to DNA double-strand breaks Miyuki Bohgakia,b,1, Toshiyuki Bohgakia,b,1, Samah El Ghamrasnia,b, Tharan Srikumara,b, Georges Mairec, Stephanie Panierd, Amélie Fradet-Turcotted, Grant S. Stewarte, Brian Raughta,b, Anne Hakema,b, and Razqallah Hakema,b,2 aOntario Cancer Institute, University Health Network and bDepartment of Medical Biophysics, University of Toronto, Toronto, M5G 2M9 ON, Canada; cThe Hospital for Sick Children, Toronto, M5G 2L3 ON, Canada; dDepartment of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8; and eCancer Research UK, Institute for Cancer Studies, Birmingham University, Birmingham B15 2TT, United Kingdom Edited* by Stephen J. Elledge, Harvard Medical School, Boston, MA, and approved November 14, 2013 (received for review October 30, 2013) Defective signaling or repair of DNA double-strand breaks has X–MDC1–RNF8 axis, RNF168 also functions in DSB signaling been associated with developmental defects and human diseases. independently of this pathway. Therefore, we postulated that The E3 ligase RING finger 168 (RNF168), mutated in the human RNF168 also might regulate DSB signaling through direct radiosensitivity, immunodeficiency, dysmorphic features, and modulation of 53BP1 functions. learning difficulties syndrome, was shown to ubiquitylate H2A- In the present study, we demonstrate that RNF168 associates γ – – type histones, and this ubiquitylation was proposed to facilitate with 53BP1 independently of the -H2A.X MDC1 RNF8 sig- the recruitment of p53-binding protein 1 (53BP1) to the sites of naling axis. RNF168 ubiquitylates 53BP1 before its localization DNA double-strand breaks. In contrast to more upstream proteins to DSB sites, and this ubiquitylation is important for the initial signaling DNA double-strand breaks (e.g., RNF8), deficiency of recruitment of 53BP1 to DSB sites and its function in non- homologous end joining (NHEJ) and activation of checkpoints. RNF168 fully prevents both the initial recruitment to and retention Collectively our data support a requirement for RNF168 at dif- of 53BP1 at sites of DNA damage; however, the mechanism for this ferent stages of the DSB-signaling cascade and highlight the difference has remained unclear. Here, we identify mechanisms central role of RNF168 ubiquitylation of 53BP1 in maintaining that regulate 53BP1 recruitment to the sites of DNA double-strand genomic integrity. breaks and provide evidence that RNF168 plays a central role in BIOCHEMISTRY the regulation of 53BP1 functions. RNF168 mediates K63-linked Results ubiquitylation of 53BP1 which is required for the initial recruit- Rnf168 Interacts with 53bp1. Cells deficient for Rnf168 fail to re- ment of 53BP1 to sites of DNA double-strand breaks and for its cruit or retain 53bp1 at sites of DNA damage (2, 3, 12). To ex- function in DNA damage repair, checkpoint activation, and geno- amine the mechanisms that mediate Rnf168 regulation of 53bp1 mic integrity. Our findings highlight the multistep roles of RNF168 recruitment to DSB sites, we tested whether Rnf168 contributes in signaling DNA damage. to 53bp1’s association with chromatin. We observed that 53bp1 was bound to chromatin in untreated (UT) WT thymocytes and ubiquitin | G2/M checkpoint | HR repair pathways | NHEJ repair pathway was enriched in chromatin-rich insoluble fractions after irradia- tion (IR) (Fig. 1A). Although deficiency of H2a.x or Rnf8 does ’ fi he DNA-damage response (DDR) is critical for genomic not affect 53bp1 saf nity for chromatin (14, 15), the levels of Tintegrity (1) and is regulated by posttranslational modifications 53bp1 in chromatin-enriched insoluble fractions were reduced in (PTMs) such as ubiquitylation of histones by the E3 ligases RING finger 8 (RNF8) and RING finger 168 (RNF168). Other Significance PTMs important for DDR include dimethylation of histone H4 (H4K20me2), which allows p53-binding protein 1 (53BP1), a DNA damage-repair mechanisms are highly regulated, and key mediator of DDR, to interact with chromatin. posttranslational modifications are one means of such regula- Mutations of RNF168 have been associated with the human tion. p53-binding protein 1 (53BP1) is important for the re- radiosensitivity, immunodeficiency, dysmorphic features, and sponse to DNA double-strand breaks (DSBs). Here we report learning difficulties (RIDDLE) syndrome, (2–4). RNF168 has an that its functions are regulated by RING finger 168 (RNF168), N-terminal RING finger domain, three ubiquitin (Ub)-binding a DNA DSB-signaling protein that is mutated in the human domains (UBDs); two motif interacting with Ub (MIU) domains; radiosensitivity, immunodeficiency, dysmorphic features, and and one Ub interacting motif (UIM)- and MIU-related (UMI) learning difficulties syndrome. We show that before their lo- UBD (3, 5, 6). Current data support RNF168 function in DNA calization to DNA DSBs, RNF168 interacts with 53BP1 and modifies double-strand break (DSB) signaling downstream of H2A.X, it through the addition of a chain of ubiquitin-polypeptides. mediator of DNA damage checkpoint 1 (MDC1), and RNF8 and We also show that Lysine 1268 of 53BP1 is important for its indicate its requirement for 53BP1 recruitment to DSB sites (3, ubiquitin modification by RNF168 and that loss of this modifi- 5). Through its UBDs, RNF168 recognizes RNF8 ubiquitylated cation impairs 53BP1 recruitment to sites of DNA damage and non-nucleosomal protein(s) at DSB-flanking sites, leading to its restrains its functions in the repair of DNA damage and main- recruitment at these sites of DNA damage (7). With the Ub- tenance of genomic stability. conjugating enzyme UBC13, RNF168 initiates ubiquitylation of lysine (K) 13 or 15 of histones H2A and H2A.X, leading to the Author contributions: M.B., T.B., B.R., A.H., and R.H. designed research; M.B., T.B., S.E.G., recruitment of DDR proteins, including 53BP1, to DSBs. Al- T.S., G.M., A.F.-T., and A.H. performed research; S.P. and G.S.S. contributed new reagents/ though H2A.X, MDC1, and RNF8 are important for the retention analytic tools; M.B., T.B., S.E.G., T.S., A.F.-T., B.R., A.H., and R.H. analyzed data; and M.B., of 53BP1 at these DSBs, its initial and transient recruitment to T.B., A.H., and R.H. wrote the paper. DNA breaks still occurs in their absence (8–11). In contrast, The authors declare no conflict of interest. deficiency of Rnf168 in mouse embryonic fibroblasts (MEFs) *This Direct Submission article had a prearranged editor. completely abolishes 53bp1 recruitment to DSB sites (12). Similar 1M.B. and T.B. contributed equally to this work. −/− −/− to 53bp1 mice, but in contrast to H2a.x mice (13), young 2To whom correspondence should be addressed. E-mail: [email protected]. Rnf168−/− males are fertile (12). These data suggest that, in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. addition to its role in DSB signaling downstream of the H2A. 1073/pnas.1320302111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1320302111 PNAS Early Edition | 1of6 Downloaded by guest on September 23, 2021 −/− Rnf168 thymocytes compared with controls, both under UT using IP and IB analysis, we identified the minimal amino acid conditions and 15 min post-IR (Fig. 1A). Because 53BP1 asso- residues (1241–1315) required for 53BP1’s binding to Rnf168 ciates with chromatin through its binding to H4K20me2 (16, 17), (Fig. 1E). These 53BP1 amino acid residues are 100% conserved we examined the level of this histone mark in UT or irradiated between humans and mice (Fig. S1D). To determine Rnf168 −/− WT and Rnf168 primary MEFs. Our data indicated no defects region(s) that bind 53BP1, we performed an in vivo binding in H4K20me2 levels in the absence of Rnf168 (Fig. S1A). These assay using FLAG-tagged truncated forms of Rnf168 (Fig. S2A). data suggested that Rnf168, unlike H2a.x and Rnf8, is important We transiently expressed each FLAG-tagged Rnf168 with HA- for 53bp1’s association with chromatin both under UT conditions tagged 53BP1-KBD in HEK293T cells. 53BP1-KBD interacted and in response to DNA damage and that H4K20me2 is not with both the truncated and full-length forms of Rnf168 (Fig. F a limiting factor for 53bp1 recruitment to DSBs in Rnf168- 1 ). We next performed in vivo binding assay using HA-tagged fi 53BP1-KBD and FLAG-tagged Rnf168ΔRING, Rnf168ΔMIU1, de cient cells. Next we examined whether Rnf168 interacts Δ Δ – Δ – physically with 53bp1. Immunoprecipitation (IP) and immuno- Rnf168 MIU2, Rnf168 MIU1/2, Rnf168(61 168), Rnf168 61 168Δ194–299, and full-length Rnf168. All Rnf168 mutants inter- blot (IB) analysis using HEK293T cells expressing exogenous A B Rnf168 revealed its interaction with 53BP1 (Fig. 1B). Endoge- acted with 53BP1-KBD (Fig. S2 and ). These results suggest nous Rnf168 and 53bp1 also were found to interact in WT that multiple regions of Rnf168 and 53BP1 might be involved in thymocytes (Fig. 1 and Fig. S1B). 53BP1 contains a kinetochore- their interaction. To examine further the region(s) of Rnf168 that mediate its binding to 53BP1, we performed an in vivo binding domain (KBD; amino acids 1235–1616) that includes C binding assay using HA-tagged 53BP1-KBD and FLAG-tagged a Tudor domain (Fig. S1 ) essential for 53BP1 binding to Rnf168 truncated mutants [Rnf168(1–299), Rnf168(1–299)Δ61– chromatin (14, 18). Thus, we assessed whether 53BP1-KBD may 168], and full-length Rnf168. Interestingly, additional deletion of interact with Rnf168. The amino acid sequence of the KBD residues 61–168 in Rnf168(1–299) prevented its interaction with domain of 53BP1 is highly conserved between humans and mice 53BP1-KBD (Fig. S2 A–C). Moreover, we performed a similar (92.9% identity) (Fig. S1D). Therefore, we used human 53BP1- fi in vivo binding assay using HA-tagged 53BP1-KBD, FLAG- KBD for these analyses and found that this domain was suf - tagged full-length Rnf168, and FLAG-tagged Rnf168 deletion D cient to mediate 53BP1 interaction with Rnf168 (Fig.
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