ATM Creates a veil of transcriptional silence.

The ATM kinase orchestrates diverse responses to DNA damage. By simultaneously monitoring transcription and DNA-damage responses in single cells, Shanbhag et al. (2010) now uncover a role of ATM in preventing transcription near DNA double-strand breaks.

RNF4 controls the extent of replication fork reversal to preserve genome stability.

Replication fork reversal occurs via a two-step process that entails reversal initiation and reversal extension. DNA topoisomerase IIalpha (TOP2A) facilitates extensive fork reversal, on one hand through resolving the topological stress generated by the initial reversal, on the other hand via its role in recruiting the SUMO-targeted DNA translocase PICH to stalled forks in a manner that is dependent on its SUMOylation by the SUMO E3 ligase ZATT. However, how TOP2A activities at stalled forks are precisely regulated remains poorly understood. Here we show that, upon replication stress, the SUMO-targeted ubiquitin E3 ligase RNF4 accumulates at stalled forks and targets SUMOylated TOP2A for ubiquitination and degradation. Downregulation of RNF4 resulted in aberrant activation of the ZATT-TOP2A-PICH complex at stalled forks, which in turn led to excessive reversal and elevated frequencies of fork collapse. These results uncover a previously unidentified regulatory mechanism that regulates TOP2A activities at stalled forks and thus the extent of fork reversal.

ATM controls the extent of DNA end resection by eliciting sequential posttranslational modifications of CtIP.

DNA end resection is a critical step in the repair of DNA double-strand breaks (DSBs) via homologous recombination (HR). However, the mechanisms governing the extent of resection at DSB sites undergoing homology-directed repair remain unclear. Here, we show that, upon DSB induction, the key resection factor CtIP is modified by the ubiquitin-like protein SUMO at lysine 578 in a PIAS4-dependent manner. CtIP SUMOylation occurs on damaged chromatin and requires prior hyperphosphorylation by the ATM protein kinase. SUMO-modified hyperphosphorylated CtIP is targeted by the SUMO-dependent E3 ubiquitin ligase RNF4 for polyubiquitination and subsequent degradation. Consequently, disruption of CtIP SUMOylation results in aberrant accumulation of CtIP at DSBs, which, in turn, causes uncontrolled excessive resection, defective HR, and increased cellular sensitivity to DSB-inducing agents. These findings reveal a previously unidentified regulatory mechanism that regulates CtIP activity at DSBs and thus the extent of end resection via ATM-dependent sequential posttranslational modification of CtIP.

A DYRK1B-dependent pathway suppresses rDNA transcription in response to DNA damage.

DNA double-strand breaks (DSBs) at ribosomal gene loci trigger inhibition of ribosomal DNA (rDNA) transcription and extensive nucleolar reorganization, including the formation of nucleolar caps where rDNA DSBs engage with canonical DSB signaling and repair factors. While these nucleolar responses underlie maintenance of rDNA stability, the molecular components that drive each of these events remain to be defined. Here we report that full suppression of rRNA synthesis requires the DYRK1B kinase, a nucleolar DSB response that can be uncoupled from ATM-mediated DSB signaling events at the nucleolar periphery. Indeed, by targeting DSBs onto rDNA arrays, we uncovered that chemical inhibition or genetic inactivation of DYRK1B led to sustained nucleolar transcription. Not only does DYRK1B exhibit robust nucleolar accumulation following laser micro-irradiation across cell nuclei, we further showed that DYRK1B is required for rDNA DSB repair and rDNA copy number maintenance, and that DYRK1B-inactivated cells are hypersensitised to DSBs induced at the rDNA arrays. Together, our findings not only identify DYRK1B as a key signaling intermediate that coordinates DSB repair and rDNA transcriptional activities, but also support the idea of specialised DSB responses that operate within the nucleolus to preserve rDNA integrity.

Screen identifies DYRK1B network as mediator of transcription repression on damaged chromatin.

DNA double-strand breaks (DSBs) trigger transient pausing of nearby transcription, an emerging ATM-dependent response that suppresses chromosomal instability. We screened a chemical library designed to target the human kinome for new activities that mediate gene silencing on DSB-flanking chromatin, and have uncovered the DYRK1B kinase as an early respondent to DNA damage. We showed that DYRK1B is swiftly and transiently recruited to laser-microirradiated sites, and that genetic inactivation of DYRK1B or its kinase activity attenuated DSB-induced gene silencing and led to compromised DNA repair. Notably, global transcription shutdown alleviated DNA repair defects associated with DYRK1B loss, suggesting that DYRK1B is strictly required for DSB repair on active chromatin. We also found that DYRK1B mediates transcription silencing in part via phosphorylating and enforcing DSB accumulation of the histone methyltransferase EHMT2. Together, our findings unveil the DYRK1B signaling network as a key branch of mammalian DNA damage response circuitries, and establish the DYRK1B-EHMT2 axis as an effector that coordinates DSB repair on transcribed chromatin.

BRCA1 and its toolbox for the maintenance of genome integrity.

The breast and ovarian cancer type 1 susceptibility protein (BRCA1) has pivotal roles in the maintenance of genome stability. Studies support that BRCA1 exerts its tumour suppression function primarily through its involvement in cell cycle checkpoint control and DNA damage repair. In addition, recent proteomic and genetic studies have revealed the presence of distinct BRCA1 complexes in vivo, each of which governs a specific cellular response to DNA damage. Thus, BRCA1 is emerging as the master regulator of the genome through its ability to execute and coordinate various aspects of the DNA damage response.

LC8/DYNLL1 is a 53BP1 effector and regulates checkpoint activation.

The tumor suppressor protein 53BP1 plays key roles in response to DNA double-strand breaks (DSBs) by serving as a master scaffold at the damaged chromatin. Current evidence indicates that 53BP1 assembles a cohort of DNA damage response (DDR) factors to distinctly execute its repertoire of DSB responses, including checkpoint activation and non-homologous end joining (NHEJ) repair. Here, we have uncovered LC8 (a.k.a. DYNLL1) as an important 53BP1 effector. We found that LC8 accumulates at laser-induced DNA damage tracks in a 53BP1-dependent manner and requires the canonical H2AX-MDC1-RNF8-RNF168 signal transduction cascade. Accordingly, genetic inactivation of LC8 or its interaction with 53BP1 resulted in checkpoint defects. Importantly, loss of LC8 alleviated the hypersensitivity of BRCA1-depleted cells to ionizing radiation and PARP inhibition, highlighting the 53BP1-LC8 module in counteracting BRCA1-dependent functions in the DDR. Together, these data establish LC8 as an important mediator of a subset of 53BP1-dependent DSB responses.

RNF169 limits 53BP1 deposition at DSBs to stimulate single-strand annealing repair.

Unrestrained 53BP1 activity at DNA double-strand breaks (DSBs) hampers DNA end resection and upsets DSB repair pathway choice. RNF169 acts as a molecular rheostat to limit 53BP1 deposition at DSBs, but how this fine balance translates to DSB repair control remains undefined. In striking contrast to 53BP1, ChIP analyses of AsiSI-induced DSBs unveiled that RNF169 exhibits robust accumulation at DNA end-proximal regions and preferentially targets resected, RPA-bound DSBs. Accordingly, we found that RNF169 promotes CtIP-dependent DSB resection and favors homology-mediated DSB repair, and further showed that RNF169 dose-dependently stimulates single-strand annealing repair, in part, by alleviating the 53BP1-imposed barrier to DSB end resection. Our results highlight the interplay of RNF169 with 53BP1 in fine-tuning choice of DSB repair pathways.

Dual-utility NLS drives RNF169-dependent DNA damage responses.

Loading of p53-binding protein 1 (53BP1) and receptor-associated protein 80 (RAP80) at DNA double-strand breaks (DSBs) drives cell cycle checkpoint activation but is counterproductive to high-fidelity DNA repair. ring finger protein 169 (RNF169) maintains the balance by limiting the deposition of DNA damage mediator proteins at the damaged chromatin. We report here that this attribute is accomplished, in part, by a predicted nuclear localization signal (NLS) that not only shuttles RNF169 into the nucleus but also promotes its stability by mediating a direct interaction with the ubiquitin-specific protease USP7. Guided by the crystal structure of USP7 in complex with the RNF169 NLS, we uncoupled USP7 binding from its nuclear import function and showed that perturbing the USP7-RNF169 complex destabilized RNF169, compromised high-fidelity DSB repair, and hypersensitized cells to poly (ADP-ribose) polymerase inhibition. Finally, expression of USP7 and RNF169 positively correlated in breast cancer specimens. Collectively, our findings uncover an NLS-mediated bipartite mechanism that supports the nuclear function of a DSB response protein.

An E2-guided E3 Screen Identifies the RNF17-UBE2U Pair as Regulator of the Radiosensitivity, Immunodeficiency, Dysmorphic Features, and Learning Difficulties (RIDDLE) Syndrome Protein RNF168.

Protein ubiquitination has emerged as a pivotal regulatory reaction that promotes cellular responses to DNA damage. With a goal to delineate the DNA damage signal transduction cascade, we systematically analyzed the human E2 ubiquitin- and ubiquitin-like-conjugating enzymes for their ability to mobilize the DNA damage marker 53BP1 onto ionizing radiation-induced DNA double strand breaks. An RNAi-based screen identified UBE2U as a candidate regulator of chromatin responses at double strand breaks. Further mining of the UBE2U interactome uncovered its cognate E3 RNF17 as a novel factor that, via the radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties (RIDDLE) syndrome protein RNF168, enforces DNA damage responses. Our screen allowed us to uncover new players in the mammalian DNA damage response and highlights the instrumental roles of ubiquitin machineries in promoting cell responses to genotoxic stress.

Overexpression of F-box only protein 31 predicts poor prognosis and deregulates p38α- and JNK-mediated apoptosis in esophageal squamous cell carcinoma.

F-box only protein 31 (FBXO31), a subunit of the Skp1-Cul1-F box ubiquitin ligase, plays a crucial role in DNA damage response and tumorigenesis. Yet its expression and function vary in different types of human cancer. The expression of FBXO31 in esophageal squamous cell carcinoma (ESCC) and its association with clinicopathological features is not well studied. The underlying mechanism by which deregulated FBXO31 contributes to ESCC tumorigenesis is largely unknown. By immunohistochemical analysis of a tissue microarray containing 85 cases of ESCC and matched adjacent noncancerous tissue and an additional 10 cases of ESCC tissue samples, we found that FBXO31 was overexpressed in ESCC, and that its expression was significantly correlated with histological grade (p = 0.04) and clinical stage (p = 0.022). Higher expression of FBXO31 was associated with poor prognosis in univariate (p = 0.013) and multivariate (p = 0.014) analyses. We found that FBXO31 functioned as an antiapoptotic molecule in ESCC cells exposed to different types of genotoxic stress. Knockdown of FBXO31 inhibited serum-starved cell viability and decreased tumorigenicity of ESCC cells. In addition, the antiapoptotic effects of FBXO31 were associated with deactivation of stress-induced MAPK p38α and JNK. Furthermore, in vitro and in vivo data showed that silencing of FBXO31-sensitized ESCC cells and tumors to cisplatin treatment. Taken together, in addition to revealing that FBXO31 is an independent prognostic marker for ESCC, our findings substantiate a novel regulatory role of FBXO31 in tumorigenesis and drug resistance of ESCC.

Regulation of chromatin architecture by the PWWP domain-containing DNA damage-responsive factor EXPAND1/MUM1.

Dynamic changes of chromatin structure facilitate diverse biological events, including DNA replication, repair, recombination, and gene transcription. Recent evidence revealed that DNA damage elicits alterations to the chromatin to facilitate proper checkpoint activation and DNA repair. Here we report the identification of the PWWP domain-containing protein EXPAND1/MUM1 as an architectural component of the chromatin, which in response to DNA damage serves as an accessory factor to promote cell survival. Depletion of EXPAND1/MUM1 or inactivation of its PWWP domain resulted in chromatin compaction. Upon DNA damage, EXPAND1/MUM1 rapidly concentrates at the vicinity of DNA damage sites via its direct interaction with 53BP1. Ablation of this interaction impaired damage-induced chromatin decondensation, which is accompanied by sustained DNA damage and hypersensitivity to genotoxic stress. Collectively, our study uncovers a chromatin-bound factor that serves an accessory role in coupling damage signaling with chromatin changes in response to DNA damage.

The Lys63-deubiquitylating Enzyme BRCC36 Limits DNA Break Processing and Repair.

Multisubunit protein assemblies offer integrated functionalities for efficient cell signal transduction control. One example of such protein assemblies, the BRCA1-A macromolecular complex, couples ubiquitin recognition and metabolism and promotes cellular responses to DNA damage. Specifically, the BRCA1-A complex not only recognizes Lys(63)-linked ubiquitin (K63-Ub) adducts at the damaged chromatin but is endowed with K63-Ub deubiquitylase (DUB) activity. To explore how the BRCA1-A DUB activity contributes to its function at DNA double strand breaks (DSBs), we used RNAi and genome editing approaches to target BRCC36, the protein subunit that confers the BRCA1-A complex its DUB activity. Intriguingly, we found that the K63-Ub DUB activity, although dispensable for maintaining the integrity of the macromolecular protein assembly, is important in enforcing the accumulation of the BRCA1-A complex onto DSBs. Inactivating BRCC36 DUB attenuated BRCA1-A functions at DSBs and led to unrestrained DSB end resection and hyperactive DNA repair. Together, our findings uncover a pivotal role of BRCC36 DUB in limiting DSB processing and repair and illustrate how cells may physically couple ubiquitin recognition and metabolizing activities for fine tuning of DNA repair processes.

ATM-dependent Phosphorylation of the Fanconi Anemia Protein PALB2 Promotes the DNA Damage Response.

The Fanconi anemia protein PALB2, also known as FANCN, protects genome integrity by regulating DNA repair and cell cycle checkpoints. Exactly how PALB2 functions may be temporally coupled with detection and signaling of DNA damage is not known. Intriguingly, we found that PALB2 is transformed into a hyperphosphorylated state in response to ionizing radiation (IR). IR treatment specifically triggered PALB2 phosphorylation at Ser-157 and Ser-376 in manners that required the master DNA damage response kinase Ataxia telangiectasia mutated, revealing potential mechanistic links between PALB2 and the Ataxia telangiectasia mutated-dependent DNA damage responses. Consistently, dysregulated PALB2 phosphorylation resulted in sustained activation of DDRs. Full-blown PALB2 phosphorylation also required the breast and ovarian susceptible gene product BRCA1, highlighting important roles of the BRCA1-PALB2 interaction in orchestrating cellular responses to genotoxic stress. In summary, our phosphorylation analysis of tumor suppressor protein PALB2 uncovers new layers of regulatory mechanisms in the maintenance of genome stability and tumor suppression.

F-box only protein 31 (FBXO31) negatively regulates p38 mitogen-activated protein kinase (MAPK) signaling by mediating lysine 48-linked ubiquitination and degradation of mitogen-activated protein kinase kinase 6 (MKK6).

The p38 MAPK signal transduction pathway plays an important role in inflammatory and stress responses. MAPKK6 (MKK6), a dual specificity protein kinase, is a p38 activator. Activation of the MKK6-p38 pathway is kept in check by multiple layers of regulations, including autoinhibition, dimerization, scaffold proteins, and Lys-63-linked polyubiquitination. However, the mechanisms underlying deactivation of MKK6-p38, which is crucial for maintaining the magnitude and duration of signal transduction, are not well understood. Lys-48-linked ubiquitination, which marks substrates for proteasomal degradation, is an important negative posttranslational regulatory machinery for signal pathway transduction. Here we report that the accumulation of F-box only protein 31 (FBXO31), a component of Skp1 · Cul1 · F-box protein E3 ligase, negatively regulated p38 activation in cancer cells upon genotoxic stresses. Our results show that FBXO31 binds to MKK6 and mediates its Lys-48-linked polyubiquitination and degradation, thereby functioning as a negative regulator of MKK6-p38 signaling and protecting cells from stress-induced cell apoptosis. Taken together, our findings uncover a new mechanism of deactivation of MKK6-p38 and substantiate a novel regulatory role of FBXO31 in stress response.

Epstein-Barr virus BZLF1 protein impairs accumulation of host DNA damage proteins at damage sites in response to DNA damage.

Epstein-Barr virus (EBV) infection is closely associated with several human malignancies including nasopharyngeal carcinoma (NPC). The EBV immediate-early protein BZLF1 is the key mediator that switches EBV infection from latent to lytic forms. The lytic form of EBV infection has been implicated in human carcinogenesis but its molecular mechanisms remain unclear. BZLF1 has been shown to be a binding partner of several DNA damage response (DDR) proteins. Its functions in host DDR remain unknown. Thus, we explore the effects of BZLF1 on cellular response to DNA damage in NPC cells. We found that expression of BZLF1 impaired the binding between RNF8 and MDC1 (mediator of DNA damage checkpoint 1), which in turn interfered with the localization of RNF8 and 53BP1 to the DNA damage sites. The RNF8-53BP1 pathway is important for repair of DNA double-strand breaks and DNA damage-induced G2/M checkpoint activation. Our results showed that, by impairing DNA damage repair as well as abrogating G2/M checkpoint, BZLF1 induced genomic instability and rendered cells more sensitive to ionizing radiation. Moreover, the blockage of 53BP1 and RNF8 foci formation was recapitulated in EBV-infected cells. Taken together, our study raises the possibility that, by causing mis-localization of important DDR proteins, BZLF1 may function as a link between lytic EBV infection and impaired DNA damage repair, thus contributing to the carcinogenesis of EBV-associated human epithelial malignancies.

Association study of stuttering candidate genes GNPTAB, GNPTG and NAGPA with dyslexia in Chinese population.

BACKGROUND: Dyslexia is a polygenic speech and language disorder characterized by an unexpected difficulty in reading in children and adults despite normal intelligence and schooling. Increasing evidence reveals that different speech and language disorders could share common genetic factors. As previous study reported association of GNPTAB, GNPTG and NAGPA with stuttering, we investigated these genes with dyslexia through association analysis. RESULTS: The study was carried out in an unrelated Chinese cohort with 502 dyslexic individuals and 522 healthy controls. In all, 21 Tag SNPs covering GNPTAB, GNPTG and NAGPA were subjected to genotyping. Association analysis was performed on all SNPs. Significant association of rs17031962 in GNPTAB and rs882294 in NAGPA with developmental dyslexia was identified after FDR correction for multiple comparisons. CONCLUSION: Our results revealed that the stuttering risk genes GNPTAB and NAGPA might also associate with developmental dyslexia in the Chinese population.

The ubiquitin specific protease USP34 promotes ubiquitin signaling at DNA double-strand breaks.

Ubiquitylation plays key roles in DNA damage signal transduction. The current model envisions that lysine63-linked ubiquitin chains, via the concerted action of E3 ubiquitin ligases RNF8-RNF168, are built at DNA double-strand breaks (DSBs) to effectively assemble DNA damage-repair factors for proper checkpoint control and DNA repair. We found that RNF168 is a short-lived protein that is stabilized by the deubiquitylating enzyme USP34 in response to DNA damage. In the absence of USP34, RNF168 is rapidly degraded, resulting in attenuated DSB-associated ubiquitylation, defective recruitment of BRCA1 and 53BP1 and compromised cell survival after ionizing radiation. We propose that USP34 promotes a feed-forward loop to enforce ubiquitin signaling at DSBs and highlight critical roles of ubiquitin dynamics in genome stability maintenance.

Assembly of checkpoint and repair machineries at DNA damage sites.

The remarkably coordinated nature of the DNA damage response pathway relies on numerous mechanisms that facilitate the assembly of checkpoint and repair factors at DNA breaks. Post-translational modifications on and around chromatin have critical roles in allowing the timely and sequential assembly of DNA damage responsive elements at the vicinity of DNA breaks. Notably, recent advances in forward genetics and proteomics-based approaches have enabled the identification of novel components within the DNA damage response pathway, providing a more comprehensive picture of the molecular network that assists in the detection and propagation of DNA damage signals.

Differential regulation of RNF8-mediated Lys48- and Lys63-based poly-ubiquitylation.

Pairing of a given E3 ubiquitin ligase with different E2s allows synthesis of ubiquitin conjugates of different topologies. While this phenomenon contributes to functional diversity, it remains largely unknown how a single E3 ubiquitin ligase recognizes multiple E2s, and whether identical structural requirements determine their respective interactions. The E3 ubiquitin ligase RNF8 that plays a critically important role in transducing DNA damage signals, interacts with E2s UBCH8 and UBC13, and catalyzes both K48- and K63-linked ubiquitin chains. Interestingly, we report here that a single-point mutation (I405A) on the RNF8 polypeptide uncouples its ability in catalyzing K48- and K63-linked ubiquitin chain formation. Accordingly, while RNF8 interacted with E2s UBCH8 and UBC13, its I405A mutation selectively disrupted its functional interaction with UBCH8, and impaired K48-based poly-ubiquitylation reactions. In contrast, RNF8 I405A preserved its interaction with UBC13, synthesized K63-linked ubiquitin chains, and assembled BRCA1 and 53BP1 at sites of DNA breaks. Together, our data suggest that RNF8 regulates K48- and K63-linked poly-ubiquitylation via differential RING-dependent interactions with its E2s UBCH8 and UBC13, respectively.