RESUMEN
Induction of DNA damage induces a dynamic repair process involving DNA repair factors and epigenetic regulators. Chromatin alterations must occur for DNA repair factors to gain access to DNA lesions and restore original chromatin configuration to preserve the gene expression profile. We characterize the novel role of CBX8, a chromodomain-containing protein with established roles in epigenetic regulation in DNA damage response. CBX8 protein rapidly accumulates at the sites of DNA damage within 30 s and progresses to accumulate until 4 min before gradually dispersing back to its predamage distribution by 15 min. CBX8 recruitment to the sites of DNA damage is dependent upon PARP1 activation and not dependent on ATM activation. CBX8 biochemically interacts with TRIM33, and its recruitment to DNA damage is also dependent on the presence of TRIM33. Knockdown of CBX8 using siRNA significantly reduces the efficiency of both homologous and the other non-homologous recombination, as well as increases sensitivity of cells to ionizing radiation. These findings demonstrate that CBX8 functions in the PARP-dependent DNA damage response partly through interaction with TRIM33 and is required for efficient DNA repair.
Asunto(s)
Daño del ADN , Complejo Represivo Polycomb 1/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Reparación del ADN , Epigénesis Genética , Humanos , Ratones , Ratones Noqueados , Proteínas de Transporte de Membrana Mitocondrial , Complejo Represivo Polycomb 1/genética , Proteínas del Grupo Polycomb/genética , Unión Proteica , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Activation of poly(ADP-ribose) polymerase (PARP) near sites of DNA breaks facilitates recruitment of DNA repair proteins and promotes chromatin relaxation in part through the action of chromatin-remodeling enzyme Amplified in Liver Cancer 1 (ALC1). Through proteomic analysis we find that ALC1 interacts after DNA damage with Tripartite Motif-containing 33 (TRIM33), a multifunctional protein implicated in transcriptional regulation, TGF-ß signaling, and tumorigenesis. We demonstrate that TRIM33 is dynamically recruited to DNA damage sites in a PARP1- and ALC1-dependent manner. TRIM33-deficient cells show enhanced sensitivity to DNA damage and prolonged retention of ALC1 at sites of DNA breaks. Conversely, overexpression of TRIM33 alleviates the DNA repair defects conferred by ALC1 overexpression. Thus, TRIM33 plays a role in PARP-dependent DNA damage response and regulates ALC1 activity by promoting its timely removal from sites of DNA damage.
Asunto(s)
Roturas del ADN , ADN Helicasas/metabolismo , Reparación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Factores de Transcripción/metabolismo , Animales , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/genética , Proteómica , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Chromatin insulators demarcate expression domains by blocking the cis effects of enhancers or silencers in a position-dependent manner. We show that the chromatin insulator protein CTCF carries a post-translational modification: poly(ADP-ribosyl)ation. Chromatin immunoprecipitation analysis showed that a poly(ADP-ribosyl)ation mark, which exclusively segregates with the maternal allele of the insulator domain in the H19 imprinting control region, requires the bases that are essential for interaction with CTCF. Chromatin immunoprecipitation-on-chip analysis documented that the link between CTCF and poly(ADP-ribosyl)ation extended to more than 140 mouse CTCF target sites. An insulator trap assay showed that the insulator function of most of these CTCF target sites is sensitive to 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase activity. We suggest that poly(ADP-ribosyl)ation imparts chromatin insulator properties to CTCF at both imprinted and nonimprinted loci, which has implications for the regulation of expression domains and their demise in pathological lesions.
Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Proteínas Represoras/metabolismo , Animales , Factor de Unión a CCCTC , Cromatina/genética , Proteínas de Unión al ADN/genética , Epigénesis Genética , Femenino , Regulación de la Expresión Génica , Impresión Genómica , Humanos , Factor II del Crecimiento Similar a la Insulina/genética , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Procesamiento Proteico-Postraduccional , ARN Largo no Codificante , ARN no Traducido/genética , Proteínas Represoras/genética , Transcripción GenéticaRESUMEN
EHTM1 (GLP) and EHMT2 (G9a) are closely related protein lysine methyltransferases often thought to function together as a heterodimer to methylate histone H3 and non-histone substrates in diverse cellular processes including transcriptional regulation, genome methylation, and DNA repair. Here we show that EHMT1/2 inhibitors cause ATM-mediated slowdown of replication fork progression, accumulation of single-stranded replication gaps, emergence of cytosolic DNA, and increased expression of STING. EHMT1/2 inhibition strongly potentiates the efficacy of alkylating chemotherapy and anti-PD-1 immunotherapy in mouse models of tripe negative breast cancer. The effects on DNA replication and alkylating agent sensitivity are largely caused by the loss of EHMT1-mediated methylation of LIG1, whereas the elevated STING expression and remarkable response to immunotherapy appear mainly elicited by the loss of EHMT2 activity. Depletion of UHRF1, a protein known to be associated with EHMT1/2 and LIG1, also induces STING expression, and depletion of either EHMT2 or UHRF1 leads to demethylation of specific CpG sites in the STING1 promoter, suggestive of a distinct EHMT2-UHRF1 axis that regulates DNA methylation and gene transcription. These results highlight distinct functions of the two EHMT paralogs and provide enlightening paradigms and corresponding molecular basis for combination therapies involving alkylating agents and immune checkpoint inhibitors.
RESUMEN
Upon induction of DNA breaks, ATM activation leads to a cascade of local chromatin modifications that promote efficient recruitment of DNA repair proteins. Errors in this DNA repair pathway lead to genomic instability and cancer predisposition. Here, we show that the protein lysine methyltransferase G9a (also known as EHMT2) and GLP1 (also known as EHMT1) are critical components of the DNA repair pathway. G9a and GLP1 rapidly localizes to DNA breaks, with GLP1 localization being dependent on G9a. ATM phosphorylation of G9a on serine 569 is required for its recruitment to DNA breaks. G9a catalytic activity is required for the early recruitment of DNA repair factors including 53BP and BRCA1 to DNA breaks. Inhibition of G9a catalytic activity disrupts DNA repair pathways and increases sensitivity to ionizing radiation. Thus, G9a is a potential therapeutic target in the DNA repair pathway.
Asunto(s)
Daño del ADN , Péptido 1 Similar al Glucagón/metabolismo , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Línea Celular , Reparación del ADN , Antígenos de Histocompatibilidad/genética , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Modelos Biológicos , Fosforilación , Unión Proteica , Transporte de Proteínas , Proteína 1 de Unión al Supresor Tumoral P53/metabolismoRESUMEN
Ionizing radiation generates a broad spectrum of oxidative DNA lesions, including oxidized base products, abasic sites, single-strand breaks and double-strand breaks. The CUX1 protein was recently shown to function as an auxiliary factor that stimulates enzymatic activities of OGG1 through its CUT domains. In the present study, we investigated the requirement for CUX1 and OGG1 in the resistance to radiation. Cancer cell survival following ionizing radiation is reduced by CUX1 knockdown and increased by higher CUX1 expression. However, CUX1 knockdown is sufficient by itself to reduce viability in many cancer cell lines that exhibit high levels of reactive oxygen species (ROS). Consequently, clonogenic results expressed relative to that of non-irradiated cells indicate that CUX1 knockdown confers no or modest radiosensitivity to cancer cells with high ROS. A recombinant protein containing only two CUT domains is sufficient for rapid recruitment to DNA damage, acceleration of DNA repair and increased survival following radiation. In agreement with these findings, OGG1 knockdown and treatment of cells with OGG1 inhibitors sensitize cancer cells to radiation. Together, these results validate CUX1 and more specifically the CUT domains as therapeutic targets.
Asunto(s)
Reparación del ADN/genética , Proteínas de Homeodominio/metabolismo , Proteínas Nucleares/metabolismo , Tolerancia a Radiación/genética , Proteínas Represoras/metabolismo , Línea Celular Tumoral , ADN Glicosilasas/metabolismo , Técnicas de Silenciamiento del Gen , Proteínas de Homeodominio/genética , Humanos , Immunoblotting , Microscopía Confocal , Proteínas Nucleares/genética , Reacción en Cadena de la Polimerasa , Especies Reactivas de Oxígeno/metabolismo , Proteínas Represoras/genética , Factores de TranscripciónRESUMEN
Prostate cancer (PCa) is a major lethal malignancy in men, but the molecular events and their interplay underlying prostate carcinogenesis remain poorly understood. Epigenetic events and the upregulation of polycomb group silencing proteins including Bmi1 have been described to occur during PCa progression. Here, we found that conditional overexpression of Bmi1 in mice induced prostatic intraepithelial neoplasia, and elicited invasive adenocarcinoma when combined with PTEN haploinsufficiency. In addition, Bmi1 and the PI3K/Akt pathway were coactivated in a substantial fraction of human high-grade tumors. We found that Akt mediated Bmi1 phosphorylation, enhancing its oncogenic potential in an Ink4a/Arf-independent manner. This process also modulated the DNA damage response and affected genomic stability. Together, our findings demonstrate the etiological role of Bmi1 in PCa, unravel an oncogenic collaboration between Bmi1 and the PI3K/Akt pathway, and provide mechanistic insights into the modulation of Bmi1 function by phosphorylation during prostate carcinogenesis.
Asunto(s)
Adenocarcinoma/metabolismo , Reparación del ADN , Proteínas Nucleares/metabolismo , Neoplasia Intraepitelial Prostática/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Adenocarcinoma/enzimología , Adenocarcinoma/patología , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica , Roturas del ADN de Doble Cadena , Activación Enzimática , Inestabilidad Genómica , Haploinsuficiencia , Histonas/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Ratones Transgénicos , Clasificación del Tumor , Trasplante de Neoplasias , Proteínas Nucleares/genética , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Fosforilación , Complejo Represivo Polycomb 1 , Próstata/metabolismo , Próstata/patología , Neoplasia Intraepitelial Prostática/enzimología , Neoplasia Intraepitelial Prostática/patología , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Transducción de Señal , UbiquitinaciónRESUMEN
DNA damage activates signaling pathways that lead to modification of local chromatin and recruitment of DNA repair proteins. Multiple DNA repair proteins having ubiquitin ligase activity are recruited to sites of DNA damage, where they ubiquitinate histones and other substrates. This DNA damage-induced histone ubiquitination is thought to play a critical role in mediating the DNA damage response. We now report that the polycomb protein BMI1 is rapidly recruited to sites of DNA damage, where it persists for more than 8 h. The sustained localization of BMI1 to damage sites is dependent on intact ATM and ATR and requires H2AX phosphorylation and recruitment of RNF8. BMI1 is required for DNA damage-induced ubiquitination of histone H2A at lysine 119. Loss of BMI1 leads to impaired repair of DNA double-strand breaks by homologous recombination and the accumulation of cells in G(2)/M. These data support a crucial role for BMI1 in the cellular response to DNA damage.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Histonas/metabolismo , Proteínas Nucleares , Proteínas Proto-Oncogénicas , Proteínas Represoras , Proteínas de la Ataxia Telangiectasia Mutada , Western Blotting , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/metabolismo , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , Complejo Represivo Polycomb 1 , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Interferente Pequeño , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Normal cellular behavior can be described as a complex, regulated network of interaction between genes and proteins. Targeted cancer therapies aim to neutralize specific proteins that are necessary for the cancer cell to remain viable in vivo. Ideally, the proteins targeted should be such that their downregulation has a major impact on the survival/fitness of the tumor cells and, at the same time, has a smaller effect on normal cells. It is difficult to use standard analysis methods on gene or protein expression levels to identify these targets because the level thresholds for tumorigenic behavior are different for different genes/proteins. We have developed a novel methodology to identify therapeutic targets by using a new paradigm called "gene centrality." The main idea is that, in addition to being overexpressed, good therapeutic targets should have a high degree of connectivity in the tumor network because one expects that suppression of its expression would affect many other genes. We propose a mathematical quantity called "centrality," which measures the degree of connectivity of genes in a network in which each edge is weighted by the expression level of the target gene. Using our method, we found that several SRC proto-oncogenes LYN, YES1, HCK, FYN, and LCK have high centrality in identifiable subsets of basal-like and HER2+ breast cancers. To experimentally validate the clinical value of this finding, we evaluated the effect of YES1 knockdown in basal-like breast cancer cell lines that overexpress this gene. We found that YES1 downregulation has a significant effect on the survival of these cell lines. Our results identify YES1 as a target for therapeutics in a subset of basal-like breast cancers.
RESUMEN
The 5'-flank of the H19 gene harbors a differentially methylated imprinting control region that represses the maternally derived Igf2 and paternally derived H19 alleles. Here we show that the H19 imprinting control region (ICR) is a potent silencer when positioned in a promoter-proximal position. The silencing effect is not alleviated by trichostatin A treatment, suggesting that it does not involve histone deacetylase functions. When the H19 ICR is separated from the promoter by more than 1.2 +/- 0.3 kb, however, trichostatin A stimulates promoter activity 10-fold. Deletion analyses revealed that the silencing feature extended throughout the ICR segment. Finally, chromatin immunopurification analyses revealed that the H19 ICR prevented trichostatin A-dependent reacetylation of histones in the promoter region in a proximal but not in a distal position. We argue that these features are likely to be side effects of the H19 ICR, rather than explaining the mechanism of silencing of the paternal H19 allele. We issue a cautionary note, therefore, that the interpretation of insulator/silencer data could be erroneous should the distance issue not be taken into consideration.
Asunto(s)
Silenciador del Gen , Antifúngicos/farmacología , Sitios de Unión , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Coriocarcinoma , Metilación de ADN , Cartilla de ADN , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Impresión Genómica , Histonas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Reacción en Cadena de la Polimerasa , Embarazo , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Largo no Codificante , ARN no Traducido/genética , Transfección , Células Tumorales Cultivadas , Neoplasias UterinasRESUMEN
All known vertebrate chromatin insulators interact with the highly conserved, multivalent 11-zinc finger nuclear factor CTCF to demarcate expression domains by blocking enhancer or silencer signals in a position-dependent manner. Recent observations document that the properties of CTCF include reading and propagating the epigenetic state of the differentially methylated H19 imprinting control region. To assess whether these findings may reflect a universal role for CTCF targets, we identified more than 200 new CTCF target sites by generating DNA microarrays of clones derived from chromatin-immunopurified (ChIP) DNA followed by ChIP-on-chip hybridization analysis. Target sites include not only known loci involved in multiple cellular functions, such as metabolism, neurogenesis, growth, apoptosis, and signalling, but potentially also heterochromatic sequences. Using a novel insulator trapping assay, we also show that the majority of these targets manifest insulator functions with a continuous distribution of stringency. As these targets are generally DNA methylation-free as determined by antibodies against 5-methylcytidine and a methyl-binding protein (MBD2), a CTCF-based network correlates with genome-wide epigenetic states.