RESUMEN
The DNA damage response (DDR) pathway generally protects against genome instability, and defects in DDR have been exploited therapeutically in cancer treatment. We have reported that histone demethylase PHF8 demethylates TOPBP1 K118 mono-methylation (K118me1) to drive the activation of ATR kinase, one of the master regulators of replication stress. However, whether dysregulation of this physiological signalling is involved in tumorigenesis remains unknown. Here, we showed PHF8-promoted TOPBP1 demethylation is clinically associated with breast tumorigenesis and patient survival. Mammary gland tumors from Phf8 knockout mice grow slowly and exhibit higher level of K118me1, lower ATR activity, and increased chromosomal instability. Importantly, we found that disruption of PHF8-TOPBP1 axis suppresses breast tumorigenesis and creates a breast tumor-specific vulnerability to PARP inhibitor (PARPi) and platinum drug. CRISPR/Cas9 mutation modelling of the deleted or truncated mutation of PHF8 in clinical tumor samples demonstrated breast tumor cells expressing the mimetic variants are more vulnerable to PARPi. Together, our study supports the pursuit of PHF8-TOPBP1 signalling pathway as promising avenues for targeted therapies of PHF8-TOPBP1 proficient tumors, and provides proof-of-concept evidence for loss-of-function of PHF8 as a therapeutic indicator of PARPis.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Histona Demetilasas/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Neoplasias de la Mama/tratamiento farmacológico , Carcinogénesis/efectos de los fármacos , Carcinogénesis/metabolismo , Línea Celular , Línea Celular Tumoral , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Femenino , Inestabilidad Genómica/efectos de los fármacos , Inestabilidad Genómica/fisiología , Células HEK293 , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Cellular feedback systems ensure genome maintenance during DNA replication. When replication forks stall, newly replicated DNA is protected by pathways that limit excessive DNA nuclease attacks. Here we show that WEE1 activity guards against nascent DNA degradation at stalled forks. Furthermore, we identify WEE1-dependent suppression of cyclin-dependent kinase 2 (CDK2) as a major activity counteracting fork degradation. We establish DNA2 as the nuclease responsible for excessive fork degradation in WEE1-inhibited cells. In addition, WEE1 appears to be unique among CDK activity suppressors in S phase because neither CHK1 nor p21 promote fork protection as WEE1 does. Our results identify a key role of WEE1 in protecting stalled forks, which is separate from its established role in safeguarding DNA replication initiation. Our findings highlight how WEE1 inhibition evokes massive genome challenges during DNA replication, and this knowledge may improve therapeutic strategies to specifically eradicate cancer cells that frequently harbor elevated DNA replication stress.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Replicación del ADN/fisiología , Proteínas Tirosina Quinasas/metabolismo , Línea Celular , Inestabilidad Genómica/fisiología , HumanosRESUMEN
DNA is susceptible to damage by various sources. When the DNA is damaged, the cell repairs the damage through an appropriate DNA repair pathway. When the cell fails to repair DNA damage, apoptosis is initiated. Although several genes are involved in five major DNA repair pathways and two major apoptosis pathways, a comprehensive understanding of those gene expression is not well-understood in chicken tissues. We performed whole-transcriptome sequencing (WTS) analysis in the chicken embryonic fibroblasts (CEFs), stage X blastoderms, and primordial germ cells (PGCs) to uncover this deficiency. Stage X blastoderms mostly consist of undifferentiated progenitor (pluripotent) cells that have the potency to differentiate into all cell types. PGCs are also undifferentiated progenitor cells that later differentiate into male and female germ cells. CEFs are differentiated and abundant somatic cells. Through WTS analysis, we identified that the DNA repair pathway genes were expressed more highly in blastoderms and high in PGCs than CEFs. Besides, the apoptosis pathway genes were expressed low in blastoderms and PGCs than CEFs. We have also examined the WTS-based expression profiling of candidate pluripotency regulating genes due to the conserved properties of blastoderms and PGCs. In the results, a limited number of pluripotency genes, especially the core transcriptional network, were detected higher in both blastoderms and PGCs than CEFs. Next, we treated the CEFs, blastoderm cells, and PGCs with hydrogen peroxide (H2O2) for 1 h to induce DNA damage. Then, the H2O2 treated cells were incubated in fresh media for 3-12 h to observe DNA repair. Subsequent analyses in treated cells found that blastoderm cells and PGCs were more likely to undergo apoptosis along with the loss of pluripotency and less likely to undergo DNA repair, contrasting with CEFs. These properties of blastoderms and PGCs should be necessary to preserve genome stability during the development of early embryos and germ cells, respectively.
Asunto(s)
Apoptosis/genética , Blastodermo/metabolismo , Pollos/genética , Reparación del ADN/genética , Inestabilidad Genómica/fisiología , Células Germinativas/metabolismo , Animales , Embrión de Pollo , Daño del ADN/efectos de los fármacos , Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Peróxido de Hidrógeno/farmacología , Células Madre Pluripotentes/metabolismo , Transcriptoma , Secuenciación del ExomaRESUMEN
Maintenance of genome integrity in the germline and in preimplantation embryos is crucial for mammalian development. Epigenetic remodeling during primordial germ cell (PGC) and preimplantation embryo development may contribute to genomic instability in these cells, since DNA methylation is an important mechanism to silence retrotransposons. Long interspersed elements 1 (LINE-1 or L1) are the most common autonomous retrotransposons in mammals, corresponding to approximately 17% of the human genome. Retrotransposition events are more frequent in germ cells and in early stages of embryo development compared with somatic cells. It has been shown that L1 activation and expression occurs in germline and is essential for preimplantation development. In this review, we focus on the role of L1 retrotransposon in mouse and human germline and early embryo development and discuss the possible relationship between L1 expression and genomic instability during these stages. Although several studies have addressed L1 expression at different stages of development, the developmental consequences of this expression remain poorly understood. Future research is still needed to highlight the relationship between L1 retrotransposition events and genomic instability during germline and early embryo development.
Asunto(s)
Desarrollo Embrionario/efectos de los fármacos , Inestabilidad Genómica , Células Germinativas , Elementos de Nucleótido Esparcido Largo , Animales , Regulación del Desarrollo de la Expresión Génica , Inestabilidad Genómica/genética , Inestabilidad Genómica/fisiología , Células Germinativas/metabolismo , Células Germinativas/fisiología , Humanos , Elementos de Nucleótido Esparcido Largo/fisiología , RatonesRESUMEN
Germline mutations underlie genetic diversity and species evolution. Previous studies have assessed the theoretical mutation rates and spectra in germ cells mostly by analyzing genetic markers and reporter genes in populations and pedigrees. This study reported the direct measurement of germline mutations by whole-genome sequencing of cultured spermatogonial stem cells in mice, namely germline stem (GS) cells, together with multipotent GS (mGS) cells that spontaneously dedifferentiated from GS cells. GS cells produce functional sperm that can generate offspring by transplantation into seminiferous tubules, whereas mGS cells contribute to germline chimeras by microinjection into blastocysts in a manner similar to embryonic stem cells. The estimated mutation rate of GS and mGS cells was approximately 0.22 × 10-9 and 1.0 × 10-9 per base per cell population doubling, respectively, indicating that GS cells have a lower mutation rate compared to mGS cells. GS and mGS cells also showed distinct mutation patterns, with C-to-T transition as the most frequent in GS cells and C-to-A transversion as the most predominant in mGS cells. By karyotype analysis, GS cells showed recurrent trisomy of chromosomes 15 and 16, whereas mGS cells frequently exhibited chromosomes 1, 6, 8, and 11 amplifications, suggesting that distinct chromosomal abnormalities confer a selective growth advantage for each cell type in vitro. These data provide the basis for studying germline mutations and a foundation for the future utilization of GS cells for reproductive technology and clinical applications.
Asunto(s)
Células Madre Embrionarias/metabolismo , Inestabilidad Genómica/fisiología , Animales , Quimera/metabolismo , Biología Computacional , Células Madre Embrionarias/citología , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , Mutación , Especies Reactivas de Oxígeno/metabolismo , Túbulos Seminíferos/metabolismo , Espermatogonias/citología , EspermatozoidesRESUMEN
Transposable elements (TEs) initially attracted attention because they comprise a major portion of the genomic sequences in plants and animals. TEs may jump around the genome and disrupt both coding genes as well as regulatory sequences to cause disease. Host cells have therefore evolved various epigenetic and functional RNA-mediated mechanisms to mitigate the disruption of genomic integrity by TEs. TE associated sequences therefore acquire the tendencies of attracting various epigenetic modifiers to induce epigenetic alterations that may spread to the neighboring genes. In addition to posting threats for (epi)genome integrity, emerging evidence suggested the physiological importance of endogenous TEs either as cis-acting control elements for controlling gene regulation or as TE-containing functional transcripts that modulate the transcriptome of the host cells. Recent advances in long-reads sequence analysis technologies, bioinformatics and genetic editing tools have enabled the profiling, precise annotation and functional characterization of TEs despite their challenging repetitive nature. The importance of specific TEs in preimplantation embryonic development, germ cell differentiation and meiosis, cell fate determination and in driving species specific differences in mammals will be discussed.
Asunto(s)
Elementos Transponibles de ADN/fisiología , Epigénesis Genética/fisiología , Regulación de la Expresión Génica/fisiología , Inestabilidad Genómica/fisiología , Animales , HumanosRESUMEN
Proteins containing breast cancer type 1 (BRCA1) C-terminal domains play crucial roles in response to and repair of DNA damage. Epithelial cell transforming factor (epithelial cell transforming sequence 2 [ECT2]) is a member of the BRCA1 C-terminal protein family, but it is not known if ECT2 directly contributes to DNA repair. In this study, we report that ECT2 is recruited to DNA lesions in a poly (ADP-ribose) polymerase 1-dependent manner. Using co-immunoprecipitation analysis, we showed that ECT2 physically associates with KU70-KU80 and BRCA1, proteins involved in nonhomologous end joining and homologous recombination, respectively. ECT2 deficiency impairs the recruitment of KU70 and BRCA1 to DNA damage sites, resulting in defective DNA double-strand break repair, an accumulation of damaged DNA, and hypersensitivity of cells to genotoxic insults. Interestingly, we demonstrated that ECT2 promotes DNA repair and genome integrity largely independently of its canonical guanine nucleotide exchange activity. Together, these results suggest that ECT2 is directly involved in DNA double-strand break repair and is an important genome caretaker.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN/fisiología , Inestabilidad Genómica/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteína BRCA1/metabolismo , Células HeLa , Recombinación Homóloga , Humanos , Autoantígeno Ku/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Proteínas Proto-Oncogénicas/metabolismoRESUMEN
The genome of living organisms frequently undergoes various types of modifications which are recognized and repaired by the relevant repair mechanisms. These repair pathways are increasingly being deciphered to understand the mechanisms. Base excision repair (BER) is indispensable to maintain genome stability. One of the enigmatic repair proteins of BER, Apurinic/Apyrimidinic Endonuclease 2 (APE2), like APE1, is truly multifunctional and demonstrates the independent and non-redundant function in maintaining the genome integrity. APE2 is involved in ATR-Chk1 mediated DNA damage response. It also resolves topoisomerase1 mediated cleavage complex intermediate which is formed while repairing misincorporated ribonucleotides in the absence of functional RNase H2 mediated excision repair pathway. BER participates in the demethylation pathway and the role of Arabidopsis thaliana APE2 is demonstrated in this process. Moreover, APE2 is synthetically lethal to BRCA1, BRCA2, and RNase H2, and its homolog, APE1 fails to complement the function. Hence, the role of APE2 is not just an alternate to the repair mechanisms but has implications in diverse functional pathways related to the maintenance of genome integrity. This review analyses genomic features of APE2 and delineates its enzyme function as error-prone as well as efficient and accurate repair protein based on the studies on mammalian or its homolog proteins from model systems such as Arabidopsis thaliana, Schizosaccharomyces pombe, Trypanosoma curzi, Xenopus laevis, Danio rerio, Mus musculus, and Homo sapiens.
Asunto(s)
Reparación del ADN/fisiología , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Inestabilidad Genómica/fisiología , Animales , Variaciones en el Número de Copia de ADN , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , Dosificación de Gen , Humanos , Mutación Puntual , Especificidad por SustratoRESUMEN
BACKGROUND/OBJECTIVES: Epidemiological evidence indicates obesity in childhood and adolescence to be an independent risk factor for cancer and premature mortality in adulthood. Pathological implications from excess adiposity may begin early in life. Obesity is concurrent with a state of chronic inflammation, a well-known aetiological factor for DNA damage. In addition, obesity has been associated with micro-nutritional deficiencies. Vitamin D has attracted attention for its anti-inflammatory properties and role in genomic integrity and stability. The aim of this study was to determine a novel approach for predicting genomic instability via the combined assessment of adiposity, DNA damage, systemic inflammation, and vitamin D status. SUBJECTS/METHODS: We carried out a cross-sectional study with 132 participants, aged 10-18, recruited from schools and paediatric obesity clinics in London. Anthropometric assessments included BMI Z-score, waist and hip circumference, and body fat percentage via bioelectrical impedance. Inflammation and vitamin D levels in saliva were assessed by enzyme-linked immunosorbent assay. Oxidative DNA damage was determined via quantification of 8-hydroxy-2'-deoxyguanosine in urine. Exfoliated cells from the oral cavity were scored for genomic instability via the buccal cytome assay. RESULTS: As expected, comparisons between participants with obesity and normal range BMI showed significant differences in anthropometric measures (p < 0.001). Significant differences were also observed in some measures of genomic instability (p < 0.001). When examining relationships between variables for all participants, markers of adiposity positively correlated with acquired oxidative DNA damage (p < 0.01) and genomic instability (p < 0.001), and negatively correlated with vitamin D (p < 0.01). Multiple regression analyses identified obesity (p < 0.001), vitamin D (p < 0.001), and oxidative DNA damage (p < 0.05) as the three significant predictors of genomic instability. CONCLUSIONS: Obesity, oxidative DNA damage, and vitamin D deficiency are significant predictors of genomic instability. Non-invasive biomonitoring and predictive modelling of genomic instability in young patients with obesity may contribute to the prioritisation and severity of clinical intervention measures.
Asunto(s)
Estrés Oxidativo/efectos de los fármacos , Obesidad Infantil/genética , Deficiencia de Vitamina D/complicaciones , Vitamina D/análisis , Adolescente , Índice de Masa Corporal , Distribución de Chi-Cuadrado , Niño , Estudios Transversales , Femenino , Inestabilidad Genómica/genética , Inestabilidad Genómica/fisiología , Humanos , Londres/epidemiología , Masculino , Obesidad Infantil/complicaciones , Obesidad Infantil/epidemiología , Medicina Estatal , Vitamina D/sangre , Deficiencia de Vitamina D/genética , Deficiencia de Vitamina D/fisiopatologíaRESUMEN
Redox homeostasis is imperative to maintain normal physiologic and metabolic functions. Radiotherapy disturbs this balance and induces genomic instability in diseased cells. However, radiation-induced effects propagate beyond the targeted cells, affecting the adjacent non-targeted cells (bystander effects). The cellular impact of radiation, thus, encompasses both targeted and non-targeted effects. Use of external modulators along with radiation can increase radio-therapeutic efficiency. The modulators' classification as protectors or sensitizers depends on interactions with damaged DNA molecules. Thus, it is necessary to realize the functions of various radio-sensitizers or radio-protectors in both irradiated and bystander cells. This review focuses on some modulators of radiation-induced bystander effects (RIBE) and their action mechanisms. Knowledge about the underlying signaling cross-talk may promote selective sensitization of radiation-targeted cells and protection of bystander cells.
Asunto(s)
Inestabilidad Genómica/fisiología , Animales , Efecto Espectador , Inestabilidad Genómica/genética , Homeostasis/genética , Homeostasis/fisiología , Humanos , Oxidación-Reducción , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
DNA replication stress is a major source of DNA damage, including double-stranded breaks that promote DNA damage response (DDR) signaling. Inefficient repair of such lesions can affect genome integrity. During DNA replication different factors act on chromatin remodeling in a coordinated way. While recent studies have highlighted individual molecular mechanisms of interaction, less is known about the orchestration of chromatin changes under replication stress. In this review we attempt to explore the complex relationship between DNA replication stress, DDR and genome integrity in mammalian cells, taking into account the role of chromatin disposition as an important modulator of DNA repair. Recent data on chromatin restoration and epigenetic re-establishment after DNA replication stress are reviewed.
Asunto(s)
Daño del ADN/fisiología , Replicación del ADN/fisiología , Inestabilidad Genómica/fisiología , Animales , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/genética , Ensamble y Desensamble de Cromatina/fisiología , Daño del ADN/genética , Replicación del ADN/genética , Inestabilidad Genómica/genética , HumanosRESUMEN
Despite being an important diagnostic and treatment modality, ionizing radiation (IR) is also known to cause genotoxicity and multiple side effects leading to secondary carcinogenesis. While modern cancer radiation therapy has improved patient recovery and enhanced survival rates, the risk of radiation-related adverse effects has become a growing challenge. It is now well-accepted that IR-induced side effects are not exclusively restricted to exposed cells but also spread to distant 'bystander' cells and even to the unexposed progeny of the irradiated cells. These 'off-targeted' effects involve a plethora of molecular events depending on the type of radiation and tumor tissue background. While the mechanisms by which off-targeted effects arise remain obscure, emerging evidence based on the non-mendelian inheritance of various manifestations of them as well as their persistence for longer periods supports a contribution of epigenetic factors. This review focuses on the major epigenetic phenomena including DNA methylation, histone modifications, and small RNA mediated silencing and their versatile role in the manifestation of IR induced off-targeted effects. As short- and long-range communication vehicles respectively, the role of gap junctions and exosomes in spreading these epigenetic-alteration driven off-targeted effects is also discussed. Furthermore, this review emphasizes the possible therapeutic potentials of these epigenetic mechanisms and how beneficial outcomes could potentially be achieved by targeting various signaling molecules involved in these mechanisms.
Asunto(s)
Epigénesis Genética/genética , Efecto Espectador/genética , Efecto Espectador/fisiología , Metilación de ADN/genética , Metilación de ADN/fisiología , Inestabilidad Genómica/genética , Inestabilidad Genómica/fisiología , Humanos , MicroARNs/genética , MicroARNs/metabolismoRESUMEN
Vigilin (Vgl1) is essential for heterochromatin formation, chromosome segregation, and mRNA stability and is associated with autism spectrum disorders and cancer: vigilin, for example, can suppress proto-oncogene c-fms expression in breast cancer. Conserved from yeast to humans, vigilin is an RNA-binding protein with 14 tandemly arranged nonidentical hnRNP K-type homology (KH) domains. Here, we report that vigilin depletion increased cell sensitivity to cisplatin- or ionizing radiation (IR)-induced cell death and genomic instability due to defective DNA repair. Vigilin depletion delayed dephosphorylation of IR-induced γ-H2AX and elevated levels of residual 53BP1 and RIF1 foci, while reducing Rad51 and BRCA1 focus formation, DNA end resection, and double-strand break (DSB) repair. We show that vigilin interacts with the DNA damage response (DDR) proteins RAD51 and BRCA1, and vigilin depletion impairs their recruitment to DSB sites. Transient hydroxyurea (HU)-induced replicative stress in vigilin-depleted cells increased replication fork stalling and blocked restart of DNA synthesis. Furthermore, histone acetylation promoted vigilin recruitment to DSBs preferentially in the transcriptionally active genome. These findings uncover a novel vigilin role in DNA damage repair with implications for autism and cancer-related disorders.
Asunto(s)
Trastorno Autístico/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Inestabilidad Genómica/fisiología , Proteína BRCA1 , Reparación del ADN/fisiología , Replicación del ADN/genética , Inestabilidad Genómica/genética , Humanos , Proto-Oncogenes Mas , Proteínas de Unión al ARN/metabolismo , Recombinasa Rad51/genéticaRESUMEN
In cultivated tetraploid potato (Solanum tuberosum), reduction to diploidy (dihaploidy) allows for hybridization to diploids and introgression breeding and may facilitate the production of inbreds. Pollination with haploid inducers (HIs) yields maternal dihaploids, as well as triploid and tetraploid hybrids. Dihaploids may result from parthenogenesis, entailing the development of embryos from unfertilized eggs, or genome elimination, entailing missegregation and the loss of paternal chromosomes. A sign of genome elimination is the occasional persistence of HI DNA in some dihaploids. We characterized the genomes of 919 putative dihaploids and 134 hybrids produced by pollinating tetraploid clones with three HIs: IVP35, IVP101, and PL-4. Whole-chromosome or segmental aneuploidy was observed in 76 dihaploids, with karyotypes ranging from 2n = 2x - 1 = 23 to 2n = 2x + 3 = 27. Of the additional chromosomes in 74 aneuploids, 66 were from the non-inducer parent and 8 from the inducer parent. Overall, we detected full or partial chromosomes from the HI parent in 0.87% of the dihaploids, irrespective of parental genotypes. Chromosomal breaks commonly affected the paternal genome in the dihaploid and tetraploid progeny, but not in the triploid progeny, correlating instability to sperm ploidy and to haploid induction. The residual HI DNA discovered in the progeny is consistent with genome elimination as the mechanism of haploid induction.
Asunto(s)
ADN/metabolismo , Solanum tuberosum/genética , Inestabilidad Genómica/genética , Inestabilidad Genómica/fisiología , Genotipo , Haploidia , PoliploidíaRESUMEN
Colorectal cancer is known to be the paramount reason for cancer deaths around the globe. It occurs due to the aggregation of epigenetic and genetic alterations in colon epithelial cells that transmute them into adenocarcinomas. Epigenetic mechanisms are interpreted as the changes in expression of the gene which is not associated with the alterations in the principal DNA sequence, while genetic changes involve modifications in oncogenes and tumor suppressor genes. The changes in the epigenetic in colon cancer that transmute colonic epithelial cells include chromatin modifications, microRNA expression, telomere length, and DNA methylation. DNA hypermethylation causes down-regulation and unsuitable expression of specific microRNA which can behave like tumor suppressor genes. Histone modifications can also influence the chromatin remodeling and gene expression, hence performs an eminent function in the silencing of the gene in colon cancer. Moreover, the telomere/telomerase interaction is a prime mechanism to embrace both cellular replicative potential and genomic instability and its malfunction plays a primary role in colon cancer. Deducing the genesis and the function of epigenetic abnormality in colon cancer pathogenesis will lead to potent prevention and therapeutic approach for colon cancer patients. Epigenetic drugs which emphasize the convertible essence of the epigenetic occurrences have accompanied the probability of epigenetic approach as a treatment alternative in colon cancer. Hence, this review is undertaken to critically envelop the recently advanced events in colorectal cancer therapies with a special emphasis on remedies targeting epigenetic modulators and future challenges towards therapeutic interventions.
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Neoplasias Colorrectales/terapia , Epigénesis Genética/fisiología , Terapias en Investigación/métodos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Metilación de ADN/fisiología , Regulación Neoplásica de la Expresión Génica , Inestabilidad Genómica/fisiología , Humanos , Terapias en Investigación/tendenciasRESUMEN
Histone chaperones mediate the assembly and disassembly of nucleosomes and participate in essentially all DNA-dependent cellular processes. In Arabidopsis thaliana, loss-of-function of FAS1 or FAS2 subunits of the H3-H4 histone chaperone complex CHROMATIN ASSEMBLY FACTOR 1 (CAF-1) has a dramatic effect on plant morphology, growth and overall fitness. CAF-1 dysfunction can lead to altered chromatin compaction, systematic loss of repetitive elements or increased DNA damage, clearly demonstrating its severity. How chromatin composition is maintained without functional CAF-1 remains elusive. Here we show that disruption of the H2A-H2B histone chaperone NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1) suppresses the FAS1 loss-of-function phenotype. The quadruple mutant fas1 nap1;1 nap1;2 nap1;3 shows wild-type growth, decreased sensitivity to genotoxic stress and suppression of telomere and 45S rDNA loss. Chromatin of fas1 nap1;1 nap1;2 nap1;3 plants is less accessible to micrococcal nuclease and the nuclear H3.1 and H3.3 histone pools change compared to fas1. Consistently, association between NAP1 and H3 occurs in the cytoplasm and nucleus in vivo in protoplasts. Altogether we show that NAP1 proteins play an essential role in DNA repair in fas1, which is coupled to nucleosome assembly through modulation of H3 levels in the nucleus.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cromatina/genética , Cromatina/metabolismo , Adenosina Trifosfatasas/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Inestabilidad Genómica/genética , Inestabilidad Genómica/fisiología , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Mutación/genéticaRESUMEN
Protecting replication fork integrity during DNA replication is essential for maintaining genome stability. Here, we report that SDE2, a PCNA-associated protein, plays a key role in maintaining active replication and counteracting replication stress by regulating the replication fork protection complex (FPC). SDE2 directly interacts with the FPC component TIMELESS (TIM) and enhances its stability, thereby aiding TIM localization to replication forks and the coordination of replisome progression. Like TIM deficiency, knockdown of SDE2 leads to impaired fork progression and stalled fork recovery, along with a failure to activate CHK1 phosphorylation. Moreover, loss of SDE2 or TIM results in an excessive MRE11-dependent degradation of reversed forks. Together, our study uncovers an essential role for SDE2 in maintaining genomic integrity by stabilizing the FPC and describes a new role for TIM in protecting stalled replication forks. We propose that TIM-mediated fork protection may represent a way to cooperate with BRCA-dependent fork stabilization.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Replicación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Ciclo Celular/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Estructuras Cromosómicas/metabolismo , Daño del ADN , Reparación del ADN , Replicación del ADN/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Inestabilidad Genómica/fisiología , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteína Homóloga de MRE11/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación , Dominios ProteicosRESUMEN
Persistent R-loops (RNA-DNA hybrids with a displaced single-stranded DNA) create DNA damage and lead to genomic instability. The 5'-3'-exoribonuclease 2 (XRN2) degrades RNA to resolve R-loops and promotes transcription termination. Previously, XRN2 was implicated in DNA double strand break (DSB) repair and in resolving replication stress. Here, using tandem affinity purification-mass spectrometry, bioinformatics, and biochemical approaches, we found that XRN2 associates with proteins involved in DNA repair/replication (Ku70-Ku80, DNA-PKcs, PARP1, MCM2-7, PCNA, RPA1) and RNA metabolism (RNA helicases, PRP19, p54(nrb), splicing factors). Novel major pathways linked to XRN2 include cell cycle control of chromosomal replication and DSB repair by non-homologous end joining. Investigating the biological implications of these interactions led us to discover that XRN2 depletion compromised cell survival after additional knockdown of specific DNA repair proteins, including PARP1. XRN2-deficient cells also showed enhanced PARP1 activity. Consistent with concurrent depletion of XRN2 and PARP1 promoting cell death, XRN2-deficient fibroblast and lung cancer cells also demonstrated sensitivity to PARP1 inhibition. XRN2 alterations (mutations, copy number/expression changes) are frequent in cancers. Thus, PARP1 inhibition could target cancers exhibiting XRN2 functional loss. Collectively, our data suggest XRN2's association with novel protein partners and unravel synthetic lethality between XRN2 depletion and PARP1 inhibition.
Asunto(s)
Exorribonucleasas/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Estructuras R-Loop/fisiología , Células A549 , Roturas del ADN de Doble Cadena , Daño del ADN/fisiología , Reparación del ADN por Unión de Extremidades/fisiología , Reparación del ADN/fisiología , Replicación del ADN/fisiología , Proteínas de Unión al ADN/genética , Exorribonucleasas/fisiología , Inestabilidad Genómica/fisiología , Células HEK293 , Células HeLa , Humanos , Poli(ADP-Ribosa) Polimerasa-1/fisiología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Estructuras R-Loop/genética , ARN Helicasas/metabolismo , Mutaciones Letales Sintéticas/genéticaRESUMEN
SCOPE: Trace element (TE) deficiencies often occur accumulated, as nutritional intake is inadequate for several TEs, concurrently. Therefore, the impact of a suboptimal supply of iron, zinc, copper, iodine, and selenium on the TE status, health parameters, epigenetics, and genomic stability in mice are studied. METHODS AND RESULTS: Male mice receive reduced or adequate amounts of TEs for 9 weeks. The TE status is analyzed mass-spectrometrically in serum and different tissues. Furthermore, gene and protein expression of TE biomarkers are assessed with focus on liver. Iron concentrations are most sensitive toward a reduced supply indicated by increased serum transferrin levels and altered hepatic expression of iron-related genes. Reduced TE supply results in smaller weight gain but higher spleen and heart weights. Additionally, inflammatory mediators in serum and liver are increased together with hepatic genomic instability. However, global DNA (hydroxy)methylation is unaffected by the TE modulation. CONCLUSION: Despite homeostatic regulation of most TEs in response to a low intake, this condition still has substantial effects on health parameters. It appears that the liver and immune system react particularly sensitive toward changes in TE intake. The reduced Fe status might be the primary driver for the observed effects.
Asunto(s)
Inestabilidad Genómica/efectos de los fármacos , Hígado/efectos de los fármacos , Oligoelementos/análisis , Oligoelementos/farmacología , Animales , Proteína C-Reactiva , Metilación de ADN/efectos de los fármacos , Metilación de ADN/fisiología , Epigénesis Genética , Heces/química , Ferritinas/sangre , Inestabilidad Genómica/fisiología , Glutatión Peroxidasa/sangre , Glutatión Peroxidasa/metabolismo , Inflamación/inmunología , Interleucina-6/sangre , Hígado/metabolismo , Masculino , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/sangre , Distribución Tisular , Transferrina/análisis , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Faithful chromosome segregation is essential for the maintenance of genomic integrity and requires functional centromeres. Centromeres are epigenetically defined by the histone H3 variant, centromere protein A (CENP-A). Here we highlight current knowledge regarding CENP-A-containing chromatin structure, specification of centromere identity, regulation of CENP-A deposition and possible contribution to cancer formation and/or progression. CENP-A overexpression is common among many cancers and predicts poor prognosis. Overexpression of CENP-A increases rates of CENP-A deposition ectopically at sites of high histone turnover, occluding CCCTC-binding factor (CTCF) binding. Ectopic CENP-A deposition leads to mitotic defects, centromere dysfunction and chromosomal instability (CIN), a hallmark of cancer. CENP-A overexpression is often accompanied by overexpression of its chaperone Holliday Junction Recognition Protein (HJURP), leading to epigenetic addiction in which increased levels of HJURP and CENP-A become necessary to support rapidly dividing p53 deficient cancer cells. Alterations in CENP-A posttranslational modifications are also linked to chromosome segregation errors and CIN. Collectively, CENP-A is pivotal to genomic stability through centromere maintenance, perturbation of which can lead to tumorigenesis.