Human RNF169 is a negative regulator of the ubiquitin-dependent response to DNA double-strand breaks

Nonproteolytic ubiquitylation of chromatin surrounding deoxyribonucleic acid double-strand breaks (DSBs), mediated by the RNF8/RNF168 ubiquitin ligases, plays a key role in recruiting repair factors, including 53BP1 and BRCA1, to reestablish genome integrity. In this paper, we show that human RNF169, an uncharacterized E3 ubiquitin ligase paralogous to RNF168, accumulated in DSB repair foci through recognition of RNF168-catalyzed ubiquitylation products by its motif interacting with ubiquitin domain. Unexpectedly, RNF169 was dispensable for chromatin ubiquitylation and ubiquitin-dependent accumulation of repair factors at DSB sites. Instead, RNF169 functionally competed with 53BP1 and RAP80-BRCA1 for association with RNF168-modified chromatin independent of its catalytic activity, limiting the magnitude of their recruitment to DSB sites. By delaying accumulation of 53BP1 and RAP80 at damaged chromatin, RNF169 stimulated homologous recombination and restrained nonhomologous end joining, affecting cell survival after DSB infliction. Our results show that RNF169 functions in a noncanonical fashion to harness RNF168-mediated protein recruitment to DSB-containing chromatin, thereby contributing to regulation of DSB repair pathway utilization.     

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.     

Posttranslational control of telomere maintenance and the telomere damage response

Telomeres help maintain genome integrity by protecting natural chromosome ends from being recognized as damaged DNA. When telomeres become dysfunctional, they limit replicative lifespan and prevent outgrowth of potentially cancerous cells by activating a DNA damage response that forces cells into senescence or apoptosis. On the other hand, chromosome ends devoid of proper telomere protection are subject to DNA repair activities that cause end-to-end fusions and, when cells divide, extensive genomic instability that can promote cancer. While telomeres represent unique chromatin structures with important roles in cancer and aging, we have limited understanding of the way telomeres and the response to their malfunction are controlled at the level of chromatin. Accumulating evidence indicates that different types of posttranslational modifications act in both telomere maintenance and the response to telomere uncapping. Here, we discuss the latest insights on posttranslational control of telomeric chromatin, with emphasis on ubiquitylation and SUMOylation events.     

Solving the RIDDLE of 53BP1 recruitment to sites of damage

The cellular response to DNA double strand breaks is a complex, integrated network of pathways, coordinated by the PI-3-kinase-like family of kinases, which includes ATM, ATR and DNA-PK, that function to preserve the integrity of the genome. Mutations in genes that control these pathways are associated with increased genomic instability, neurodegeneration, immunodeficiency, premature aging and tumour predisposition. Indeed a significant proportion of our understanding regarding the mechanisms controlling DNA double strand break (DSB) repair has come from the study of cells derived from patients with inherited mutations in these genes. The discovery of the E3 ubiquitin ligase, RNF8, as a regulator of DNA DSB repair has brought to light a critical role for the ubiquitin system in regulating the cellular DSBs. Recently, identification of mutations in a second E3 ubiquitin ligase, RNF168, as the underlying genetic cause of the DNA repair deficiency disorder, RIDDLE syndrome, has provided the first link between ubiquitin-dependent DSB repair and immune system development in man. The finding that RNF168 functions downstream of RNF8 to orchestrate the recruitment of repair proteins, such as BRCA1 and 53BP1, to sites of DNA damage suggests that these two E3 ligases define a ubiquitylation cascade that regulates the spatial relocalisation of DSB repair proteins.     

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.     

BRCA1 and CtIP promote alternative non-homologous end-joining at uncapped telomeres

Loss of telomere protection occurs during physiological cell senescence and ageing, due to attrition of telomeric repeats and insufficient retention of the telomere-binding factor TRF2. Subsequently formed telomere fusions trigger rampant genomic instability leading to cell death or tumorigenesis. Mechanistically, telomere fusions require either the classical non-homologous end-joining (C-NHEJ) pathway dependent on Ku70/80 and LIG4, or the alternative non-homologous end-joining (A-NHEJ), which relies on PARP1 and LIG3. Here, we show that the tumour suppressor BRCA1, together with its interacting partner CtIP, both acting in end resection, also promotes end-joining of uncapped telomeres. BRCA1 and CtIP do not function in the ATM-dependent telomere damage signalling, nor in telomere overhang removal, which are critical for telomere fusions by C-NHEJ. Instead, BRCA1 and CtIP act in the same pathway as LIG3 to promote joining of de-protected telomeres by A-NHEJ. Our work therefore ascribes novel roles for BRCA1 and CtIP in end-processing and fusion reactions at uncapped telomeres, underlining the complexity of DNA repair pathways that act at chromosome ends lacking protective structures. Moreover, A-NHEJ provides a mechanism of previously unanticipated significance in telomere dysfunction-induced genome instability.     

The Cell Division Cycle Puts Up with Unprotected Telomeres: Cell Cycle Regulated Telomere Uncapping as a Means to Achieve Telomere Homeostasis

Telomeres have unique properties that distinguish natural chromosomal ends from accidental DNA double-strand interruptions arising elsewhere in the genome. However, the slightest perturbation in their unique organization may obliterate this distinction, channelling chromosomal ends into unwarranted repair events, eventually causing genome instability. Recent results revealed that the processing of both dysfunctional telomeres and accidental DNA double-strand breaks (DSB) by DNA repair activities is tightly regulated in a cell cycle-dependent manner by the S phase-promoting cell cycle kinase CDK1 (Clb-Cdc28p). Surprisingly, the cell cycle determinants and the timing of processing at unprotected telomeres closely match the requirements of other transactions that occur at telomeres. In particular, the replenishment of telomeric repeats by telomerase is tightly linked to cell cycle progression and occurs in the same interval. Furthermore, cell survival in the absence of essential telomeric proteins being dependent on telomere-telomere recombination mechanisms may require a similar regulation. Thus, a temporally limited state of telomere dysfunction leading to chromosome end processing may represent a well-governed cell cycle event that constitutes an integral part of the assembly of a new functional telomere.     

The ubiquitin- and SUMO-dependent signaling response to DNA double-strand breaks

DNA double-strand breaks (DSBs) represent the most destructive type of chromosomal lesion and trigger rapid chromatin restructuring accompanied by accumulation of proteins in the vicinity of the DSB. Non-proteolytic ubiquitylation of chromatin surrounding DSBs, mediated by the RNF8/RNF168 ubiquitin ligase cascade, has emerged as a key mechanism for restoration of genome integrity by licensing the DSB-modified chromatin to concentrate genome caretaker proteins such as 53BP1 and BRCA1 near the lesions. In parallel, SUMOylation of upstream DSB regulators is also required for execution of this ubiquitin-dependent chromatin response, but its molecular basis is currently unclear. Here, we discuss recent insights into how ubiquitin- and SUMO-dependent signaling processes cooperate to orchestrate protein interactions with sites of DNA damage to facilitate DSB repair.     

Homologous recombination-mediated irreversible genome damage underlies telomere-induced senescence

Loss of telomeric DNA leads to telomere uncapping, which triggers a persistent, p53-centric DNA damage response that sustains a stable senescence-associated proliferation arrest. Here, we show that in normal cells telomere uncapping triggers a focal telomeric DNA damage response accompanied by a transient cell cycle arrest. Subsequent cell division with dysfunctional telomeres resulted in sporadic telomeric sister chromatid fusions that gave rise to next-mitosis genome instability, including non-telomeric DNA lesions responsible for a stable, p53-mediated, senescence-associated proliferation arrest. Unexpectedly, the blocking of Rad51/RPA-mediated homologous recombination, but not non-homologous end joining (NHEJ), prevented senescence despite multiple dysfunctional telomeres. When cells approached natural replicative senescence, interphase senescent cells displayed genome instability, whereas near-senescent cells that underwent mitosis despite the presence of uncapped telomeres did not. This suggests that these near-senescent cells had not yet acquired irreversible telomeric fusions. We propose a new model for telomere-initiated senescence where tolerance of telomere uncapping eventually results in irreversible non-telomeric DNA lesions leading to stable senescence. Paradoxically, our work reveals that senescence-associated tumor suppression from telomere shortening requires irreversible genome instability at the single-cell level, which suggests that interventions to repair telomeres in the pre-senescent state could prevent senescence and genome instability.     

The Role of ATM in the Deficiency in Nonhomologous End-Joining near Telomeres in a Human Cancer Cell Line

Telomeres distinguish chromosome ends from double-strand breaks (DSBs) and prevent chromosome fusion. However, telomeres can also interfere with DNA repair, as shown by a deficiency in nonhomologous end joining (NHEJ) and an increase in large deletions at telomeric DSBs. The sensitivity of telomeric regions to DSBs is important in the cellular response to ionizing radiation and oncogene-induced replication stress, either by preventing cell division in normal cells, or by promoting chromosome instability in cancer cells. We have previously proposed that the telomeric protein TRF2 causes the sensitivity of telomeric regions to DSBs, either through its inhibition of ATM, or by promoting the processing of DSBs as though they are telomeres, which is independent of ATM. Our current study addresses the mechanism responsible for the deficiency in repair of DSBs near telomeres by combining assays for large deletions, NHEJ, small deletions, and gross chromosome rearrangements (GCRs) to compare the types of events resulting from DSBs at interstitial and telomeric DSBs. Our results confirm the sensitivity of telomeric regions to DSBs by demonstrating that the frequency of GCRs is greatly increased at DSBs near telomeres and that the role of ATM in DSB repair is very different at interstitial and telomeric DSBs. Unlike at interstitial DSBs, a deficiency in ATM decreases NHEJ and small deletions at telomeric DSBs, while it increases large deletions. These results strongly suggest that ATM is functional near telomeres and is involved in end protection at telomeric DSBs, but is not required for the extensive resection at telomeric DSBs. The results support our model in which the deficiency in DSB repair near telomeres is a result of ATM-independent processing of DSBs as though they are telomeres, leading to extensive resection, telomere loss, and GCRs involving alternative NHEJ.     

Close encounters of the RNF8th kind: when chromatin meets DNA repair

Cells counteract the adverse effects of chromosome breakage by activating the DNA damage response (DDR), which entails a coordinated series of events that regulate cell cycle progression and repair of DNA lesions. The packaging of genomic DNA into condensed, often inaccessible chromatin severely complicates efficient DNA damage repair in living cells. Recent studies implicate a large number of chromatin-modifying enzymes in the DDR, suggesting a stepwise model in which chromatin is continually reconfigured to accommodate the association and action of repair factors during the different stages of the DDR. Emerging evidence suggests that the histone ubiquitin ligases RNF8/RNF168 act in concert with ATP-dependent chromatin remodelling enzymes to orchestrate the signalling and repair of DNA lesions in specific chromatin topologies.     

Ectopic expression of RNF168 and 53BP1 increases mutagenic but not physiological non-homologous end joining

DNA double strand breaks (DSBs) formed during S phase are preferentially repaired by homologous recombination (HR), whereas G₁ DSBs, such as those occurring during immunoglobulin class switch recombination (CSR), are repaired by non-homologous end joining (NHEJ). The DNA damage response proteins 53BP1 and BRCA1 regulate the balance between NHEJ and HR. 53BP1 promotes CSR in part by mediating synapsis of distal DNA ends, and in addition, inhibits 5’ end resection. BRCA1 antagonizes 53BP1 dependent DNA end-blocking activity during S phase, which would otherwise promote mutagenic NHEJ and genome instability. Recently, it was shown that supra-physiological levels of the E3 ubiquitin ligase RNF168 results in the hyper-accumulation of 53BP1/BRCA1 which accelerates DSB repair. Here, we ask whether increased expression of RNF168 or 53BP1 impacts physiological versus mutagenic NHEJ. We find that the anti-resection activities of 53BP1 are rate-limiting for mutagenic NHEJ but not for physiological CSR. As heterogeneity in the expression of RNF168 and 53BP1 is found in human tumors, our results suggest that deregulation of the RNF168/53BP1 pathway could alter the chemosensitivity of BRCA1 deficient tumors.     

Chromatin regulation and non-coding RNAs at mammalian telomeres

In eukaryotes, terminal chromosome repeats are bound by a specialized nucleoprotein complex that controls telomere length and protects chromosome ends from DNA repair and degradation. In mammals the “shelterin” complex mediates these central functions at telomeres. In the recent years it has become evident that also the heterochromatic structure of mammalian telomeres is implicated in telomere length regulation. Impaired telomeric chromatin compaction results in a loss of telomere length control. Progressive telomere shortening affects chromatin compaction at telomeric and subtelomeric repeats and activates alternative telomere maintenance mechanisms. Dynamics of chromatin structure of telomeres during early mammalian development and nuclear reprogramming further indicates a central role of telomeric heterochromatin in organismal development. In addition, the recent discovery that telomeres are transcribed, giving rise to UUAGGG-repeat containing TelRNAs/TERRA, opens a new level of chromatin regulation at telomeres. Understanding the links between the epigenetic status of telomeres, TERRA/TelRNA and telomere homeostasis will open new avenues for our understanding of organismal development, cancer and ageing.     

Autophagy regulates DNA repair through SQSTM1/p62

Macroautophagy/autophagy is primarily a degradative pathway that clears malfunctioning cellular components in response to various types of stress. Recent studies have indicated that autophagy also plays an important role in maintaining genome stability. Loss of autophagy is associated with increased damage to DNA, inappropriate amplification of genomic regions and abnormal chromosome number. In a recent paper by Wang et al. the authors uncover a mechanism through which autophagy regulates the ubiquitination of chromatin. In particular, the autophagy receptor and substrate SQSTM1/p62 inhibits the E3 ligase RNF168-dependent ubiquitination of histone in response to DNA double-strand breaks. Dysregulation of this process leads to a reduced ability to repair DNA and a corresponding increase in the sensitivity of cells to radiation-induced damage.     

RNF126 Quenches RNF168 Function in the DNA Damage Response

DNA damage response (DDR) is essential for maintaining genome stability and protecting cells from tumorigenesis. Ubiquitin and ubiquitin-like modifications play an important role in DDR, from signaling DNA damage to mediating DNA repair. In this report, we found that the E3 ligase ring finger protein 126 (RNF126) was recruited to UV laser micro-irradiation-induced stripes in a RNF8-dependent manner. RNF126 directly interacted with and ubiquitinated another E3 ligase, RNF168. Overexpression of wild type RNF126, but not catalytically-inactive mutant RNF126 (CC229/232AA), diminished ubiquitination of H2A histone family member X (H2AX), and subsequent bleomycin-induced focus formation of total ubiquitin FK2, TP53-binding protein 1 (53BP1), and receptor-associated protein 80 (RAP80). Interestingly, both RNF126 overexpression and RNF126 downregulation compromised homologous recombination (HR)-mediated repair of DNA double-strand breaks (DSBs). Taken together, our findings demonstrate that RNF126 negatively regulates RNF168 function in DDR and its appropriate cellular expression levels are essential for HR-mediated DSB repair.     

Fanconi anemia proteins in telomere maintenance

Mammalian chromosome ends are protected by nucleoprotein structures called telomeres. Telomeres ensure genome stability by preventing chromosome termini from being recognized as DNA damage. Telomere length homeostasis is inevitable for telomere maintenance because critical shortening or over-lengthening of telomeres may lead to DNA damage response or delay in DNA replication, and hence genome instability. Due to their repetitive DNA sequence, unique architecture, bound shelterin proteins, and high propensity to form alternate/secondary DNA structures, telomeres are like common fragile sites and pose an inherent challenge to the progression of DNA replication, repair, and recombination apparatus. It is conceivable that longer the telomeres are, greater is the severity of such challenges. Recent studies have linked excessively long telomeres with increased tumorigenesis. Here we discuss telomere abnormalities in a rare recessive chromosomal instability disorder called Fanconi Anemia and the role of the Fanconi Anemia pathway in telomere biology. Reports suggest that Fanconi Anemia proteins play a role in maintaining long telomeres, including processing telomeric joint molecule intermediates. We speculate that ablation of the Fanconi Anemia pathway would lead to inadequate aberrant structural barrier resolution at excessively long telomeres, thereby causing replicative burden on the cell.     

An RNF168 fragment defective for focal accumulation at DNA damage is proficient for inhibition of homologous recombination in BRCA1 deficient cells

The E3 ubiquitin ligase RNF168 is a DNA damage response (DDR) factor that promotes monoubiquitination of H2A/H2AX at K13/15, facilitates recruitment of other DDR factors (e.g. 53BP1) to DNA damage, and inhibits homologous recombination (HR) in cells deficient in the tumor suppressor BRCA1. We have examined the domains of RNF168 important for these DDR events, including chromosomal HR that is induced by several nucleases (I-SceI, CAS9-WT and CAS9-D10A), since the inducing nuclease affects the relative frequency of distinct repair outcomes. We found that an N-terminal fragment of RNF168 (1-220/N221*) efficiently inhibits HR induced by each of these nucleases in BRCA1 depleted cells, and promotes recruitment of 53BP1 to DNA damage and H2AX monoubiquitination at K13/15. Each of these DDR events requires a charged residue in RNF168 (R57). Notably, RNF168-N221* fails to self-accumulate into ionizing radiation induced foci (IRIF). Furthermore, expression of RNF168 WT and N221* can significantly bypass the role of another E3 ubiquitin ligase, RNF8, for inhibition of HR in BRCA1 depleted cells, and for promotion of 53BP1 IRIF. We suggest that the ability for RNF168 to promote H2A/H2AX monoubiquitination and 53BP1 IRIF, but not RNF168 self-accumulation into IRIF, is important for inhibition of HR in BRCA1 deficient cells.