The Deubiquitylating Enzyme USP44 Counteracts the DNA Double-strand Break Response Mediated by the RNF8 and RNF168 Ubiquitin Ligases

Protein recruitment to DNA double-strand breaks (DSBs) relies on ubiquitylation of the surrounding chromatin by the RING finger ubiquitin ligases RNF8 and RNF168. Flux through this pathway is opposed by several deubiquitylating enzymes (DUBs), including OTUB1 and USP3. By analyzing the effect of individually overexpressing the majority of human DUBs on RNF8/RNF168-mediated 53BP1 retention at DSB sites, we found that USP44 and USP29 powerfully inhibited this response at the level of RNF168 accrual. Both USP44 and USP29 promoted efficient deubiquitylation of histone H2A, but unlike USP44, USP29 displayed nonspecific reactivity toward ubiquitylated substrates. Moreover, USP44 but not other H2A DUBs was recruited to RNF168-generated ubiquitylation products at DSB sites. Individual depletion of these DUBs only mildly enhanced accumulation of ubiquitin conjugates and 53BP1 at DSBs, suggesting considerable functional redundancy among cellular DUBs that restrict ubiquitin-dependent protein assembly at DSBs. Our findings implicate USP44 in negative regulation of the RNF8/RNF168 pathway and illustrate the usefulness of DUB overexpression screens for identification of antagonizers of ubiquitin-dependent cellular responses.     

Ring Finger Protein RNF169 Antagonizes the Ubiquitin-dependent Signaling Cascade at Sites of DNA Damage

Ubiquitin signals emanating from DNA double-strand breaks (DSBs) trigger the ordered assembly of DNA damage mediator and repair proteins. This highly orchestrated process is accomplished, in part, through the concerted action of the RNF8 and RNF168 E3 ligases, which have emerged as core signaling intermediates that promote DSB-associated ubiquitylation events. In this study, we report the identification of RNF169 as a negative regulator of the DNA damage signaling cascade. We found that RNF169 interacted with ubiquitin structures and relocalized to DSBs in an RNF8/RNF168-dependent manner. Moreover, ectopic expression of RNF169 attenuated ubiquitin signaling and compromised 53BP1 accumulation at DNA damage sites, suggesting that RNF169 antagonizes RNF168 functions at DSBs. Our study unveils RNF169 as a component in DNA damage signal transduction and adds to the complexity of regulatory ubiquitylation in genome stability maintenance.     

The de-ubiquitylating enzymes USP26 and USP37 regulate homologous recombination by counteracting RAP80

The faithful repair of DNA double-strand breaks (DSBs) is essential to safeguard genome stability. DSBs elicit a signaling cascade involving the E3 ubiquitin ligases RNF8/RNF168 and the ubiquitin-dependent assembly of the BRCA1-Abraxas-RAP80-MERIT40 complex. The association of BRCA1 with ubiquitin conjugates through RAP80 is known to be inhibitory to DSB repair by homologous recombination (HR). However, the precise regulation of this mechanism remains poorly understood. Through genetic screens we identified USP26 and USP37 as key de-ubiquitylating enzymes (DUBs) that limit the repressive impact of RNF8/RNF168 on HR. Both DUBs are recruited to DSBs where they actively remove RNF168-induced ubiquitin conjugates. Depletion of USP26 or USP37 disrupts the execution of HR and this effect is alleviated by the simultaneous depletion of RAP80. We demonstrate that USP26 and USP37 prevent excessive spreading of RAP80-BRCA1 from DSBs. On the other hand, we also found that USP26 and USP37 promote the efficient association of BRCA1 with PALB2. This suggests that these DUBs limit the ubiquitin-dependent sequestration of BRCA1 via the BRCA1-Abraxas-RAP80-MERIT40 complex, while promoting complex formation and cooperation of BRCA1 with PALB2-BRCA2-RAD51 during HR. These findings reveal a novel ubiquitin-dependent mechanism that regulates distinct BRCA1-containing complexes for efficient repair of DSBs by HR.     

Ubiquitin-H2AX fusions render 53BP1 recruitment to DNA damage sites independent of RNF8 or RNF168

The mammalian E3 ubiquitin ligases RNF8 and RNF168 facilitate recruitment of the DNA damage response protein 53BP1 to sites of DNA double-strand breaks (DSBs). The mechanism involves recruitment of RNF8, followed by recruitment of RNF168, which ubiquitinates histones H2A/H2AX on K15. 53BP1 then binds to nucleosomes at sites of DNA DSBs by recognizing, in addition to methyl marks, histone H2A/H2AX ubiquitinated on K15. We report here that expressing H2AX fusion proteins with N-terminal bulky moieties can rescue 53BP1 recruitment to sites of DNA DSBs in cells lacking RNF8 or RNF168 or in cells treated with proteasome inhibitors, in which histone ubiquitination at sites of DNA DSBs is compromised. The rescue required S139 at the C-terminus of the H2AX fusion protein and was occasionally accompanied by partial rescue of ubiquitination at sites of DNA DSBs. We conclude that recruitment of 53BP1 to sites of DNA DSBs is possible in the absence of RNF8 or RNF168, but still dependent on chromatin ubiquitination.     

Ataxin-3 consolidates the MDC1-dependent DNA double-strand break response by counteracting the SUMO-targeted ubiquitin ligase RNF4

The SUMO-targeted ubiquitin ligase RNF4 functions at the crossroads of the SUMO and ubiquitin systems. Here, we report that the deubiquitylation enzyme (DUB) ataxin-3 counteracts RNF4 activity during the DNA double-strand break (DSB) response. We find that ataxin-3 negatively regulates ubiquitylation of the checkpoint mediator MDC1, a known RNF4 substrate. Loss of ataxin-3 markedly decreases the chromatin dwell time of MDC1 at DSBs, which can be fully reversed by co-depletion of RNF4. Ataxin-3 is recruited to DSBs in a SUMOylation-dependent fashion, and in vitro it directly interacts with and is stimulated by recombinant SUMO, defining a SUMO-dependent mechanism for DUB activity toward MDC1. Loss of ataxin-3 results in reduced DNA damage-induced ubiquitylation due to impaired MDC1-dependent recruitment of the ubiquitin ligases RNF8 and RNF168, and reduced recruitment of 53BP1 and BRCA1. Finally, ataxin-3 is required for efficient MDC1-dependent DSB repair by non-homologous end-joining and homologous recombination. Consequently, loss of ataxin-3 sensitizes cells to ionizing radiation and poly(ADP-ribose) polymerase inhibitor. We propose that the opposing activities of RNF4 and ataxin-3 consolidate robust MDC1-dependent signaling and repair of DSBs.     

USP14 regulates DNA damage repair by targeting RNF168-dependent ubiquitination

Recent reports have made important revelations, uncovering direct regulation of DNA damage response (DDR)-associated proteins and chromatin ubiquitination (Ubn) by macroautophagy/autophagy. Here, we report a previously unexplored connection between autophagy and DDR, via a deubiquitnase (DUB), USP14. Loss of autophagy in prostate cancer cells led to unrepaired DNA double-strand breaks (DSBs) as indicated by persistent ionizing radiation (IR)-induced foci (IRIF) formation for γH2AFX, and decreased protein levels and IRIF formation for RNF168, an E3-ubiquitin ligase essential for chromatin Ubn and recruitment of critical DDR effector proteins in response to DSBs, including TP53BP1. Consistently, RNF168-associated Ubn signaling and TP53BP1 IRIF formation were reduced in autophagy-deficient cells. An activity assay identified several DUBs, including USP14, which showed higher activity in autophagy-deficient cells. Importantly, inhibiting USP14 could overcome DDR defects in autophagy-deficient cells. USP14 IRIF formation and protein stability were increased in autophagy-deficient cells. Co-immunoprecipitation and colocalization of USP14 with MAP1LC3B and the UBA-domain of SQSTM1 identified USP14 as a substrate of autophagy and SQSTM1. Additionally, USP14 directly interacted with RNF168, which depended on the MIU1 domain of RNF168. These findings identify USP14 as a novel substrate of autophagy and regulation of RNF168-dependent Ubn and TP53BP1 recruitment by USP14 as a critical link between DDR and autophagy. Given the role of Ubn signaling in non-homologous end joining (NHEJ), the major pathway for repair of IR-induced DNA damage, these findings provide unique insights into the link between autophagy, DDR-associated Ubn signaling and NHEJ DNA repair.

Abbreviations: ATG7: autophagy related 7; CQ: chloroquine; DDR: DNA damage response; DUB: deubiquitinase; HR: homologous recombination; IR: ionizing radiation; IRIF: ionizing radiation-induced foci; LAMP2: lysosomal associated membrane protein 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MIU1: motif interacting with ubiquitin; NHEJ: non homologous end-joining; PCa: prostate cancer; TP53BP1/53BP1: tumor protein p53 binding protein 1; RNF168: ring finger protein 168; SQSTM1/p62 sequestosome 1; γH2AFX/γH2AX: H2A histone family member X: phosphorylated, UBA: ubiquitin-associated; Ub: ubiquitin; Ubn: ubiquitination; USP14: ubiquitin specific peptidase 14.     

Regulatory ubiquitylation in response to DNA double-strand breaks

DNA double-strand breaks (DSBs) are highly cytolethal DNA lesions. In response to DSBs, cells initiate a complex response that minimizes their deleterious impact on cellular and organismal physiology. In this review, we discuss the discovery of a regulatory ubiquitylation system that modifies the chromatin that surrounds DNA lesions. This pathway is under the control of RNF8 and RNF168, two E3 ubiquitin ligases that cooperate with UBC13 to promote the relocalization of 53BP1 and BRCA1 to sites of DNA damage. RNF8 and RNF168 orchestrate the recruitment of DNA damage response proteins by catalyzing the ubiquitylation of H2A-type histones and the formation of K63-linked ubiquitin chains on damaged chromatin. Finally, we identify some unresolved issues raised by the discovery of this pathway and discuss the implications of DNA damage-induced ubiquitylation in human disease and development.     

Critical roles of ring finger protein RNF8 in replication stress responses

Histone ubiquitylation is emerging as an important protective component in cellular responses to DNA damage. The ubiquitin ligases RNF8 and RNF168 assemble ubiquitin chains onto histone molecules surrounding DNA breaks and facilitate retention of DNA repair proteins. Although RNF8 and RNF168 play important roles in repair of DNA double strand breaks, their requirement for cell protection from replication stress is largely unknown. In this study, we uncovered RNF168-independent roles of RNF8 in repair of replication inhibition-induced DNA damage. We showed that RNF8 depletion, but not RNF168 depletion, hyper-sensitized cells to hydroxyurea and aphidicolin treatment. Consistently, hydroxyurea induced persistent single strand DNA lesions and sustained CHK1 activation in RNF8-depleted cells. In line with strict requirement for RAD51-dependent repair of hydroxyurea-stalled replication forks, RNF8 depletion compromised RAD51 accumulation onto single strand DNA lesions, suggesting that impaired replication fork repair may underlie the enhanced cellular sensitivity to replication arrest observed in RNF8-depleted cells. In total, our study highlights the differential requirement for the ubiquitin ligase RNF8 in facilitating repair of replication stress-associated DNA damage.     

A Small Molecule Inhibitor of Polycomb Repressive Complex 1 Inhibits Ubiquitin Signaling at DNA Double-strand Breaks

Polycomb-repressive complex 1 (PRC1)-mediated histone ubiquitylation plays an important role in aberrant gene silencing in human cancers and is a potential target for cancer therapy. Here we show that 2-pyridine-3-yl-methylene-indan-1,3-dione (PRT4165) is a potent inhibitor of PRC1-mediated H2A ubiquitylation in vivo and in vitro. The drug also inhibits the accumulation of all detectable ubiquitin at sites of DNA double-strand breaks (DSBs), the retention of several DNA damage response proteins in foci that form around DSBs, and the repair of the DSBs. In vitro E3 ubiquitin ligase activity assays revealed that PRT4165 inhibits both RNF2 and RING 1A, which are partially redundant paralogues that together account for the E3 ubiquitin ligase activity found in PRC1 complexes, but not RNF8 nor RNF168. Because ubiquitylation is completely inhibited despite the efficient recruitment of RNF8 to DSBs, our results suggest that PRC1-mediated monoubiquitylation is required for subsequent RNF8- and/or RNF168-mediated polyubiquitylation. Our results demonstrate the unique feature of PRT4165 as a novel chromatin-remodeling compound and provide a new tool for the inhibition of ubiquitylation signaling at DNA double-strand breaks.     

A small ubiquitin binding domain inhibits ubiquitin-dependent protein recruitment to DNA repair foci

The rapid ubiquitination of chromatin surrounding DNA double-stranded breaks (DSB) drives the formation of large structures called ionizing radiation-induced foci (IRIF), comprising many DNA damage response (DDR) proteins. This process is regulated by RNF8 and RNF168 ubiquitin ligases and is thought to be necessary for DNA repair and activation of signaling pathways involved in regulating cell cycle checkpoints. Here we demonstrate that it is possible to interfere with ubiquitin-dependent recruitment of DDR factors by expressing proteins containing ubiquitin binding domains (UBDs) that bind to lysine 63-linked polyubiquitin chains. Expression of the E3 ubiquitin ligase RAD18 prevented chromatin spreading of 53BP1 at DSBs, and this phenomenon was dependent upon the integrity of the RAD18 UBD. An isolated RAD18 UBD interfered with 53BP1 chromatin spreading, as well as other important DDR mediators, including RAP80 and the BRCA1 tumor suppressor protein, consistent with the model that the RAD18 UBD is blocking access of proteins to ubiquitinated chromatin. Using the RAD18 UBD as a tool to impede localization of 53BP1 and BRCA1 to repair foci, we found that DDR signaling, DNA DSB repair, and radiosensitivity were unaffected. We did find that activated ATM (S1981P) and phosphorylated SMC1 (a specific target of ATM) were not detectable in DNA repair foci, in addition to upregulated homologous recombination repair, revealing 2 DDR responses that are dependent upon chromatin spreading of certain DDR factors at DSBs. These data demonstrate that select UBDs containing targeting motifs may be useful probes in determining the biological significance of protein–ubiquitin interactions.     

USP3 counteracts RNF168 via deubiquitinating H2A and γH2AX at lysine 13 and 15

Histone ubiquitination plays a vital role in DNA damage response (DDR), which is important for maintaining genomic integrity in eukaryotic cells. In DDR, ubiquitination of histone H2A and γH2AX by the concerted action of ubiquitin (Ub) ligases, RNF168 and RNF8, generates a cascade of ubiquitination signaling. However, little is known about deubiquitinating enzymes (DUBs) that may catalyze the removal of Ub from these histones. This study demonstrated that USP3, an apparent DUB for mono-ubiquitinated H2A, is indeed the enzyme for deubiquitinating Ub conjugates of γH2AX and H2A from lysine sites, where the ubiquitination is initiated by RNF168. Here, we showed that ectopic expression of USP3 led to the deubiquitination of both H2A and γH2AX in response to UV-induced DNA damage. Moreover, ectopic USP3 expression abrogated FK2 antibody-reactive Ub-conjugate foci, which co-localize with damage-induced γH2AX foci. In addition, USP3 overexpression impaired the accumulation of downstream repair factors BRCA1 and 53BP1 at the damage sites in response to both UV and γ-irradiation. We further identified that the USP3 removes Ub at lysine 13 and 15 of H2A and γH2AX, as well as lysine 118 and 119 of H2AX in response to DNA damage. Taken together, the results suggested that USP3 is a negative regulator of ubiquitination signaling, counteracting RNF168- and RNF8-mediated ubiquitination.     

Structural basis for role of ring finger protein RNF168 RING domain

Ubiquitin adducts surrounding DNA double-strand breaks (DSBs) have emerged as molecular platforms important for the assembly of DNA damage mediator and repair proteins. Central to these chromatin modifications lies the E2 UBC13, which has been implicated in a bipartite role in priming and amplifying lys63-linked ubiquitin chains on histone molecules through coupling with the E3 RNF8 and RNF168. However, unlike the RNF8-UBC13 holoenyzme, exactly how RNF168 work in concert with UBC13 remains obscure. To provide a structural perspective for the RNF168-UBC13 complex, we solved the crystal structure of the RNF168 RING domain. Interestingly, while the RNF168 RING adopts a typical RING finger fold with two zinc ions coordinated by several conserved cystine and histine residues arranged in a C3HC4 “cross-brace” manner, structural superimposition of RNF168 RING with other UBC13-binding E3 ubiquitin ligases revealed substantial differences at its corresponding UBC13-binding interface. Consistently, and in stark contrast to that between RNF8 and UBC13, RNF168 did not stably associate with UBC13 in vitro or in vivo. Moreover, domain-swapping experiments indicated that the RNF8 and RNF168 RING domains are not functionally interchangeable. We propose that RNF8 and RNF168 operate in different modes with their cognate E2 UBC13 at DSBs.     

TRIP12 and UBR5 Suppress Spreading of Chromatin Ubiquitylation at Damaged Chromosomes

Histone ubiquitylation is a prominent response to DNA double-strand breaks (DSBs), but how these modifications are confined to DNA lesions is not understood. Here, we show that TRIP12 and UBR5, two HECT domain ubiquitin E3 ligases, control accumulation of RNF168, a rate-limiting component of?a pathway that ubiquitylates histones after DNA breakage. We find that RNF168 can be saturated by increasing amounts of DSBs. Depletion of TRIP12 and UBR5 allows accumulation of RNF168 to supraphysiological levels, followed by massive spreading of ubiquitin conjugates and hyperaccumulation of ubiquitin-regulated genome caretakers such as 53BP1 and BRCA1. Thus, regulatory and proteolytic ubiquitylations are wired in a self-limiting circuit that promotes histone ubiquitylation near the DNA lesions but at the same time counteracts its excessive spreading to undamaged chromosomes. We provide evidence that this mechanism is vital for the homeostasis of ubiquitin-controlled events after DNA breakage and can be subverted during tumorigenesis.     

A new non-catalytic role for ubiquitin ligase RNF8 in unfolding higher-order chromatin structure

The ubiquitin ligases RNF8 and RNF168 orchestrate DNA damage signalling through the ubiquitylation of histone H2A and the recruitment of downstream repair factors. Here, we demonstrate that RNF8, but not RNF168 or the canonical H2A ubiquitin ligase RNF2, mediates extensive chromatin decondensation. Our data show that CHD4, the catalytic subunit of the NuRD complex, interacts with RNF8 and is essential for RNF8-mediated chromatin unfolding. The chromatin remodelling activity of CHD4 promotes efficient ubiquitin conjugation and assembly of RNF168 and BRCA1 at DNA double-strand breaks. Interestingly, RNF8-mediated recruitment of CHD4 and subsequent chromatin remodelling were independent of the ubiquitin-ligase activity of RNF8, but involved a non-canonical interaction with the forkhead-associated (FHA) domain. Our study reveals a new mechanism of chromatin remodelling-assisted ubiquitylation, which involves the cooperation between CHD4 and RNF8 to create a local chromatin environment that is permissive to the assembly of checkpoint and repair machineries at DNA lesions.     

UMI, a novel RNF168 ubiquitin binding domain involved in the DNA damage signaling pathway

Ubiquitination regulates important cellular processes, including the DNA damage response (DDR) and DNA repair. The complexity of the ubiquitin-mediated signals is decoded by ubiquitin receptors, which contain protein modules named ubiquitin binding domains (UBDs). We previously identified a new ubiquitin ligase, RNF168, involved in DDR and endowed with two UBDs named MIU (motif interacting with ubiquitin). Here we have provided the identification of a novel UBD, the UMI (UIM- and MIU-related UBD), present in RNF168, and characterized the interaction surface with ubiquitin, centered on two Leu residues. We have demonstrated that integrity of the UMI, in addition to the MIUs, is necessary for the proper localization of RNF168 and for ubiquitination of nuclear proteins, including histone H2A. Finally, we have shown that simultaneous inactivation of UMI and MIUs prevents the recruitment to DDR foci of the crucial downstream mediator 53BP1.     

Give me a break,but not in mitosis

DNA double-strand breaks (DSBs) are extremely cytotoxic with a single unrepaired DSB being sufficient to induce cell death. A complex signalling cascade, termed the DNA damage response (DDR), is in place to deal with such DNA lesions and maintain genome stability. Recent work by us and others has found that the signalling cascade activated by DSBs in mitosis is truncated, displaying apical, but not downstream, components of the DDR. The E3 Ubiquitin ligases RNF8, RNF168 and BRCA1, along with the DDR mediator 53BP1, are not recruited to DSB sites in mitosis, and activation of downstream checkpoint kinases is also impaired. Here, we show that RNF8 and RNF168 are recruited to DNA damage foci in late mitosis, presumably to prime sites for 53BP1 recruitment in early G1. Interestingly, we show that, although RNF8, RNF168 and 53BP1 are excluded from DSB sites during most of mitosis, they associate with mitotic structures such as the kinetochore, suggesting roles for these DDR factors during mitotic cell division. We discuss these and other recent findings and suggest how these novel data collectively contribute to our understanding of mitosis and how cells deal with DNA damage during this crucial cell cycle stage.     

When cleavage is not attractive: non-catalytic inhibition of ubiquitin chains at DNA double-strand breaks by OTUB1

Non-degradative ubiquitylation plays a crucial role in many cellular signaling pathways, including the DNA damage response. Two ubiquitin ligases, RNF8 and RNF168, in combination with the E2 ubiquitin conjugating enzyme UBC13 catalyze the formation of K63-linked ubiquitin chains at sites of DNA double-strand breaks to promote their faithful repair. However, little is known about their negative regulation. A recent study identifies a deubiquitylating enzyme, OTUB1, which counteracts RNF8/RNF168-dependent ubiquitin chain formation at break sites. Surprisingly, this enzyme carries out its function not by cleavage of polyubiquitin chains, but by targeting UBC13. This non-canonical role for a deubiquitylating enzyme has implications for the regulation of ubiquitylation not just in DNA repair, but potentially in many other cellular signaling processes.     

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

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.