RESUMO
PURPOSE: This study investigated how isoform switching affects the cellular response to ionizing radiation (IR), an understudied area despite its relevance to radiation therapy in cancer treatment. We aimed to identify changes in transcript isoform expression post-IR exposure and the proteins mediating these changes, with a focus on their potential to modulate radiosensitivity. METHODS AND MATERIALS: Using RNA sequencing, we analyzed the B-cell lines derived from 10 healthy individuals at 3 timepoints, applying the mixture of isoforms algorithm to quantify alternative splicing. We examined RNA binding protein motifs within the sequences of IR-responsive isoforms and validated the serine/arginine-rich splicing factor 1 (SRSF1) as a predominant mediator through RNA immunoprecipitation. We further investigated the effects of SRSF1 on radiosensitivity by RNA interference and by analyzing publicly available data on patients with cancer. RESULTS: We identified â¼1900 radiation-responsive alternatively spliced isoforms. Many isoforms were differentially expressed without changes in their overall gene expression. Over a third of these transcripts underwent exon skipping, while others used proximal last exons. These IR-responsive isoforms tended to be shorter transcripts missing vital domains for preventing apoptosis and promoting cell division but retaining those necessary for DNA repair. Our combined computational, genetic, and molecular analyses identified the proto-oncogene SRSF1 as a mediator of these radiation-induced isoform-switching events that promote apoptosis. After exposure to DNA double-strand break-inducing agents, SRSF1 expression decreased. A reduction in SRSF1 increased radiosensitivity in vitro and among patients with cancer. CONCLUSIONS: We establish a pivotal role for isoform switching in the cellular response to IR and propose SRSF1 as a promising biomarker for assessing radiation therapy effectiveness.
Assuntos
Processamento Alternativo , Isoformas de Proteínas , Proto-Oncogene Mas , Tolerância a Radiação , Radiação Ionizante , Fatores de Processamento de Serina-Arginina , Fatores de Processamento de Serina-Arginina/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Humanos , Tolerância a Radiação/genética , ÉxonsRESUMO
Since genome instability can drive cancer initiation and progression, cells have evolved highly effective and ubiquitous DNA damage response (DDR) programs. However, some cells (for example, in skin) are normally exposed to high levels of DNA-damaging agents. Whether such high-risk cells possess lineage-specific mechanisms that tailor DNA repair to the tissue remains largely unknown. Using melanoma as a model, we show here that the microphthalmia-associated transcription factor MITF, a lineage addition oncogene that coordinates many aspects of melanocyte and melanoma biology, plays a nontranscriptional role in shaping the DDR. On exposure to DNA-damaging agents, MITF is phosphorylated at S325, and its interactome is dramatically remodeled; most transcription cofactors dissociate, and instead MITF interacts with the MRE11-RAD50-NBS1 (MRN) complex. Consequently, cells with high MITF levels accumulate stalled replication forks and display defects in homologous recombination-mediated repair associated with impaired MRN recruitment to DNA damage. In agreement with this, high MITF levels are associated with increased single-nucleotide and copy number variant burdens in melanoma. Significantly, the SUMOylation-defective MITF-E318K melanoma predisposition mutation recapitulates the effects of DNA-PKcs-phosphorylated MITF. Our data suggest that a nontranscriptional function of a lineage-restricted transcription factor contributes to a tissue-specialized modulation of the DDR that can impact cancer initiation.
Assuntos
Melanoma , Humanos , Melanoma/genética , Fator de Transcrição Associado à Microftalmia/genética , Dano ao DNA , Instabilidade Genômica/genética , DNARESUMO
SETD2-dependent H3 Lysine-36 trimethylation (H3K36me3) has been recently linked to the deposition of de-novo DNA methylation. SETD2 is frequently mutated in cancer, however, the functional impact of SETD2 loss and depletion on DNA methylation across cancer types and tumorigenesis is currently unknown. Here, we perform a pan-cancer analysis and show that both SETD2 mutation and reduced expression are associated with DNA methylation dysregulation across 21 out of the 24 cancer types tested. In renal cancer, these DNA methylation changes are associated with altered gene expression of oncogenes, tumour suppressors, and genes involved in neoplasm invasiveness, including TP53, FOXO1, and CDK4. This suggests a new role for SETD2 loss in tumorigenesis and cancer aggressiveness through DNA methylation dysregulation. Moreover, using a robust machine learning methodology, we develop and validate a 3-CpG methylation signature which is sufficient to predict SETD2 mutation status with high accuracy and correlates with patient prognosis.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Metilação de DNA , Histonas/metabolismo , Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , Carcinogênese/genética , Transformação Celular Neoplásica/genéticaRESUMO
Since genome instability can drive cancer initiation and progression, cells have evolved highly effective and ubiquitous DNA Damage Response (DDR) programs. However, some cells, in skin for example, are normally exposed to high levels of DNA damaging agents. Whether such high-risk cells possess lineage-specific mechanisms that tailor DNA repair to the tissue remains largely unknown. Here we show, using melanoma as a model, that the microphthalmia-associated transcription factor MITF, a lineage addition oncogene that coordinates many aspects of melanocyte and melanoma biology, plays a non-transcriptional role in shaping the DDR. On exposure to DNA damaging agents, MITF is phosphorylated by ATM/DNA-PKcs, and unexpectedly its interactome is dramatically remodelled; most transcription (co)factors dissociate, and instead MITF interacts with the MRE11-RAD50-NBS1 (MRN) complex. Consequently, cells with high MITF levels accumulate stalled replication forks, and display defects in homologous recombination-mediated repair associated with impaired MRN recruitment to DNA damage. In agreement, high MITF levels are associated with increased SNV burden in melanoma. Significantly, the SUMOylation-defective MITF-E318K melanoma predisposition mutation recapitulates the effects of ATM/DNA-PKcs-phosphorylated MITF. Our data suggest that a non-transcriptional function of a lineage-restricted transcription factor contributes to a tissue-specialised modulation of the DDR that can impact cancer initiation.
RESUMO
Chromosomal instability (CIN) drives cell-to-cell heterogeneity, and the development of genetic diseases, including cancer. Impaired homologous recombination (HR) has been implicated as a major driver of CIN, however, the underlying mechanism remains unclear. Using a fission yeast model system, we establish a common role for HR genes in suppressing DNA double-strand break (DSB)-induced CIN. Further, we show that an unrepaired single-ended DSB arising from failed HR repair or telomere loss is a potent driver of widespread CIN. Inherited chromosomes carrying a single-ended DSB are subject to cycles of DNA replication and extensive end-processing across successive cell divisions. These cycles are enabled by Cullin 3-mediated Chk1 loss and checkpoint adaptation. Subsequent propagation of unstable chromosomes carrying a single-ended DSB continues until transgenerational end-resection leads to fold-back inversion of single-stranded centromeric repeats and to stable chromosomal rearrangements, typically isochromosomes, or to chromosomal loss. These findings reveal a mechanism by which HR genes suppress CIN and how DNA breaks that persist through mitotic divisions propagate cell-to-cell heterogeneity in the resultant progeny.
Assuntos
Schizosaccharomyces , Humanos , Instabilidade Cromossômica , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Recombinação Homóloga , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMO
We constructed a panel of S. pombe strains expressing DNA polymerase ε variants associated with cancer, specifically POLES297F, POLEV411L, POLEL424V, POLES459F, and used these to compare mutation rates determined by canavanine resistance with other selective methods. Canavanine-resistance mutation rates are broadly similar to those seen with reversion of the ade-485 mutation to adenine prototrophy, but lower than 5-fluoroorotic acid (FOA)-resistance rates (inactivation of ura4+ or ura5+ genes). Inactivation of several genes has been associated with canavanine resistance in S. pombe but surprisingly whole genome sequencing showed that 8/8 spontaneous canavanine-resistant mutants have an R175C mutation in the any1/arn1 gene. This gene encodes an α-arrestin-like protein involved in mediating Pub1 ubiquitylation of target proteins, and the phenotypic resistance to canavanine by this single mutation is similar to that shown by the original "can1-1" strain, which also has the any1R175C mutation. Some of the spontaneous mutants have additional mutations in arginine transporters, suggesting that this may marginally increase resistance to canavanine. The any1R175C strain showed internalisation of the Cat1 arginine transporter as previously reported, explaining the canavanine-resistance phenotype.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Canavanina/farmacologia , Canavanina/metabolismo , Taxa de Mutação , Proteínas de Schizosaccharomyces pombe/metabolismo , Mutação , Arginina/metabolismo , Arrestinas/metabolismoRESUMO
Little is known about the pathways regulating MHC antigen presentation and the identity of treatment-specific T cell antigens induced by ionizing radiation. For this reason, we investigated the radiation-specific changes in the colorectal tumor cell proteome. We found an increase in DDX58 and ZBP1 protein expression, two nucleic acid sensing molecules likely involved in induction of the dominant interferon response signature observed after genotoxic insult. We further observed treatment-induced changes in key regulators and effector proteins of the antigen processing and presentation machinery. Differential regulation of MHC allele expression was further driving the presentation of a significantly broader MHC-associated peptidome postirradiation, defining a radiation-specific peptide repertoire. Interestingly, treatment-induced peptides originated predominantly from proteins involved in catecholamine synthesis and metabolic pathways. A nuanced relationship between protein expression and antigen presentation was observed where radiation-induced changes in proteins do not correlate with increased presentation of associated peptides. Finally, we detected an increase in the presentation of a tumor-specific neoantigen derived from Mtch1. This study provides new insights into how radiation enhances antigen processing and presentation that could be suitable for the development of combinatorial therapies. Data are available via ProteomeXchange with identifier PXD032003.
Assuntos
Apresentação de Antígeno , Proteoma , Proteoma/metabolismo , Peptídeos/metabolismo , Proteômica , Radiação IonizanteRESUMO
Histone Post-Translational Modifications (PTMs) play fundamental roles in mediating DNA-related processes such as transcription, replication and repair. The histone mark H3K36me3 and its associated methyltransferase SETD2 (Set2 in yeast) are archetypical in this regard, performing critical roles in each of these DNA transactions. Here, we present an overview of H3K36me3 regulation and the roles of its writers, readers and erasers in maintaining genome stability through facilitating DNA double-strand break (DSB) repair, checkpoint signalling and replication stress responses. Further, we consider how loss of SETD2 and H3K36me3, frequently observed in a number of different cancer types, can be specifically targeted in the clinic through exploiting loss of particular genome stability functions.
Assuntos
Instabilidade Genômica , Histonas , DNA , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Histonas/metabolismo , Humanos , Saccharomyces cerevisiae/genéticaRESUMO
PURPOSE: Outcomes in RAS-mutant metastatic colorectal cancer (mCRC) remain poor and patients have limited therapeutic options. Adavosertib is the first small-molecule inhibitor of WEE1 kinase. We hypothesized that aberrations in DNA replication seen in mCRC with both RAS and TP53 mutations would sensitize tumors to WEE1 inhibition. METHODS: Patients with newly diagnosed mCRC were registered into FOCUS4 and tested for TP53 and RAS mutations. Those with both mutations who were stable or responding after 16 weeks of chemotherapy were randomly assigned 2:1 between adavosertib and active monitoring (AM). Adavosertib (250 mg or 300 mg) was taken orally once on days 1-5 and days 8-12 of a 3-week cycle. The primary outcome was progression-free survival (PFS), with a target hazard ratio (HR) of 0.5 and 80% power with a one-sided 0.025 significance level. RESULTS: FOCUS4-C was conducted between April 2017 and Mar 2020 during which time 718 patients were registered; 247 (34%) were RAS/TP53-mutant. Sixty-nine patients were randomly assigned from 25 UK hospitals (adavosertib = 44; AM = 25). Adavosertib was associated with a PFS improvement over AM (median 3.61 v 1.87 months; HR = 0.35; 95% CI, 0.18 to 0.68; P = .0022). Overall survival (OS) was not improved with adavosertib versus AM (median 14.0 v 12.8 months; HR = 0.92; 95% CI, 0.44 to 1.94; P = .93). In prespecified subgroup analysis, adavosertib activity was greater in left-sided tumors (HR = 0.24; 95% CI, 0.11 to 0.51), versus right-sided (HR = 1.02; 95% CI, 0.41 to 2.56; interaction P = .043). Adavosertib was well-tolerated; grade 3 toxicities were diarrhea (9%), nausea (5%), and neutropenia (7%). CONCLUSION: In this phase II randomized trial, adavosertib improved PFS compared with AM and demonstrates potential as a well-tolerated therapy for RAS/TP53-mutant mCRC. Further testing is required in this sizable population of unmet need.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Neoplasias Colorretais/tratamento farmacológico , Mutação , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/uso terapêutico , Pirimidinonas/uso terapêutico , Proteína Supressora de Tumor p53/genética , Conduta Expectante/estatística & dados numéricos , Proteínas ras/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Prognóstico , Qualidade de Vida , Taxa de SobrevidaRESUMO
Somatic and germline mutations in the proofreading domain of the replicative DNA polymerase ε (POLE-exonuclease domain mutations, POLE-EDMs) are frequently found in colorectal and endometrial cancers and, occasionally, in other tumours. POLE-associated cancers typically display hypermutation, and a unique mutational signature, with a predominance of C > A transversions in the context TCT and C > T transitions in the context TCG. To understand better the contribution of hypermutagenesis to tumour development, we have modelled the most recurrent POLE-EDM (POLE-P286R) in Schizosaccharomyces pombe. Whole-genome sequencing analysis revealed that the corresponding pol2-P287R allele also has a strong mutator effect in vivo, with a high frequency of base substitutions and relatively few indel mutations. The mutations are equally distributed across different genomic regions, but in the immediate vicinity there is an asymmetry in AT frequency. The most abundant base-pair changes are TCT > TAT transversions and, in contrast to human mutations, TCG > TTG transitions are not elevated, likely due to the absence of cytosine methylation in fission yeast. The pol2-P287R variant has an increased sensitivity to elevated dNTP levels and DNA damaging agents, and shows reduced viability on depletion of the Pfh1 helicase. In addition, S phase is aberrant and RPA foci are elevated, suggestive of ssDNA or DNA damage, and the pol2-P287R mutation is synthetically lethal with rad3 inactivation, indicative of checkpoint activation. Significantly, deletion of genes encoding some translesion synthesis polymerases, most notably Pol κ, partially suppresses pol2-P287R hypermutation, indicating that polymerase switching contributes to this phenotype.
Assuntos
DNA Polimerase II/genética , Replicação do DNA , Mutação , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Quinase do Ponto de Checagem 2/genética , DNA Helicases/genética , DNA Polimerase II/metabolismo , Genoma Fúngico , Humanos , Neoplasias/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Fase S/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Preclinical models of cancer have demonstrated enhanced efficacy of cell-cycle checkpoint kinase inhibitors when used in combination with genotoxic agents. This combination therapy is predicted to be exquisitely toxic to cells with a deficient G1-S checkpoint or cells with a genetic predisposition leading to intrinsic DNA replication stress, as these cancer cells become fully dependent on the intra-S and G2-M checkpoints for DNA repair and cellular survival. Therefore, abolishing remaining cell-cycle checkpoints after damage leads to increased cell death in a tumor cell-specific fashion. However, the preclinical success of these drug combinations is not consistently replicated in clinical trials. Here, we provide a perspective on the translation of preclinical studies into rationally designed clinical studies. We will discuss successes and failures of current treatment combinations and drug regimens and provide a detailed overview of all clinical trials using ATR, CHK1, or WEE1 inhibitors in combination with genotoxic agents. This highlights the need for revised patient stratification and the use of appropriate pharmacodynamic biomarkers to improve the success rate of clinical trials.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Quimiorradioterapia/métodos , Neoplasias/terapia , Inibidores de Proteínas Quinases/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas de Ciclo Celular/antagonistas & inibidores , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Ensaios Clínicos como Assunto , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Neoplasias/genética , Neoplasias/mortalidade , Neoplasias/patologia , Intervalo Livre de Progressão , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidoresRESUMO
Repair of DNA damage protects genomic integrity, which is key to tissue functional integrity. In cancer, the type and fidelity of DNA damage response is the fundamental basis for clinical response to cytotoxic therapy. Here we consider the contribution of transforming growth factor-beta (TGFß), a ubiquitous, pleotropic cytokine that is abundant in the tumor microenvironment, to therapeutic response. The action of TGFß is best illustrated in head and neck squamous cell carcinoma (HNSCC). Survival of HNSCC patients with human papilloma virus (HPV) positive cancer is more than double compared to those with HPV-negative HNSCC. Notably, HPV infection profoundly impairs TGFß signaling. HPV blockade of TGFß signaling, or pharmaceutical TGFß inhibition that phenocopies HPV infection, shifts cancer cells from error-free homologous-recombination DNA double-strand-break (DSB) repair to error-prone alternative end-joining (altEJ). Cells using altEJ are more sensitive to standard of care radiotherapy and cisplatin, and are sensitized to PARP inhibitors. Hence, HPV-positive HNSCC is an experiment of nature that provides a strong rationale for the use of TGFß inhibitors for optimal therapeutic combinations that improve patient outcome.
RESUMO
Replication stress is a common feature of cancer cells, and thus a potentially important therapeutic target. Here, we show that cyclin-dependent kinase (CDK)-induced replication stress, resulting from Wee1 inactivation, is synthetic lethal with mutations disrupting dNTP homeostasis in fission yeast. Wee1 inactivation leads to increased dNTP demand and replication stress through CDK-induced firing of dormant replication origins. Subsequent dNTP depletion leads to inefficient DNA replication, DNA damage and to genome instability. Cells respond to this replication stress by increasing dNTP supply through histone methyltransferase Set2-dependent MBF-induced expression of Cdc22, the catalytic subunit of ribonucleotide reductase (RNR). Disrupting dNTP synthesis following Wee1 inactivation, through abrogating Set2-dependent H3K36 tri-methylation or DNA integrity checkpoint inactivation results in critically low dNTP levels, replication collapse and cell death, which can be rescued by increasing dNTP levels. These findings support a 'dNTP supply and demand' model in which maintaining dNTP homeostasis is essential to prevent replication catastrophe in response to CDK-induced replication stress.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Nucleotídeos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Pontos de Checagem do Ciclo Celular , Dano ao DNA , Replicação do DNA , Código das Histonas , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Homeostase , Metilação , Schizosaccharomyces/metabolismo , Mutações Sintéticas Letais , Fatores de Transcrição/metabolismoRESUMO
The nuclear pore complex (NPC) machinery is emerging as an important determinant in the maintenance of genome integrity and sensitivity to DNA double-strand break (DSB)-inducing agents, such as ionising radiation (IR). In this study, using a high-throughput siRNA screen, we identified the central channel NPC protein Nup54, and concomitantly its molecular partners Nup62 and Nup58, as novel factors implicated in radiosensitivity. Nup54 depletion caused an increase in cell death by mitotic catastrophe after IR, and specifically enhanced both the duration of the G2 arrest and the radiosensitivity of cells that contained replicated DNA at the time of IR exposure. Nup54-depleted cells also exhibited increased formation of chromosome aberrations arisen from replicated DNA. Interestingly, we found that Nup54 is epistatic with the homologous recombination (HR) factor Rad51. Moreover, using specific DNA damage repair reporters, we observed a decreased HR repair activity upon Nup54 knockdown. In agreement with a role in HR repair, we also demonstrated a decreased formation of HR-linked DNA synthesis foci and sister chromatid exchanges after IR in cells depleted of Nup54. Our study reveals a novel role for Nup54 in the response to IR and the maintenance of HR-mediated genome integrity.
Assuntos
Replicação do DNA , DNA/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Reparo de DNA por Recombinação , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , DNA/genética , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Células HeLa , Humanos , Células MCF-7 , Poro Nuclear/genética , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Interferência de RNA , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Radiação Ionizante , Troca de Cromátide Irmã/efeitos da radiaçãoRESUMO
Chromatin modification through histone H3 lysine 36 methylation by the SETD2 tumor suppressor plays a key role in maintaining genome stability. Here, we describe a role for Set2-dependent H3K36 methylation in facilitating DNA replication and the transcriptional responses to both replication stress and DNA damage through promoting MluI cell-cycle box (MCB) binding factor (MBF)-complex-dependent transcription in fission yeast. Set2 loss leads to reduced MBF-dependent ribonucleotide reductase (RNR) expression, reduced deoxyribonucleoside triphosphate (dNTP) synthesis, altered replication origin firing, and a checkpoint-dependent S-phase delay. Accordingly, prolonged S phase in the absence of Set2 is suppressed by increasing dNTP synthesis. Furthermore, H3K36 is di- and tri-methylated at these MBF gene promoters, and Set2 loss leads to reduced MBF binding and transcription in response to genotoxic stress. Together, these findings provide new insights into how H3K36 methylation facilitates DNA replication and promotes genotoxic stress responses in fission yeast.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Replicação do DNA , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Pontos de Checagem do Ciclo Celular/genética , Dano ao DNA/genética , Replicação do DNA/genética , DNA Fúngico/metabolismo , Regulação para Baixo/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Mutação/genética , Nucleotídeos/metabolismo , Origem de Replicação/genética , Fase S/genéticaRESUMO
The partner and localiser of BRCA2 (PALB2) plays important roles in the maintenance of genome integrity and protection against cancer. Although PALB2 is commonly described as a repair factor recruited to sites of DNA breaks, recent studies provide evidence that PALB2 also associates with unperturbed chromatin. Here, we investigated the previously poorly described role of chromatin-associated PALB2 in undamaged cells. We found that PALB2 associates with active genes through its major binding partner, MRG15, which recognizes histone H3 trimethylated at lysine 36 (H3K36me3) by the SETD2 methyltransferase. Missense mutations that ablate PALB2 binding to MRG15 confer elevated sensitivity to the topoisomerase inhibitor camptothecin (CPT) and increased levels of aberrant metaphase chromosomes and DNA stress in gene bodies, which were suppressed by preventing DNA replication. Remarkably, the level of PALB2 at genic regions was frequently decreased, rather than increased, upon CPT treatment. We propose that the steady-state presence of PALB2 at active genes, mediated through the SETD2/H3K36me3/MRG15 axis, ensures an immediate response to DNA stress and therefore effective protection of these regions during DNA replication. This study provides a conceptual advance in demonstrating that the constitutive chromatin association of repair factors plays a key role in the maintenance of genome stability and furthers our understanding of why PALB2 defects lead to human genome instability syndromes.
Assuntos
Cromatina/ultraestrutura , Dano ao DNA , Proteína do Grupo de Complementação N da Anemia de Fanconi/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Fatores de Transcrição/metabolismo , Proteína BRCA2/genética , Linhagem Celular Tumoral , Cromossomos/ultraestrutura , Reparo do DNA , Replicação do DNA , Genoma Humano , Células HEK293 , Células HeLa , Humanos , Concentração Inibidora 50 , Mutação , Ligação Proteica , Proteômica , Transcrição Gênica , Proteínas Supressoras de Tumor/metabolismoRESUMO
DNA double-strand breaks (DSBs) are toxic lesions, which if improperly repaired can result in cell death or genomic instability. DSB repair is usually facilitated by the classical non-homologous end joining (C-NHEJ), or homologous recombination (HR) pathways. However, a mutagenic alternative NHEJ pathway, microhomology-mediated end joining (MMEJ), can also be deployed. While MMEJ is suppressed by C-NHEJ, the relationship between HR and MMEJ is less clear. Here, we describe a role for HR genes in suppressing MMEJ in human cells. By monitoring DSB mis-repair using a sensitive HPRT assay, we found that depletion of HR proteins, including BRCA2, BRCA1 or RPA, resulted in a distinct mutational signature associated with significant increases in break-induced mutation frequencies, deletion lengths and the annealing of short regions of microhomology (2-6 bp) across the break-site. This signature was dependent on CtIP, MRE11, POLQ and PARP, and thus indicative of MMEJ. In contrast to CtIP or MRE11, depletion of BRCA1 resulted in increased partial resection and MMEJ, thus revealing a functional distinction between these early acting HR factors. Together these findings indicate that HR factors suppress mutagenic MMEJ following DSB resection.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA/metabolismo , Reparo de DNA por Recombinação , Proteína de Replicação A/genética , Proteína BRCA1/antagonistas & inibidores , Proteína BRCA1/metabolismo , Proteína BRCA2/antagonistas & inibidores , Proteína BRCA2/metabolismo , Sequência de Bases , Bioensaio , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Endodesoxirribonucleases , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Proteína Homóloga a MRE11 , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína de Replicação A/antagonistas & inibidores , Proteína de Replicação A/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , DNA Polimerase tetaRESUMO
The formation of RNA-DNA hybrids, referred to as R-loops, can promote genome instability and cancer development. Yet the mechanisms by which R-loops compromise genome instability are poorly understood. Here, we establish roles for the evolutionarily conserved Nrl1 protein in pre-mRNA splicing regulation, R-loop suppression and in maintaining genome stability. nrl1Δ mutants exhibit endogenous DNA damage, are sensitive to exogenous DNA damage, and have defects in homologous recombination (HR) repair. Concomitantly, nrl1Δ cells display significant changes in gene expression, similar to those induced by DNA damage in wild-type cells. Further, we find that nrl1Δ cells accumulate high levels of R-loops, which co-localize with HR repair factors and require Rad51 and Rad52 for their formation. Together, our findings support a model in which R-loop accumulation and subsequent DNA damage sequesters HR factors, thereby compromising HR repair at endogenously or exogenously induced DNA damage sites, leading to genome instability.
Assuntos
Processamento Alternativo/genética , Instabilidade Genômica/genética , Recombinação Homóloga/genética , Precursores de RNA/genética , Proteínas de Schizosaccharomyces pombe/genética , DNA/química , DNA/genética , Reparo do DNA/genética , RNA/química , RNA/genética , Rad51 Recombinase/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Schizosaccharomyces/genética , Spliceossomos/genética , Spliceossomos/metabolismoRESUMO
Histone H3K36 trimethylation (H3K36me3) is frequently lost in multiple cancer types, identifying it as an important therapeutic target. Here we identify a synthetic lethal interaction in which H3K36me3-deficient cancers are acutely sensitive to WEE1 inhibition. We show that RRM2, a ribonucleotide reductase subunit, is the target of this synthetic lethal interaction. RRM2 is regulated by two pathways here: first, H3K36me3 facilitates RRM2 expression through transcription initiation factor recruitment; second, WEE1 inhibition degrades RRM2 through untimely CDK activation. Therefore, WEE1 inhibition in H3K36me3-deficient cells results in RRM2 reduction, critical dNTP depletion, S-phase arrest, and apoptosis. Accordingly, this synthetic lethality is suppressed by increasing RRM2 expression or inhibiting RRM2 degradation. Finally, we demonstrate that WEE1 inhibitor AZD1775 regresses H3K36me3-deficient tumor xenografts.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Histonas/metabolismo , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Nucleotídeos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Humanos , Lisina/genética , Lisina/metabolismo , Metilação/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Camundongos Nus , Dados de Sequência Molecular , Neoplasias/genética , Neoplasias/prevenção & controle , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Nucleotídeos/genética , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Pirazóis/farmacologia , Pirimidinas/farmacologia , Pirimidinonas , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleosídeo Difosfato Redutase/genética , Ribonucleosídeo Difosfato Redutase/metabolismo , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Understanding the mechanisms of chromosomal double-strand break repair (DSBR) provides insight into genome instability, oncogenesis and genome engineering, including disease gene correction. Research into DSBR exploits rare-cutting endonucleases to cleave exogenous reporter constructs integrated into the genome. Multiple reporter constructs have been developed to detect various DSBR pathways. Here, using a single endogenous reporter gene, the X-chromosomal disease gene encoding hypoxanthine phosphoribosyltransferase (HPRT), we monitor the relative utilization of three DSBR pathways following cleavage by I-SceI or CRISPR/Cas9 nucleases. For I-SceI, our estimated frequencies of accurate or mutagenic non-homologous end-joining and gene correction by homologous recombination are 4.1, 1.5 and 0.16%, respectively. Unexpectedly, I-SceI and Cas9 induced markedly different DSBR profiles. Also, using an I-SceI-sensitive HPRT minigene, we show that gene correction is more efficient when using long double-stranded DNA than single- or double-stranded oligonucleotides. Finally, using both endogenous HPRT and exogenous reporters, we validate novel cell cycle phase-specific I-SceI derivatives for investigating cell cycle variations in DSBR. The results obtained using these novel approaches provide new insights into template design for gene correction and the relationships between multiple DSBR pathways at a single endogenous disease gene.