Human Rad52 Promotes XPG-Mediated R-loop Processing to Initiate Transcription-Associated Homologous Recombination Repair.

Given that genomic DNA exerts its function by being transcribed, it is critical for the maintenance of homeostasis that DNA damage, such as double-strand breaks (DSBs), within transcriptionally active regions undergoes accurate repair. However, it remains unclear how this is achieved. Here, we describe a mechanism for transcription-associated homologous recombination repair (TA-HRR) in human cells. The process is initiated by R-loops formed upon DSB induction. We identify Rad52, which is recruited to the DSB site in a DNA-RNA-hybrid-dependent manner, as playing pivotal roles in promoting XPG-mediated R-loop processing and initiating subsequent repair by HRR. Importantly, dysfunction of TA-HRR promotes DSB repair via non-homologous end joining, leading to a striking increase in genomic aberrations. Thus, our data suggest that the presence of R-loops around DSBs within transcriptionally active regions promotes accurate repair of DSBs via processing by Rad52 and XPG to protect genomic information in these critical regions from gene alterations.

Condensates induced by transcription inhibition localize active chromatin to nucleoli.

The proper function of the genome relies on spatial organization of DNA, RNA, and proteins, but how transcription contributes to the organization is unclear. Here, we show that condensates induced by transcription inhibition (CITIs) drastically alter genome spatial organization. CITIs are formed by SFPQ, NONO, FUS, and TAF15 in nucleoli upon inhibition of RNA polymerase II (RNAPII). Mechanistically, RNAPII inhibition perturbs ribosomal RNA (rRNA) processing, releases rRNA-processing factors from nucleoli, and enables SFPQ to bind rRNA. While accumulating in CITIs, SFPQ/TAF15 remain associated with active genes and tether active chromatin to nucleoli. In the presence of DNA double-strand breaks (DSBs), the altered chromatin compartmentalization induced by RNAPII inhibition increases gene fusions in CITIs and stimulates the formation of fusion oncogenes. Thus, proper RNAPII transcription and rRNA processing prevent the altered compartmentalization of active chromatin in CITIs, suppressing the generation of gene fusions from DSBs.

DNA damage promotes HLA class I presentation by stimulating a pioneer round of translation-associated antigen production.

Antigen presentation by the human leukocyte antigen (HLA) on the cell surface is critical for the transduction of the immune signal toward cytotoxic T lymphocytes. DNA damage upregulates HLA class I presentation; however, the mechanism is unclear. Here, we show that DNA-damage-induced HLA (di-HLA) presentation requires an immunoproteasome, PSMB8/9/10, and antigen-transporter, TAP1/2, demonstrating that antigen production is essential. Furthermore, we show that di-HLA presentation requires ATR, AKT, mTORC1, and p70-S6K signaling. Notably, the depletion of CBP20, a factor initiating the pioneer round of translation (PRT) that precedes nonsense-mediated mRNA decay (NMD), abolishes di-HLA presentation, suggesting that di-antigen production requires PRT. RNA-seq analysis demonstrates that DNA damage reduces NMD transcripts in an ATR-dependent manner, consistent with the requirement for ATR in the initiation of PRT/NMD. Finally, bioinformatics analysis identifies that PRT-derived 9-mer peptides bind to HLA and are potentially immunogenic. Therefore, DNA damage signaling produces immunogenic antigens by utilizing the machinery of PRT/NMD.

CARM1 regulates replication fork speed and stress response by stimulating PARP1.

DNA replication forks use multiple mechanisms to deal with replication stress, but how the choice of mechanisms is made is still poorly understood. Here, we show that CARM1 associates with replication forks and reduces fork speed independently of its methyltransferase activity. The speeding of replication forks in CARM1-deficient cells requires RECQ1, which resolves reversed forks, and RAD18, which promotes translesion synthesis. Loss of CARM1 reduces fork reversal and increases single-stranded DNA (ssDNA) gaps but allows cells to tolerate higher replication stress. Mechanistically, CARM1 interacts with PARP1 and promotes PARylation at replication forks. In vitro, CARM1 stimulates PARP1 activity by enhancing its DNA binding and acts jointly with HPF1 to activate PARP1. Thus, by stimulating PARP1, CARM1 slows replication forks and promotes the use of fork reversal in the stress response, revealing that CARM1 and PARP1 function as a regulatory module at forks to control fork speed and the choice of stress response mechanisms.

Hypoxia- and Postirradiation reoxygenation-induced HMHA1/ARHGAP45 expression contributes to cancer cell invasion in a HIF-dependent manner.

BACKGROUND: Cancer cells in severely hypoxic regions have been reported to invade towards tumour blood vessels after surviving radiotherapy in a postirradiation reoxygenation- and hypoxia-inducible factor (HIF)-dependent manner and cause recurrence. However, how HIF induces invasiveness of irradiated and reoxygenated cancer cells remains unclear. METHODS: Here, we identified human minor histocompatibility antigen 1 (HMHA1), which has been suggested to function in cytoskeleton dynamics and cellular motility, as a responsible factor and elucidated its mechanism of action using molecular and cellular biology techniques. RESULTS: HMHA1 expression was found to be induced at the transcription initiation level in a HIF-dependent manner under hypoxia. Boyden chamber invasion assay revealed that the induction of HMHA1 expression is required for the increase in invasion of hypoxic cancer cells. Reoxygenation treatment after ionising radiation in vitro that mimics dynamic changes of a microenvironment in hypoxic regions of tumour tissues after radiation therapy further enhanced HMHA1 expression and invasive potential of HMHA1 wildtype cancer cells in ROS- and HIF-dependent manners, but not of HMHA1 knockout cells. CONCLUSION: These results together provide insights into a potential molecular mechanism of the acquisition of invasiveness by hypoxic cancer cells after radiotherapy via the activation of the ROS/HIF/HMHA1 axis.

Mechanism of chromosome rearrangement arising from single-strand breaks.

Chromosome rearrangements, which are structural chromosomal abnormalities commonly found in human cancer, result from the misrejoining between two or more DNA double-strand breaks arising at different genomic regions. Consequently, chromosome rearrangements can generate fusion genes that promote tumorigenesis. The mechanisms of chromosome rearrangement have been studied using exogenous double-strand break inducers, such as radiation and nucleases. However, the mechanism underlying the occurrence of chromosome rearrangements in the absence of exogenous double-strand break-inducing stimuli is unclear. This study aimed to identify the major source of chromosome rearrangements and the DNA repair pathway that suppresses them. DNA repair factors that potentially suppress gene fusion were screened using The Cancer Genome Atlas dataset. In total, 22 repair factors whose expression levels were negatively correlated with the frequency of gene fusion were identified. More than 60% of these repair factors are involved in homologous recombination, a major double-strand break repair pathway. We hypothesized that DNA single-strand breaks are the source of double-strand breaks that lead to chromosome rearrangements. This study demonstrated that hydrogen peroxide (H2O2)-induced single-strand breaks gave rise to double-strand breaks in a replication-dependent manner. Additionally, H2O2 induced the formation of RPA and RAD51 foci, which indicated that double-strand breaks derived from single-strand breaks were repaired through homologous recombination. Moreover, treatment with H2O2 promoted the formation of radial chromosomes, a type of chromosome rearrangements, only upon the downregulation of homologous recombination factors, such as BRCA1 and CtIP. Thus, single-strand breaks are the major source of chromosome rearrangements when the expression of homologous recombination factors is downregulated.

An ATR-PrimPol pathway confers tolerance to oncogenic KRAS-induced and heterochromatin-associated replication stress.

Activation of the KRAS oncogene is a source of replication stress, but how this stress is generated and how it is tolerated by cancer cells remain poorly understood. Here we show that induction of KRASG12V expression in untransformed cells triggers H3K27me3 and HP1-associated chromatin compaction in an RNA transcription dependent manner, resulting in replication fork slowing and cell death. Furthermore, elevated ATR expression is necessary and sufficient for tolerance of KRASG12V-induced replication stress to expand replication stress-tolerant cells (RSTCs). PrimPol is phosphorylated at Ser255, a potential Chk1 substrate site, under KRASG12V-induced replication stress and promotes repriming to maintain fork progression and cell survival in an ATR/Chk1-dependent manner. However, ssDNA gaps are generated at heterochromatin by PrimPol-dependent repriming, leading to genomic instability. These results reveal a role of ATR-PrimPol in enabling precancerous cells to survive KRAS-induced replication stress and expand clonally with accumulation of genomic instability.

RAP80 suppresses the vulnerability of R-loops during DNA double-strand break repair.

Single-stranded DNA (ssDNA) arising as an intermediate of cellular processes on DNA is a potential vulnerability of the genome unless it is appropriately protected. Recent evidence suggests that R-loops, consisting of ssDNA and DNA-RNA hybrids, can form in the proximity of DNA double-strand breaks (DSBs) within transcriptionally active regions. However, how the vulnerability of ssDNA in R-loops is overcome during DSB repair remains unclear. Here, we identify RAP80 as a factor suppressing the vulnerability of ssDNA in R-loops, chromosome translocations, and deletions during DSB repair. Mechanistically, RAP80 prevents unscheduled nucleolytic processing of ssDNA in R-loops by CtIP. This mechanism promotes efficient DSB repair via transcription-associated end joining dependent on BRCA1, Polθ, and LIG1/3. Thus, RAP80 suppresses the vulnerability of R-loops during DSB repair, thereby precluding genomic abnormalities in a critical component of the genome caused by deleterious R-loop processing.

Active RB causes visible changes in nuclear organization.

RB restricts G1/S progression by inhibiting E2F. Here, we show that sustained expression of active RB, and prolonged G1 arrest, causes visible changes in chromosome architecture that are not directly associated with E2F inhibition. Using FISH probes against two euchromatin RB-associated regions, two heterochromatin domains that lack RB-bound loci, and two whole-chromosome probes, we found that constitutively active RB (ΔCDK-RB) promoted a more diffuse, dispersed, and scattered chromatin organization. These changes were RB dependent, were driven by specific isoforms of monophosphorylated RB, and required known RB-associated activities. ΔCDK-RB altered physical interactions between RB-bound genomic loci, but the RB-induced changes in chromosome architecture were unaffected by dominant-negative DP1. The RB-induced changes appeared to be widespread and influenced chromosome localization within nuclei. Gene expression profiles revealed that the dispersion phenotype was associated with an increased autophagy response. We infer that, after cell cycle arrest, RB acts through noncanonical mechanisms to significantly change nuclear organization, and this reorganization correlates with transitions in cellular state.

Impacts of chromatin dynamics and compartmentalization on DNA repair.

The proper spatial organization of DNA, RNA, and proteins is critical for a variety of cellular processes. The genome is organized into numerous functional units, such as topologically associating domains (TADs), the formation of which is regulated by both proteins and RNA. In addition, a group of chromatin-bound proteins with the ability to undergo liquid-liquid phase separation (LLPS) can affect the spatial organization and compartmentalization of chromatin, RNA, and proteins by forming condensates, conferring unique properties to specific chromosomal regions. Although the regulation of DNA repair by histone modifications and chromatin accessibility is well established, the impacts of higher-order chromatin and protein organization on the DNA damage response (DDR) have not been appreciated until recently. In this review, we will focus on the movement of chromatin during the DDR, the compartmentalization of DDR proteins via LLPS, and the roles of membraneless nuclear bodies and transcription in DNA repair. With this backdrop, we will discuss the importance of the spatial organization of chromatin and proteins for the maintenance of genome integrity.

High linear energy transfer carbon-ion irradiation upregulates PD-L1 expression more significantly than X-rays in human osteosarcoma U2OS cells.

Programmed death ligand 1 (PD-L1) expression on the surface of cancer cells affects the efficacy of anti-PD-1/PD-L1 immune checkpoint therapy. However, the mechanism underlying PD-L1 expression in cancer cells is not fully understood, particularly after ionizing radiation (IR). Here, we examined the impact of high linear energy transfer (LET) carbon-ion irradiation on the expression of PD-L1 in human osteosarcoma U2OS cells. We found that the upregulation of PD-L1 expression after high LET carbon-ion irradiation was greater than that induced by X-rays at the same physical and relative biological effectiveness (RBE) dose, and that the upregulation of PD-L1 induced by high LET carbon-ion irradiation was predominantly dependent on ataxia telangiectasia and Rad3-related (ATR) kinase activity. Moreover, we showed that the downstream signaling, e.g. STAT1 phosphorylation and IRF1 expression, was upregulated to a greater extent after high LET carbon-ion irradiation than X-rays, and that IRF1 upregulation was also ATR dependent. Finally, to visualize PD-L1 molecules on the cell surface in 3D, we applied immunofluorescence-based super-resolution imaging. The three-dimensional structured illumination microscopy (3D-SIM) analyses revealed substantial increases in the number of presented PD-L1 molecules on the cell surface after high LET carbon-ion irradiation compared with X-ray irradiation.

DNA Repair and Signaling in Immune-Related Cancer Therapy.

Cancer therapy using immune checkpoint inhibitors (ICIs) is a promising clinical strategy for patients with multiple types of cancer. The expression of programmed cell death ligand-1 (PD-L1), an immune-suppressor ligand, in cancer cells is a factor that influences the efficacy of ICI therapy, particularly in the anti-programmed cell death protein-1 (PD-1)/PD-L1 antibody therapy. PD-L1 expression in cancer cells are associated with tumor mutation burden including microsatellite instability because the accumulation of mutations in the cancer genome can produce abnormal proteins via mutant mRNAs, resulting in neoantigen production and HLA-neoantigen complex presentation in cancer cells. HLA-neoantigen presentation promotes immune activity within tumor environment; therefore, known as hot tumor. Thus, as the fidelity of DNA repair affects the generation of genomic mutations, the status of DNA repair and signaling in cancer cells can be considered prior to ICI therapy. The Cancer Genome Atlas (TCGA) and The Cancer Immunome Atlas (TCIA) database analysis showed that tumor samples harboring mutations in any non-homologous end joining, homologous recombination, or DNA damage signaling genes exhibit high neoantigen levels. Alternatively, an urgent task is to understand how the DNA damage-associated cancer treatments change the status of immune activity in patients because multiple clinical trials on combination therapy are ongoing. Recent studies demonstrated that multiple pathways regulate PD-L1 expression in cancer cells. Here, we summarize the regulation of the immune response to ICI therapy, including PD-L1 expression, and also discuss the potential strategies to improve the efficacy of ICI therapy for poor responders from the viewpoint of DNA damage response before or after DNA damage-associated cancer treatment.

RNF8 promotes high linear energy transfer carbon-ion-induced DNA double-stranded break repair in serum-starved human cells.

The cell-killing effect of radiotherapy largely depends on unrepaired DNA double-stranded breaks (DSBs) or lethal chromosome aberrations induced by DSBs. Thus, the capability of DSB repair is critically important for the cancer-cell-killing effect of ionizing radiation. Here, we investigated the involvement of the DNA damage signaling factors ataxia telangiectasia mutated (ATM), ring finger protein 8 (RNF8), and RNF168 in quiescent G0/G1 cells, which are expressed in the majority of cell populations in tumors, after high linear energy transfer (LET) carbon-ion irradiation. Interestingly, ATM inhibition caused a substantial DSB repair defect after high-LET carbon-ion irradiation. Similarly, RNF8 or RNF168 depletion caused a substantial DSB repair defect. ATM inhibition did not exert an additive effect in RNF8-depleted cells, suggesting that ATM and RNF8 function in the same pathway. Importantly, we found that the RNF8 RING mutant showed a similar DSB repair defect, suggesting the requirement of ubiquitin ligase activity in this repair pathway. The RNF8 FHA domain was also required for DSB repair in this axis. Furthermore, the p53-binding protein 1 (53BP1), which is an important downstream factor in RNF8-dependent DSB repair, was also required for this repair. Importantly, either ATM inhibition or RNF8 depletion increased the frequency of chromosomal breaks, but reduced dicentric chromosome formation, demonstrating that ATM/RNF8 is required for the rejoining of DSB ends for the formation of dicentric chromosomes. Finally, we showed that RNF8 depletion augmented radiosensitivity after high-LET carbon-ion irradiation. This study suggests that the inhibition of RNF8 activity or its downstream pathway may augment the efficacy of high-LET carbon-ion therapy.

The role of R-loops in transcription-associated DNA double-strand break repair.

The roles of RNA in the DNA damage response are emerging. We highlight findings from our recent study demonstrating the mechanism for transcription-associated homologous recombination repair (TA-HRR) of DNA double-strand breaks and the critical role of R-loops in TA-HRR.

p53 deficiency augments nucleolar instability after ionizing irradiation.

Ribosomes are important cellular components that maintain cellular homeostasis through overall protein synthesis. The nucleolus is a prominent subnuclear structure that contains ribosomal DNA (rDNA) encoding ribosomal RNA (rRNA), an essential component of ribosomes. Despite the significant role of the rDNA­rRNA­ribosome axis in cellular homeostasis, the stability of rDNA in the context of the DNA damage response has not been fully investigated. In the present study, the number and morphological changes of nucleolin, a marker of the nucleolus, were examined following ionizing radiation (IR) in order to investigate the impact of DNA damage on nucleolar stability. An increase in the number of nucleoli per cell was found in HCT116 and U2OS cells following IR. Interestingly, the IR­dependent increase in nucleolar fragmentation was enhanced by p53 deficiency. In addition, the morphological analysis revealed several distinct types of nucleolar fragmentation following IR. The pattern of nucleolar morphology differed between HCT116 and U2OS cells, and the p53 deficiency altered the pattern of nucleolar morphology. Finally, a significant decrease in rRNA synthesis was observed in HCT116 p53­/­ cells following IR, suggesting that severe nucleolar fragmentation downregulates rRNA transcription. The findings of the present study suggest that p53 plays a key role in protecting the transcriptional activity of rDNA in response to DNA damage.

Base excision repair regulates PD-L1 expression in cancer cells.

Programmed death-ligand 1 (PD-L1) is a key factor influencing cancer immunotherapy; however, the regulation of PD-L1 expression in cancer cells remains unclear, particularly regarding DNA damage, repair and its signalling. Herein, we demonstrate that oxidative DNA damage induced by exogenously applied hydrogen peroxide (H2O2) upregulates PD-L1 expression in cancer cells. Further, depletion of the base excision repair (BER) enzyme DNA glycosylase augments PD-L1 upregulation in response to H2O2. PD-L1 upregulation in BER-depleted cells requires ATR/Chk1 kinase activities, demonstrating that PD-L1 upregulation is mediated by DNA damage signalling. Further analysis of The Cancer Genome Atlas revealed that the expression of PD-L1 is negatively correlated with that of the BER/single-strand break repair (SSBR) and tumours with low BER/SSBR gene expression show high microsatellite instability and neoantigen production. Hence, these results suggest that PD-L1 expression is regulated in cancer cells via the DNA damage signalling and neoantigen-interferon-γ pathway under oxidative stress.

3D-structured illumination microscopy reveals clustered DNA double-strand break formation in widespread γH2AX foci after high LET heavy-ion particle radiation.

DNA double-strand breaks (DSBs) induced by ionising radiation are considered the major cause of genotoxic mutations and cell death. While DSBs are dispersed throughout chromatin after X-rays or γ-irradiation, multiple types of DNA damage including DSBs, single-strand breaks and base damage can be generated within 1-2 helical DNA turns, defined as a complex DNA lesion, after high Linear Energy Transfer (LET) particle irradiation. In addition to the formation of complex DNA lesions, recent evidence suggests that multiple DSBs can be closely generated along the tracks of high LET particle irradiation. Herein, by using three dimensional (3D)-structured illumination microscopy, we identified the formation of 3D widespread γH2AX foci after high LET carbon-ion irradiation. The large γH2AX foci in G2-phase cells encompassed multiple foci of replication protein A (RPA), a marker of DSBs undergoing resection during homologous recombination. Furthermore, we demonstrated by 3D analysis that the distance between two individual RPA foci within γH2AX foci was approximately 700 nm. Together, our findings suggest that high LET heavy-ion particles induce clustered DSB formation on a scale of approximately 1 µm3. These closely localised DSBs are considered to be a risk for the formation of chromosomal rearrangement after heavy-ion irradiation.

DNA double-strand break repair pathway regulates PD-L1 expression in cancer cells.

Accumulating evidence suggests that exogenous cellular stress induces PD-L1 upregulation in cancer. A DNA double-strand break (DSB) is the most critical type of genotoxic stress, but the involvement of DSB repair in PD-L1 expression has not been investigated. Here we show that PD-L1 expression in cancer cells is upregulated in response to DSBs. This upregulation requires ATM/ATR/Chk1 kinases. Using an siRNA library targeting DSB repair genes, we discover that BRCA2 depletion enhances Chk1-dependent PD-L1 upregulation after X-rays or PARP inhibition. In addition, we show that Ku70/80 depletion substantially enhances PD-L1 upregulation after X-rays. The upregulation by Ku80 depletion requires Chk1 activation following DNA end-resection by Exonuclease 1. DSBs activate STAT1 and STAT3 signalling, and IRF1 is required for DSB-dependent PD-L1 upregulation. Thus, our findings reveal the involvement of DSB repair in PD-L1 expression and provide mechanistic insight into how PD-L1 expression is regulated after DSBs.

High RAD54B expression: an independent predictor of postoperative distant recurrence in colorectal cancer patients.

We recently reported a specific mechanism that RAD54B, an important factor in homologous recombination, promotes genomic instability via the degradation of p53 protein in vitro. However, clinical significance of RAD54Bin colorectal cancer (CRC) remains unclear. Thus we analyzed RAD54B geneexpression in CRC patients. Using the training set (n = 123), the optimal cut-off value for stratification was determined, and validated in another cohort (n = 89). Kaplan-Meier plots showed that distant recurrence free survival was significantly lesser in high RAD54B expression group compared with that of low expression group in both training (P = 0.0013) and validation (P = 0.024) set. Multivariate analysis using Cox proportional-hazards model showed that high RAD54B expression was an independent predictor in both training (hazard ratio, 4.31; 95% CI, 1.53-13.1; P = 0.0060) and validation (hazard ratio, 3.63; 95% CI, 1.23-10.7; P = 0.021) set. In addition, a negative significant correlation between RAD54B and CDKN1A, a target gene of p53, was partially confirmed, suggesting that RAD54B functions via the degradation of p53 protein even in clinical samples. This study first demonstrated RAD54B expression has potential to serve as a novel prognostic biomarker, particularly for distant recurrence in CRC patients.

Rad54B serves as a scaffold in the DNA damage response that limits checkpoint strength.

The strength of the DNA damage checkpoint critically influences cell fate, yet the mechanisms behind the fine tuning of checkpoint strength during the DNA damage response (DDR) are poorly understood. Here we show that Rad54B--a SNF2 helicase-like DNA-repair protein--limits the strength of both the G1/S and G2/M checkpoints. We find that Rad54B functions as a scaffold for p53 degradation via its direct interaction with the MDM2-MDMX ubiquitin-ligase complex. During the early phases of the DDR, Rad54B is upregulated, thereby maintaining low checkpoint strength and facilitating cell cycle progression. Once the p53-mediated checkpoint is established, Rad54B is downregulated, and high checkpoint strength is maintained. Constitutive upregulation of Rad54B activity, which is frequently observed in tumours, promotes genomic instability because of checkpoint override. Thus, the scaffolding function of Rad54B dynamically regulates the maintenance of genome integrity by limiting checkpoint strength.