RESUMO
Obesity is a major risk factor for cancer. Conventional thought suggests that elevated adiposity predisposes to heightened inflammatory stress and potentiates tumor growth, yet underlying mechanisms remain ill-defined. Here, we show that tumors from patients with a body mass index >35 carry a high burden of senescent cells. In mouse syngeneic tumor models, we correlated a pronounced accretion of senescent cancer cells with poorly immunogenic tumors when mice were subjected to diet-induced obesity (DIO). Highly immunogenic tumors showed lesser senescence burden suggesting immune-mediated elimination of senescent cancer cells, likely targeted as a consequence of their senescence-associated secretory phenotype. Treatment with the senolytic BH3 mimetic small molecule inhibitor ABT-263 selectively stalled tumor growth in mice with DIO to rates comparable to regular diet-fed mice. Thus, consideration of body adiposity in the selection of cancer therapy may be a critical determinant for disease outcome in poorly immunogenic malignancies.
Assuntos
Senescência Celular , Neoplasias , Camundongos , Animais , Obesidade/complicaçõesRESUMO
Helix-destabilizing DNA lesions induced by environmental mutagens such as UV light cause genomic instability by strongly blocking the progression of DNA replication forks (RFs). At blocked RF, single-stranded DNA (ssDNA) accumulates and is rapidly bound by Replication Protein A (RPA) complexes. Such stretches of RPA-ssDNA constitute platforms for recruitment/activation of critical factors that promote DNA synthesis restart. However, during periods of severe replicative stress, RPA availability may become limiting due to inordinate sequestration of this multifunctional complex on ssDNA, thereby negatively impacting multiple vital RPA-dependent processes. Here, we performed a genome-wide screen to identify factors that restrict the accumulation of RPA-ssDNA during UV-induced replicative stress. While this approach revealed some expected "hits" acting in pathways such as nucleotide excision repair, translesion DNA synthesis, and the intra-S phase checkpoint, it also identified SCAI, whose role in the replicative stress response was previously unappreciated. Upon UV exposure, SCAI knock-down caused elevated accumulation of RPA-ssDNA during S phase, accompanied by reduced cell survival and compromised RF progression. These effects were independent of the previously reported role of SCAI in 53BP1-dependent DNA double-strand break repair. We also found that SCAI is recruited to UV-damaged chromatin and that its depletion promotes nascent DNA degradation at stalled RF. Finally, we (i) provide evidence that EXO1 is the major nuclease underlying ssDNA formation and DNA replication defects in SCAI knockout cells and, consistent with this, (ii) demonstrate that SCAI inhibits EXO1 activity on a ssDNA gap in vitro. Taken together, our data establish SCAI as a novel regulator of the UV-induced replicative stress response in human cells.
Assuntos
DNA de Cadeia Simples , Proteína de Replicação A , Humanos , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , DNA de Cadeia Simples/genética , Raios Ultravioleta/efeitos adversos , Replicação do DNA/genética , Cromatina , DNA , MutagênicosRESUMO
Nucleotide excision repair (NER) eliminates highly genotoxic solar UV-induced DNA photoproducts that otherwise stimulate malignant melanoma development. Here, a genome-wide loss-of-function screen, coupling CRISPR/Cas9 technology with a flow cytometry-based DNA repair assay, was used to identify novel genes required for efficient NER in primary human fibroblasts. Interestingly, the screen revealed multiple genes encoding proteins, with no previously known involvement in UV damage repair, that significantly modulate NER uniquely during S phase of the cell cycle. Among these, we further characterized Dyrk1A, a dual specificity kinase that phosphorylates the proto-oncoprotein cyclin D1 on threonine 286 (T286), thereby stimulating its timely cytoplasmic relocalization and proteasomal degradation, which is required for proper regulation of the G1-S phase transition and control of cellular proliferation. We demonstrate that in UV-irradiated HeLa cells, depletion of Dyrk1A leading to overexpression of cyclin D1 causes inhibition of NER uniquely during S phase and reduced cell survival. Consistently, expression/nuclear accumulation of nonphosphorylatable cyclin D1 (T286A) in melanoma cells strongly interferes with S phase NER and enhances cytotoxicity post-UV. Moreover, the negative impact of cyclin D1 (T286A) overexpression on repair is independent of cyclin-dependent kinase activity but requires cyclin D1-dependent upregulation of p21 expression. Our data indicate that inhibition of NER during S phase might represent a previously unappreciated noncanonical mechanism by which oncogenic cyclin D1 fosters melanomagenesis.
Assuntos
Ciclina D1 , Inibidor de Quinase Dependente de Ciclina p21 , Reparo do DNA , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases , Humanos , Ciclina D1/genética , Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA/efeitos da radiação , Células HeLa , Proteínas Tirosina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/efeitos da radiação , Fase S , Fase G1 , Melanoma/genética , Melanoma/patologia , Células Cultivadas , Raios Ultravioleta/efeitos adversos , Carcinogênese/genética , Carcinogênese/patologia , Carcinogênese/efeitos da radiação , Quinases DyrkRESUMO
Cellular adaptation to low oxygen tension triggers primitive pathways that ensure proper cell function. Conditions of hypoxia and low glucose are characteristic of injured tissues and hence successive waves of inflammatory cells must be suited to function under low oxygen tension and metabolic stress. While Hypoxia-Inducible Factor (HIF)-1α has been shown to be essential for the inflammatory response of myeloid cells by regulating the metabolic switch to glycolysis, less is known about how HIF1α is triggered in inflammation. Here, we demonstrate that cells of the innate immune system require activity of the inositol-requiring enzyme 1α (IRE1α/XBP1) axis in order to initiate HIF1α-dependent production of cytokines such as IL1ß, IL6 and VEGF-A. Knockout of either HIF1α or IRE1α in myeloid cells ameliorates vascular phenotypes in a model of retinal pathological angiogenesis driven by sterile inflammation. Thus, pathways associated with ER stress, in partnership with HIF1α, may co-regulate immune adaptation to low oxygen.
Assuntos
Endorribonucleases , Proteínas Serina-Treonina Quinases , Humanos , Proteínas Serina-Treonina Quinases/genética , Hipóxia , Oxigênio/metabolismo , Células Mieloides/metabolismo , Inflamação/metabolismo , Subunidade alfa do Fator 1 Induzível por HipóxiaRESUMO
Production of type I interferons (IFN-I) is a crucial innate immune mechanism against viral infections. IFN-I induction is subject to negative regulation by both viral and cellular factors, but the underlying mechanism remains unclear. We report that the noncanonical NF-κB pathway was stimulated along with innate immune cell differentiation and viral infections and had a vital role in negatively regulating IFN-I induction. Genetic deficiencies in major components of the noncanonical NF-κB pathway caused IFN-I hyperinduction and rendered cells and mice substantially more resistant to viral infection. Noncanonical NF-κB suppressed signal-induced histone modifications at the Ifnb promoter, an action that involved attenuated recruitment of the transcription factor RelA and a histone demethylase, JMJD2A. These findings reveal an unexpected function of the noncanonical NF-κB pathway and highlight an important mechanism regulating antiviral innate immunity.
Assuntos
Imunidade Inata , Interferon Tipo I/biossíntese , NF-kappa B/metabolismo , Viroses/imunologia , Viroses/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Ativação Enzimática , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Histona Desmetilases/metabolismo , Histonas/metabolismo , Imunidade Inata/efeitos dos fármacos , Interferon beta/genética , Interferon beta/metabolismo , Ligantes , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Toll-Like/metabolismo , Fator de Transcrição RelA/metabolismo , Viroses/genética , Quinase Induzida por NF-kappaBRESUMO
Translation plays a crucial role in shaping the proteome during adaptation to various types of stress. A recent study by Gameiro and Struhl identified an inflammatory response which comprises coordination of transcriptional and translational programs, and which appears to be required for recovery from nutrient deprivation.
Assuntos
Nutrientes , Proteoma , Humanos , InflamaçãoRESUMO
Metabolic dysfunction is a major driver of tumorigenesis. The serine/threonine kinase mechanistic target of rapamycin (mTOR) constitutes a key central regulator of metabolic pathways promoting cancer cell proliferation and survival. mTOR activity is regulated by metabolic sensors as well as by numerous factors comprising the phosphatase and tensin homolog/PI3K/AKT canonical pathway, which are often mutated in cancer. However, some cancers displaying constitutively active mTOR do not carry alterations within this canonical pathway, suggesting alternative modes of mTOR regulation. Since DEPTOR, an endogenous inhibitor of mTOR, was previously found to modulate both mTOR complexes 1 and 2, we investigated the different post-translational modification that could affect its inhibitory function. We found that tyrosine (Tyr) 289 phosphorylation of DEPTOR impairs its interaction with mTOR, leading to increased mTOR activation. Using proximity biotinylation assays, we identified SYK (spleen tyrosine kinase) as a kinase involved in DEPTOR Tyr 289 phosphorylation in an ephrin (erythropoietin-producing hepatocellular carcinoma) receptor-dependent manner. Altogether, our work reveals that phosphorylation of Tyr 289 of DEPTOR represents a novel molecular switch involved in the regulation of both mTOR complex 1 and mTOR complex 2.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosforilação , Serina-Treonina Quinases TOR/genética , Tirosina/genética , Tirosina/metabolismoRESUMO
Senescent cells display senescence-associated (SA) phenotypic programs such as stable proliferation arrest (SAPA) and a secretory phenotype (SASP). Senescence-inducing persistent DNA double-strand breaks (pDSBs) cause an immediate DNA damage response (DDR) and SAPA, but the SASP requires days to develop. Here, we show that following the immediate canonical DDR, a delayed chromatin accumulation of the ATM and MRN complexes coincides with the expression of SASP factors. Importantly, histone deacetylase inhibitors (HDACi) trigger SAPA and SASP in the absence of DNA damage. However, HDACi-induced SASP also requires ATM/MRN activities and causes their accumulation on chromatin, revealing a DNA damage-independent, non-canonical DDR activity that underlies SASP maturation. This non-canonical DDR is required for the recruitment of the transcription factor NF-κB on chromatin but not for its nuclear translocation. Non-canonical DDR further does not require ATM kinase activity, suggesting structural ATM functions. We propose that delayed chromatin recruitment of SASP modulators is the result of non-canonical DDR signaling that ensures SASP activation only in the context of senescence and not in response to transient DNA damage-induced proliferation arrest.
Assuntos
Senescência Celular , NF-kappa B , Senescência Celular/genética , Cromatina/genética , Dano ao DNA , NF-kappa B/metabolismo , Transdução de SinaisRESUMO
Diabetic macular edema is a major complication of diabetes resulting in loss of central vision. Although heightened vessel leakiness has been linked to glial and neuronal-derived factors, relatively little is known on the mechanisms by which mature endothelial cells exit from a quiescent state and compromise barrier function. Here we report that endothelial NOTCH1 signaling in mature diabetic retinas contributes to increased vascular permeability. By providing both human and mouse data, we show that NOTCH1 ligands JAGGED1 and DELTA LIKE-4 are up-regulated secondary to hyperglycemia and activate both canonical and rapid noncanonical NOTCH1 pathways that ultimately disrupt endothelial adherens junctions in diabetic retinas by causing dissociation of vascular endothelial-cadherin from ß-catenin. We further demonstrate that neutralization of NOTCH1 ligands prevents diabetes-induced retinal edema. Collectively, these results identify a fundamental process in diabetes-mediated vascular permeability and provide translational rational for targeting the NOTCH pathway (primarily JAGGED1) in conditions characterized by compromised vascular barrier function.
Assuntos
Permeabilidade Capilar , Retinopatia Diabética/patologia , Receptor Notch1/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Proteínas de Ligação ao Cálcio/biossíntese , Ativação Enzimática , Hiperglicemia/metabolismo , Proteína Jagged-1/biossíntese , Camundongos , Óxido Nítrico/biossíntese , Vasos Retinianos/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Quinases da Família src/metabolismoRESUMO
PURPOSE: JARID2, located on chromosome 6p22.3, is a regulator of histone methyltransferase complexes that is expressed in human neurons. So far, 13 individuals sharing clinical features including intellectual disability (ID) were reported with de novo heterozygous deletions in 6p22-p24 encompassing the full length JARID2 gene (OMIM 601594). However, all published individuals to date have a deletion of at least one other adjoining gene, making it difficult to determine if JARID2 is the critical gene responsible for the shared features. We aim to confirm JARID2 as a human disease gene and further elucidate the associated clinical phenotype. METHODS: Chromosome microarray analysis, exome sequencing, and an online matching platform (GeneMatcher) were used to identify individuals with single-nucleotide variants or deletions involving JARID2. RESULTS: We report 16 individuals in 15 families with a deletion or single-nucleotide variant in JARID2. Several of these variants are likely to result in haploinsufficiency due to nonsense-mediated messenger RNA (mRNA) decay. All individuals have developmental delay and/or ID and share some overlapping clinical characteristics such as facial features with those who have larger deletions involving JARID2. CONCLUSION: We report that JARID2 haploinsufficiency leads to a clinically distinct neurodevelopmental syndrome, thus establishing gene-disease validity for the purpose of diagnostic reporting.
Assuntos
Deficiência Intelectual , Transtornos do Neurodesenvolvimento , Haploinsuficiência/genética , Heterozigoto , Humanos , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Transtornos do Neurodesenvolvimento/diagnóstico , Transtornos do Neurodesenvolvimento/genética , Fenótipo , Complexo Repressor Polycomb 2/genética , Síndrome , Sequenciamento do ExomaRESUMO
Constitutive activation of growth factor signaling pathways paradoxically triggers a cell cycle arrest known as cellular senescence. In primary cells expressing oncogenic ras, this mechanism effectively prevents cell transformation. Surprisingly, attenuation of ERK/MAP kinase signaling by genetic inactivation of Erk2, RNAi-mediated knockdown of ERK1 or ERK2, or MEK inhibitors prevented the activation of the senescence mechanism, allowing oncogenic ras to transform primary cells. Mechanistically, ERK-mediated senescence involved the proteasome-dependent degradation of proteins required for cell cycle progression, mitochondrial functions, cell migration, RNA metabolism, and cell signaling. This senescence-associated protein degradation (SAPD) was observed not only in cells expressing ectopic ras, but also in cells that senesced due to short telomeres. Individual RNAi-mediated inactivation of SAPD targets was sufficient to restore senescence in cells transformed by oncogenic ras or trigger senescence in normal cells. Conversely, the anti-senescence viral oncoproteins E1A, E6, and E7 prevented SAPD. In human prostate neoplasms, high levels of phosphorylated ERK were found in benign lesions, correlating with other senescence markers and low levels of STAT3, one of the SAPD targets. We thus identified a mechanism that links aberrant activation of growth signaling pathways and short telomeres to protein degradation and cellular senescence.
Assuntos
Senescência Celular/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Proteólise , Animais , Linhagem Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/enzimologia , Técnicas de Silenciamento de Genes , Humanos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas ras/metabolismoRESUMO
Aberrant expression of noncoding RNAs plays a critical role during tumorigenesis. To uncover novel functions of long non-coding RNA (lncRNA) in lung adenocarcinoma, we used a microarray-based screen identifying LINC00673 with elevated expression in matched tumor versus normal tissue. We report that loss of LINC00673 is sufficient to trigger cellular senescence, a tumor suppressive mechanism associated with permanent cell cycle arrest, both in lung cancer and normal cells in a p53-dependent manner. LINC00673-depleted cells fail to efficiently transit from G1- to S-phase. Using a quantitative proteomics approach, we confirm the modulation of senescence-associated genes as a result of LINC00673 knockdown. In addition, we uncover that depletion of p53 in normal and tumor cells is sufficient to overcome LINC00673-mediated cell cycle arrest and cellular senescence. Furthermore, we report that overexpression of LINC00673 reduces p53 translation and contributes to the bypass of Ras-induced senescence. In summary, our findings highlight LINC00673 as a crucial regulator of proliferation and cellular senescence in lung cancer.
Assuntos
Senescência Celular/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , RNA Longo não Codificante/genética , Adenocarcinoma/genética , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Técnicas de Silenciamento de Genes , Humanos , Modelos Biológicos , Mutação , Interferência de RNA , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
SOCS1 is lost in many human tumors, but its tumor suppression activities are not well understood. We report that SOCS1 is required for transcriptional activity, DNA binding, and serine 15 phosphorylation of p53 in the context of STAT5 signaling. In agreement, inactivation of SOCS1 disabled p53-dependent senescence in response to oncogenic STAT5A and radiation-induced apoptosis in T cells. In addition, SOCS1 was sufficient to induce p53-dependent senescence in fibroblasts. The mechanism of activation of p53 by SOCS1 involved a direct interaction between the SH2 domain of SOCS1 and the N-terminal transactivation domain of p53, while the C-terminal domain of SOCS1 containing the SOCS Box mediated interaction with the DNA damage-regulated kinases ATM/ATR. Also, SOCS1 colocalized with ATM at DNA damage foci induced by oncogenic STAT5A. Collectively, these results add another component to the p53 and DNA damage networks and reveal a mechanism by which SOCS1 functions as a tumor suppressor.
Assuntos
Senescência Celular , Proteínas Supressoras da Sinalização de Citocina/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/análise , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
G protein pathway suppressor 2 (GPS2) is a multifunctional protein involved in the regulation of a number of metabolic organs. First identified as part of the NCoR-SMRT corepressor complex, GPS2 is known to play an important role in the nucleus in the regulation of gene transcription and meiotic recombination. In addition, we recently reported a non-transcriptional role of GPS2 as an inhibitor of the proinflammatory TNFα pathway in the cytosol. Although this suggests that the control of GPS2 localization may be an important determinant of its molecular functions, a clear understanding of GPS2 differential targeting to specific cellular locations is still lacking. Here we show that a fine balance between protein stabilization and degradation tightly regulates GPS2 nuclear function. Our findings indicate that GPS2 is degraded upon polyubiquitination by the E3 ubiquitin ligase Siah2. Unexpectedly, interaction with the exchange factor TBL1 is required to protect GPS2 from degradation, with methylation of GPS2 by arginine methyltransferase PRMT6 regulating the interaction with TBL1 and inhibiting proteasome-dependent degradation. Overall, our findings indicate that regulation of GPS2 by posttranslational modifications provides an effective strategy for modulating its molecular function within the nuclear compartment.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína-Arginina N-Metiltransferases/fisiologia , Transducina/fisiologia , Transporte Ativo do Núcleo Celular , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Metilação , Sinais de Localização Nuclear , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteólise , UbiquitinaçãoRESUMO
The deubiquitinase (DUB) and tumor suppressor BAP1 catalyzes ubiquitin removal from histone H2A Lys-119 and coordinates cell proliferation, but how BAP1 partners modulate its function remains poorly understood. Here, we report that BAP1 forms two mutually exclusive complexes with the transcriptional regulators ASXL1 and ASXL2, which are necessary for maintaining proper protein levels of this DUB. Conversely, BAP1 is essential for maintaining ASXL2, but not ASXL1, protein stability. Notably, cancer-associated loss of BAP1 expression results in ASXL2 destabilization and hence loss of its function. ASXL1 and ASXL2 use their ASXM domains to interact with the C-terminal domain (CTD) of BAP1, and these interactions are required for ubiquitin binding and H2A deubiquitination. The deubiquitination-promoting effect of ASXM requires intramolecular interactions between catalytic and non-catalytic domains of BAP1, which generate a composite ubiquitin-binding interface (CUBI). Notably, the CUBI engages multiple interactions with ubiquitin involving (i) the ubiquitin carboxyl hydrolase catalytic domain of BAP1, which interacts with the hydrophobic patch of ubiquitin, and (ii) the CTD domain, which interacts with a charged patch of ubiquitin. Significantly, we identified cancer-associated mutations of BAP1 that disrupt the CUBI and notably an in-frame deletion in the CTD that inhibits its interaction with ASXL1/2 and DUB activity and deregulates cell proliferation. Moreover, we demonstrated that BAP1 interaction with ASXL2 regulates cell senescence and that ASXL2 cancer-associated mutations disrupt BAP1 DUB activity. Thus, inactivation of the BAP1/ASXL2 axis might contribute to cancer development.
Assuntos
Proliferação de Células , Neoplasias/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Células HEK293 , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Neoplasias/genética , Neoplasias/patologia , Proteínas Repressoras/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Proteases Específicas de Ubiquitina/genéticaRESUMO
In response to DNA damage, cells initiate complex signalling cascades leading to growth arrest and DNA repair. The recruitment of 53BP1 to damaged sites requires the activation of the ubiquitination cascade controlled by the E3 ubiquitin ligases RNF8 and RNF168, and methylation of histone H4 on lysine 20. However, molecular events that regulate the accessibility of methylated histones, to allow the recruitment of 53BP1 to DNA breaks, are unclear. Here, we show that like 53BP1, the JMJD2A (also known as KDM4A) tandem tudor domain binds dimethylated histone H4K20; however, JMJD2A is degraded by the proteasome following the DNA damage in an RNF8-dependent manner. We demonstrate that JMJD2A is ubiquitinated by RNF8 and RNF168. Moreover, ectopic expression of JMJD2A abrogates 53BP1 recruitment to DNA damage sites, indicating a role in antagonizing 53BP1 for methylated histone marks. The combined knockdown of JMJD2A and JMJD2B significantly rescued the ability of RNF8- and RNF168-deficient cells to form 53BP1 foci. We propose that the RNF8-dependent degradation of JMJD2A regulates DNA repair by controlling the recruitment of 53BP1 at DNA damage sites.
Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Reparo do DNA , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Modelos Biológicos , Dados de Sequência Molecular , Ligação Proteica , Transdução de Sinais , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
Arginine methylation of histones is a well-known regulator of gene expression. Protein arginine methyltransferase 6 (PRMT6) has been shown to function as a transcriptional repressor by methylating the histone H3 arginine 2 [H3R2(me2a)] repressive mark; however, few targets are known. To define the physiological role of PRMT6 and to identify its targets, we generated PRMT6(-/-) mouse embryo fibroblasts (MEFs). We observed that early passage PRMT6(-/-) MEFs had growth defects and exhibited the hallmarks of cellular senescence. PRMT6(-/-) MEFs displayed high transcriptional levels of p53 and its targets, p21 and PML. Generation of PRMT6(-/-); p53(-/-) MEFs prevented the premature senescence, suggesting that the induction of senescence is p53-dependent. Using chromatin immunoprecipitation assays, we observed an enrichment of PRMT6 and H3R2(me2a) within the upstream region of Trp53. The PRMT6 association and the H3R2(me2a) mark were lost in PRMT6(-/-) MEFs and an increase in the H3K4(me3) activator mark was observed. Our findings define a new regulator of p53 transcriptional regulation and define a role for PRMT6 and arginine methylation in cellular senescence.
Assuntos
Proteínas de Drosophila/fisiologia , Regulação da Expressão Gênica , Proteína-Arginina N-Metiltransferases/fisiologia , Transcrição Gênica , Proteína Supressora de Tumor p53/genética , Animais , Células Cultivadas , Senescência Celular/genética , Proteínas de Drosophila/genética , Camundongos , Camundongos Knockout , Proteína-Arginina N-Metiltransferases/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas ras/genéticaRESUMO
Compromised vascular endothelial barrier function is a salient feature of diabetic complications such as sight-threatening diabetic macular edema (DME). Current standards of care for DME manage aspects of the disease, but require frequent intravitreal administration and are poorly effective in large subsets of patients. Here we provide evidence that an elevated burden of senescent cells in the retina triggers cardinal features of DME pathology and conduct an initial test of senolytic therapy in patients with DME. In cell culture models, sustained hyperglycemia provoked cellular senescence in subsets of vascular endothelial cells displaying perturbed transendothelial junctions associated with poor barrier function and leading to micro-inflammation. Pharmacological elimination of senescent cells in a mouse model of DME reduces diabetes-induced retinal vascular leakage and preserves retinal function. We then conducted a phase 1 single ascending dose safety study of UBX1325 (foselutoclax), a senolytic small-molecule inhibitor of BCL-xL, in patients with advanced DME for whom anti-vascular endothelial growth factor therapy was no longer considered beneficial. The primary objective of assessment of safety and tolerability of UBX1325 was achieved. Collectively, our data suggest that therapeutic targeting of senescent cells in the diabetic retina with a BCL-xL inhibitor may provide a long-lasting, disease-modifying intervention for DME. This hypothesis will need to be verified in larger clinical trials. ClinicalTrials.gov identifier: NCT04537884 .
Assuntos
Diabetes Mellitus , Retinopatia Diabética , Edema Macular , Animais , Camundongos , Humanos , Edema Macular/tratamento farmacológico , Edema Macular/etiologia , Retinopatia Diabética/tratamento farmacológico , Inibidores da Angiogênese/uso terapêutico , Células Endoteliais , Senoterapia , Senescência CelularRESUMO
The inaugural Canadian Conferences on Translational Geroscience were held as 2 complementary sessions in October and November 2023. The conferences explored the profound interplay between the biology of aging, social determinants of health, the potential societal impact of geroscience, and the maintenance of health in aging individuals. Although topics such as cellular senescence, molecular and genetic determinants of aging, and prevention of chronic disease were addressed, the conferences went on to emphasize practical applications for enhancing older people's quality of life. This article summarizes the proceeding and underscores the synergy between clinical and fundamental studies. Future directions highlight national and global collaborations and the crucial integration of early-career investigators. This work charts a course for a national framework for continued innovation and advancement in translational geroscience in Canada.