RESUMO
An organism encounters an extensive range of dietary choices in its niche. The macronutrient and micronutrient quotients of these diets instruct downstream molecular events, eventually orchestrating complex physiological responses that determine their life-history traits. In response to diverse nutritional inputs, organisms deploy multiple adaptive mechanisms to maintain optimal life span and health. However, we know little about these processes. Although we have come to appreciate the complex relationship between diet and genes in modulating life span in response to diverse nutritional inputs, it can be difficult to identify these interactions in the laboratory, as an organism maintains a normal life span on a wide range of diet. But in rare cases, a mutant laboratory animal, with the mutation attributable to a single gene, may display altered physiological traits on only one diet but not on others. These 'diet-gene pairs' contribute immensely to our understanding of how diet quality influences physiological traits like longevity and health.
Assuntos
Dieta , Longevidade , Animais , Longevidade/genética , NutrientesRESUMO
Germ line integrity is crucial for progeny fitness. Organisms deploy the DNA damage response (DDR) signaling to protect the germ line from genotoxic stress, facilitating the cell-cycle arrest of germ cells and DNA repair or their apoptosis. Cell-autonomous regulation of germ line quality in response to DNA damage is well studied; however, how quality is enforced cell non-autonomously on sensing somatic DNA damage is less known. Using Caenorhabditis elegans, we show that DDR disruption, only in the uterus, when insulin/IGF-1 signaling (IIS) is low, arrests oogenesis in the pachytene stage of meiosis I, in a FOXO/DAF-16 transcription factor-dependent manner. Without FOXO/DAF-16, germ cells of the IIS mutant escape the arrest to produce poor-quality oocytes, showing that the transcription factor imposes strict quality control during low IIS. Activated FOXO/DAF-16 senses DDR perturbations during low IIS to lower ERK/MPK-1 signaling below a threshold to promote germ line arrest. Altogether, we elucidate a new surveillance role for activated FOXO/DAF-16 that ensures optimal germ cell quality and progeny fitness in response to somatic DNA damage.
Assuntos
Proteínas de Caenorhabditis elegans , Animais , Feminino , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Caenorhabditis elegans/metabolismo , Insulina/metabolismo , Dano ao DNA/genética , Oogênese/genética , Longevidade/fisiologiaRESUMO
Aging is associated with changes in circulating levels of various molecules, some of which remain undefined. We find that concentrations of circulating taurine decline with aging in mice, monkeys, and humans. A reversal of this decline through taurine supplementation increased the health span (the period of healthy living) and life span in mice and health span in monkeys. Mechanistically, taurine reduced cellular senescence, protected against telomerase deficiency, suppressed mitochondrial dysfunction, decreased DNA damage, and attenuated inflammaging. In humans, lower taurine concentrations correlated with several age-related diseases and taurine concentrations increased after acute endurance exercise. Thus, taurine deficiency may be a driver of aging because its reversal increases health span in worms, rodents, and primates and life span in worms and rodents. Clinical trials in humans seem warranted to test whether taurine deficiency might drive aging in humans.
Assuntos
Envelhecimento , Taurina , Animais , Humanos , Camundongos , Envelhecimento/sangue , Envelhecimento/efeitos dos fármacos , Envelhecimento/metabolismo , Senescência Celular , Haplorrinos , Longevidade/efeitos dos fármacos , Longevidade/fisiologia , Taurina/sangue , Taurina/deficiência , Taurina/farmacologia , Suplementos Nutricionais , Dano ao DNA/efeitos dos fármacos , Telomerase/metabolismoRESUMO
Treatment of triple-negative breast cancer (TNBC) is challenging because of its "COLD" tumor immunosuppressive microenvironment (TIME). Here, we present a hydrogel-mediated localized delivery of a combination of docetaxel (DTX) and carboplatin (CPT) (called DTX-CPT-Gel therapy) that ensured enhanced anticancer effect and tumor regression on multiple murine syngeneic and xenograft tumor models. DTX-CPT-Gel therapy modulated the TIME by an increase of antitumorigenic M1 macrophages, attenuation of myeloid-derived suppressor cells, and increase of granzyme B+CD8+ T cells. DTX-CPT-Gel therapy elevated ceramide levels in tumor tissues that activated the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)-mediated unfolded protein response (UPR). This UPR-mediated activation of apoptotic cell death led to release of damage-associated molecular patterns, thereby activating the immunogenic cell death that could even clear the metastatic tumors. This study provides a promising hydrogel-mediated platform for DTX-CPT therapy that induces tumor regression and effective immune modulation and, therefore, can be explored further for treatment of TNBC.
Assuntos
Hidrogéis , Neoplasias de Mama Triplo Negativas , Humanos , Animais , Camundongos , Morte Celular Imunogênica , Linfócitos T CD8-Positivos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Ceramidas , Modelos Animais de Doenças , Imunossupressores , Resposta a Proteínas não Dobradas , Microambiente TumoralRESUMO
Advanced glycation end-products (AGEs) form when glucose reacts non-enzymatically with proteins, leading to abnormal protein function, oxidative stress, and inflammation. AGEs are associated with aging and age-related diseases; their formation is aggravated during diabetes. Therefore, drugs preventing AGE formation can potentially treat diabetic complications, positively affecting health. Earlier, we demonstrated that rifampicin and its analogs have potent anti-glycating activities and increase the life span of Caenorhabditis elegans. This study aimed to investigate the effects of rifampicin during hyperglycemia in C. elegans and in a mouse model of obesity-induced type 2 diabetes. The effects of rifampicin were assessed by determining the life span of C. elegans cultured in the presence of glucose and by measuring HbA1c, AGE levels, and glucose excursions in the diabetic mouse model. Our results show that rifampicin protects C. elegans from glucose-induced toxicity and increases life span. In mice, rifampicin reduces HbA1c and AGEs, improves insulin sensitivity, and reduces indications of diabetic nephropathy without inducing hepatotoxicity. Rifampicin quinone, an analog with lower anti-microbial activity, also reduces HbA1c levels, improves glucose homeostasis and insulin sensitivity, and lowers indications of diabetic nephropathy, without adversely affecting the liver of the diabetic mice. Altogether, our results indicate that rifampicin and its analog have protective roles during diabetes without inflicting hepatic damage and may potentially be considered for repositioning to treat hyperglycemia-related complications in patients.
RESUMO
Diet regulates complex life-history traits such as longevity. For optimal lifespan, organisms employ intricate adaptive mechanisms whose molecular underpinnings are less known. We show that Caenorhabditis elegans FLR-4 kinase prevents lifespan differentials on the bacterial diet having higher Vitamin B12 levels. The flr-4 mutants are more responsive to the higher B12 levels of Escherichia coli HT115 diet, and consequently, have enhanced flux through the one-carbon cycle. Mechanistically, a higher level of B12 transcriptionally downregulates the phosphoethanolamine methyltransferase pmt-2 gene, which modulates phosphatidylcholine (PC) levels. Pmt-2 downregulation activates cytoprotective gene expression through the p38-MAPK pathway, leading to increased lifespan only in the mutant. Evidently, preventing bacterial B12 uptake or inhibiting one-carbon metabolism reverses all the above phenotypes. Conversely, supplementation of B12 to E. coli OP50 or genetically reducing PC levels in the OP50-fed mutant extends lifespan. Together, we reveal how worms maintain adaptive capacity to diets having varying micronutrient content to ensure a normal lifespan.
Assuntos
Proteínas de Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/efeitos dos fármacos , Dieta , Longevidade/efeitos dos fármacos , Vitamina B 12/uso terapêutico , Animais , Vitamina B 12/farmacologiaRESUMO
Insulin/IGF-1-like signaling (IIS) plays a crucial, conserved role in development, growth, reproduction, stress tolerance, and longevity. In Caenorhabditis elegans, the enhanced longevity under reduced insulin signaling (rIIS) is primarily regulated by the transcription factors (TFs) DAF-16/FOXO, SKN-1/Nrf-1, and HSF1/HSF-1. The specific and coordinated regulation of gene expression by these TFs under rIIS has not been comprehensively elucidated. Here, using RNA-sequencing analysis, we report a systematic study of the complexity of TF-dependent target gene interactions during rIIS under analogous genetic and experimental conditions. We found that DAF-16 regulates only a fraction of the C. elegans transcriptome but controls a large set of genes under rIIS; SKN-1 and HSF-1 show the opposite trend. Both of the latter TFs function as activators and repressors to a similar extent, while DAF-16 is predominantly an activator. For expression of the genes commonly regulated by TFs under rIIS conditions, DAF-16 is the principal determining factor, dominating over the other two TFs, irrespective of whether they activate or repress these genes. The functional annotations and regulatory networks presented in this study provide novel insights into the complexity of the gene regulatory networks downstream of the IIS pathway that controls diverse phenotypes, including longevity.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/genética , Fatores de Transcrição Forkhead/genética , Genoma Helmíntico , Insulina/metabolismo , Fatores de Transcrição/genética , Transcriptoma , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Ontologia Genética , Redes Reguladoras de Genes , Longevidade/genética , Anotação de Sequência Molecular , Fenótipo , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
Meta-analysis of transcripts in colon adenocarcinoma patient tissues led to the identification of a DNA damage responsive miR signature called DNA damage sensitive miRs (DDSMs). DDSMs were experimentally validated in the cancerous colon tissues obtained from an independent cohort of colon cancer patients and in multiple cellular systems with high levels of endogenous DNA damage. All the tested DDSMs were transcriptionally upregulated by a common intestine-specific transcription factor, CDX2. Reciprocally, DDSMs were repressed via the recruitment of HDAC1/2-containing complexes onto the CDX2 promoter. These miRs downregulated multiple key targets in the DNA damage response (DDR) pathway, namely BRCA1, ATM, Chk1 (also known as CHEK1) and RNF8. CDX2 directly regulated the DDSMs, which led to increased tumor volume and metastasis in multiple preclinical models. In colon cancer patient tissues, the DDSMs negatively correlated with BRCA1 levels, were associated with decreased probability of survival and thereby could be used as a prognostic biomarker. This article has an associated First Person interview with the first author of the paper.
Assuntos
Adenocarcinoma , Neoplasias do Colo , MicroRNAs , Fator de Transcrição CDX2/genética , Neoplasias do Colo/genética , Dano ao DNA/genética , Proteínas de Ligação a DNA/genética , Humanos , MicroRNAs/genética , Fatores de Transcrição , Ubiquitina-Proteína LigasesRESUMO
Global dysregulation of RNA splicing and imbalanced sphingolipid metabolism has emerged as promoters of cancer cell transformation. Here, we present specific signature of alternative splicing (AS) events of sphingolipid genes for each breast cancer subtype from the TCGA-BRCA dataset. We show that ceramide synthase 2 (CERS2) undergoes a unique cassette exon event specifically in Luminal B subtype tumors. We validated this exon 8 skipping event in Luminal B cancer cells compared to normal epithelial cells, and in patient-derived tumor tissues compared to matched normal tissues. Differential AS-based survival analysis shows that this AS event of CERS2 is a poor prognostic factor for Luminal B patients. As Exon 8 corresponds to catalytic Lag1p domain, overexpression of AS transcript of CERS2 in Luminal B cancer cells leads to a reduction in the level of very-long-chain ceramides compared to overexpression of protein-coding (PC) transcript of CERS2. We further demonstrate that this AS event-mediated decrease of very-long-chain ceramides leads to enhanced cancer cell proliferation and migration. Therefore, our results show subtype-specific AS of sphingolipid genes as a regulatory mechanism that deregulates sphingolipids like ceramides in breast tumors, and can be explored further as a suitable therapeutic target.
Assuntos
Processamento Alternativo , Neoplasias da Mama/enzimologia , Movimento Celular , Proliferação de Células , Ceramidas/metabolismo , Proteínas de Membrana/metabolismo , Esfingosina N-Aciltransferase/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Bases de Dados Genéticas , Feminino , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Proteínas de Membrana/genética , Invasividade Neoplásica , Transdução de Sinais , Esfingosina N-Aciltransferase/genética , Transcriptoma , Proteínas Supressoras de Tumor/genéticaRESUMO
In this study, we describe the engineering of sub-100â nm nanomicelles (DTX-PC NMs) derived from phosphocholine derivative of docetaxel (DTX)-conjugated lithocholic acid (DTX-PC) and poly(ethylene glycol)-tethered lithocholic acid. Administration of DTX-PC NMs decelerate tumor progression and increase the mice survivability compared to Taxotere (DTX-TS), the FDA-approved formulation of DTX. Unlike DTX-TS, DTX-PC NMs do not cause any systemic toxicity and slow the decay rate of plasma DTX concentration in rodents and non-rodent species including non-human primates. We further demonstrate that DTX-PC NMs target demethylation of CpG islands of Sparcl1 (a tumor suppressor gene) by suppressing DNA methyltransferase activity and increase the expression of Sparcl1 that leads to tumor regression. Therefore, this unique system has the potential to improve the quality of life in cancer patients and can be translated as a next-generation chemotherapeutic.
Assuntos
Antineoplásicos/uso terapêutico , Docetaxel/uso terapêutico , Epigênese Genética/efeitos dos fármacos , Ácido Litocólico/análogos & derivados , Ácido Litocólico/uso terapêutico , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular Tumoral , Ilhas de CpG , Desmetilação , Progressão da Doença , Docetaxel/síntese química , Docetaxel/farmacocinética , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Ácido Litocólico/farmacocinética , Camundongos Endogâmicos BALB C , Micelas , Neoplasias/fisiopatologia , Tensoativos/síntese química , Tensoativos/farmacocinética , Tensoativos/uso terapêuticoRESUMO
Dietary restriction (DR) increases life span and improves health in most model systems tested, including non-human primates. In C. elegans, as in other models, DR leads to reprogramming of metabolism, improvements in mitochondrial health, large changes in expression of cytoprotective genes and better proteostasis. Understandably, multiple global transcriptional regulators like transcription factors FOXO/DAF-16, FOXA/PHA-4, HSF1/HSF-1 and NRF2/SKN-1 are important for DR longevity. Considering the wide-ranging effects of p53 on organismal biology, we asked whether the C. elegans ortholog, CEP-1 is required for DR-mediated longevity assurance. We employed the widely-used TJ1 strain of cep-1(gk138). We show that cep-1(gk138) suppresses the life span extension of two genetic paradigms of DR, but two non-genetic modes of DR remain unaffected in this strain. We find that two aspects of DR, increased autophagy and up-regulation of the expression of cytoprotective xenobiotic detoxification program (cXDP) genes, are dampened in cep-1(gk138). Importantly, we find that background mutation(s) in the strain may be the actual cause for the phenotypic differences that we observed and cep-1 may not be directly involved in genetic DR-mediated longevity assurance in worms. Identifying these mutation(s) may reveal a novel regulator of longevity required specifically by genetic modes of DR.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Restrição Calórica , Longevidade/genética , Mutação/genética , Proteína Supressora de Tumor p53/genética , Animais , Autofagia/genética , Citoproteção/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica , Regulação para Cima/genéticaRESUMO
The metabolic state of an organism instructs gene expression modalities, leading to changes in complex life history traits, such as longevity. Dietary restriction (DR), which positively affects health and life span across species, leads to metabolic reprogramming that enhances utilisation of fatty acids for energy generation. One direct consequence of this metabolic shift is the upregulation of cytoprotective (CyTP) genes categorized in the Gene Ontology (GO) term of "Xenobiotic Detoxification Program" (XDP). How an organism senses metabolic changes during nutritional stress to alter gene expression programs is less known. Here, using a genetic model of DR, we show that the levels of polyunsaturated fatty acids (PUFAs), especially linoleic acid (LA) and eicosapentaenoic acid (EPA), are increased following DR and these PUFAs are able to activate the CyTP genes. This activation of CyTP genes is mediated by the conserved p38 mitogen-activated protein kinase (p38-MAPK) pathway. Consequently, genes of the PUFA biosynthesis and p38-MAPK pathway are required for multiple paradigms of DR-mediated longevity, suggesting conservation of mechanism. Thus, our study shows that PUFAs and p38-MAPK pathway function downstream of DR to help communicate the metabolic state of an organism to regulate expression of CyTP genes, ensuring extended life span.
Assuntos
Ácidos Graxos Insaturados/genética , Ácidos Graxos Insaturados/metabolismo , Regulação da Expressão Gênica , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Fenômenos Bioquímicos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Ácido Eicosapentaenoico/análogos & derivados , Regulação da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Ácido Linoleico/metabolismo , Longevidade , Redes e Vias Metabólicas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Unfolded protein response (UPR) of the endoplasmic reticulum (UPRER) helps maintain proteostasis in the cell. The ability to mount an effective UPRER to external stress (iUPRER) decreases with age and is linked to the pathophysiology of multiple age-related disorders. Here, we show that a transient pharmacological ER stress, imposed early in development on Caenorhabditis elegans, enhances proteostasis, prevents iUPRER decline with age, and increases adult life span. Importantly, dietary restriction (DR), that has a conserved positive effect on life span, employs this mechanism of ER hormesis for longevity assurance. We found that only the IRE-1-XBP-1 branch of UPRER is required for the longevity effects, resulting in increased ER-associated degradation (ERAD) gene expression and degradation of ER resident proteins during DR. Further, both ER hormesis and DR protect against polyglutamine aggregation in an IRE-1-dependent manner. We show that the DR-specific FOXA transcription factor PHA-4 transcriptionally regulates the genes required for ER homeostasis and is required for ER preconditioning-induced life span extension. Finally, we show that ER hormesis improves proteostasis and viability in a mammalian cellular model of neurodegenerative disease. Together, our study identifies a mechanism by which DR offers its benefits and opens the possibility of using ER-targeted pharmacological interventions to mimic the prolongevity effects of DR.
Assuntos
Restrição Calórica , Retículo Endoplasmático/metabolismo , Longevidade , Resposta a Proteínas não Dobradas , Envelhecimento , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Estresse do Retículo Endoplasmático , Homeostase , Longevidade/genéticaRESUMO
Chromatin immunoprecipitation (ChIP) coupled to quantitative real-time PCR (ChIP-qPCR) or Next-Generation Sequencing (ChIP-seq) enables us to study the dynamics of chromatin recruitment of transcription factors (TFs). The popular model system Caenorhabditis elegans has provided us with fundamental understanding of the role of Insulin/IGF-1-like signaling (IIS) in metabolism and aging. The FOXO TF DAF-16 is the major output of the pathway that regulates most of the phenotypes associated with the IIS pathway. Here, we describe a ChIP protocol to study FOXO recruitment dynamics in whole C. elegans extracts. We discuss detailed practical procedures, including optimization, growth, harvesting, formaldehyde fixation, sonication of worms, TF immunoprecipitation for further downstream processing using qPCR as well as NGS for the analysis of FOXO-bound DNA.
Assuntos
Sítios de Ligação , Imunoprecipitação da Cromatina , Fatores de Transcrição Forkhead/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Regiões Promotoras Genéticas , Animais , Caenorhabditis elegans , Imunoprecipitação da Cromatina/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ligação Proteica , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Diet profoundly affects metabolism and incidences of age-related diseases. Animals adapt their physiology to different food-types, modulating complex life-history traits like aging. The molecular mechanisms linking adaptive capacity to diet with aging are less known. We identify FLR-4 kinase as a novel modulator of aging in C. elegans, depending on bacterial diet. FLR-4 functions to prevent differential activation of the p38MAPK pathway in response to diverse food-types, thereby maintaining normal life span. In a kinase-dead flr-4 mutant, E. coli HT115 (K12 strain), but not the standard diet OP50 (B strain), is able to activate p38MAPK, elevate expression of cytoprotective genes through the nuclear hormone receptor NHR-8 and enhance life span. Interestingly, flr-4 and dietary restriction utilize similar pathways for longevity assurance, suggesting cross-talks between cellular modules that respond to diet quality and quantity. Together, our study discovers a new C. elegans gene-diet pair that controls the plasticity of aging.
Assuntos
Envelhecimento/genética , Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Dieta , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Proteínas de Caenorhabditis elegans/genética , Regulação da Expressão Gênica , Longevidade , Proteínas Serina-Treonina Quinases/genética , RNA de Helmintos/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/fisiologia , Transdução de Sinais , Transcriptoma , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
Alternative splicing (AS) coupled to nonsense-mediated decay (AS-NMD) is a conserved mechanism for post-transcriptional gene regulation. Here we show that, during dietary restriction (DR), AS is enhanced in Caenorhabditis elegans and mice. A splicing mediator hrpu-1 regulates a significant part of these AS events in C. elegans; knocking it down suppresses DR-mediated longevity. Concurrently, due to increased AS, NMD pathway genes are upregulated and knocking down UPF1 homologue smg-2 suppresses DR lifespan. Knockdown of NMD during DR significantly increases the inclusion of PTC-containing introns and the lengths of the 3'UTRs. Finally, we demonstrate that PHA-4/FOXA transcriptionally regulates the AS-NMD genes. Our study suggests that DR uses AS to amplify the proteome, supporting physiological remodelling required for enhanced longevity. This increases the dependence on NMD, but also helps fine-tune the expression of metabolic and splicing mediators. AS-NMD may thus provide an energetically favourable level of dynamic gene expression control during dietary restriction.Alternative splicing coupled to nonsense-mediated decay (AS-NMD) is a conserved mechanism for post-transcriptional gene regulation. Here, the authors provide evidence that AS-NMD is enhanced during dietary restriction (DR) and is required for DR-mediated longevity assurance in C. elegans.
Assuntos
Processamento Alternativo , Restrição Calórica , Longevidade/genética , Degradação do RNAm Mediada por Códon sem Sentido , RNA Mensageiro/genética , Regiões 3' não Traduzidas/genética , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Dieta , Éxons/genética , Perfilação da Expressão Gênica/métodos , Íntrons/genética , Camundongos , Mutação , RNA Mensageiro/metabolismoRESUMO
Ncb2, the ß subunit of NC2 complex, a heterodimeric regulator of transcription was earlier shown to be involved in the activated transcription of CDR1 gene in azole resistant isolate (AR) of Candida albicans. This study examines its genome-wide role by profiling Ncb2 occupancy between genetically matched pair of azole sensitive (AS) and AR clinical isolates. A comparison of Ncb2 recruitment between the two isolates displayed that 29 genes had higher promoter occupancy of Ncb2 in the AR isolate. Additionally, a host of genes exhibited exclusive occupancy of Ncb2 at promoters of either AR or AS isolate. The analysis also divulged new actors of multi-drug resistance, whose transcription was activated owing to the differential occupancy of Ncb2. The conditional, sequence-specific positional escape of Ncb2 from the core promoter in AS isolate and its preferential recruitment to the core promoter of certain genes in AR isolates was most noteworthy means of transcription regulation. Together, we show that positional rearrangement of Ncb2 resulting in either activation or repression of gene expression in response to drug-induced stress, represents a novel regulatory mechanism that opens new opportunities for therapeutic intervention to prevent development of drug tolerance in C. albicans cells.
Assuntos
Antifúngicos/farmacologia , Candida albicans/genética , Candida albicans/fisiologia , Proteínas Fúngicas/metabolismo , Regiões Promotoras Genéticas , Estresse Fisiológico , Transcrição Gênica , Azóis/farmacologia , Sequência de Bases , Candida albicans/efeitos dos fármacos , Candida albicans/isolamento & purificação , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genoma Fúngico , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , TATA Box/genética , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genéticaRESUMO
Reductive stress leads to the loss of disulfide bond formation and induces the unfolded protein response of the endoplasmic reticulum (UPR(ER)), necessary to regain proteostasis in the compartment. Here we show that peroxide accumulation during reductive stress attenuates UPR(ER) amplitude by altering translation without any discernible effect on transcription. Through a comprehensive genetic screen in Saccharomyces cerevisiae, we identify modulators of reductive stress-induced UPR(ER) and demonstrate that oxidative quality control (OQC) genes modulate this cellular response in the presence of chronic but not acute reductive stress. Using a combination of microarray and relative quantitative proteomics, we uncover a non-canonical translation attenuation mechanism that acts in a bipartite manner to selectively downregulate highly expressed proteins, decoupling the cell's transcriptional and translational response during reductive ER stress. Finally, we demonstrate that PERK, a canonical translation attenuator in higher eukaryotes, helps in bypassing a ROS-dependent, non-canonical mode of translation attenuation.
Assuntos
Estresse do Retículo Endoplasmático/genética , Retículo Endoplasmático/genética , Homeostase/genética , Biossíntese de Proteínas/genética , Animais , Caenorhabditis elegans/genética , Regulação para Baixo/genética , Eucariotos/genética , Peróxidos/metabolismo , Proteômica/métodos , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Transcrição Gênica/genética , Resposta a Proteínas não Dobradas/genética , eIF-2 Quinase/genéticaRESUMO
Insulin/IGF-1-like signalling (IIS) and dietary restriction (DR) are the two major modulatory pathways controlling longevity across species. Here, we show that both pathways license a common chromatin modifier, ZFP-1/AF10. The downstream transcription factors of the IIS and select DR pathways, DAF-16/FOXO or PHA-4/FOXA, respectively, both transcriptionally regulate the expression of zfp-1. ZFP-1, in turn, negatively regulates the expression of DAF-16/FOXO and PHA-4/FOXA target genes, apparently forming feed-forward loops that control the amplitude as well as the duration of gene expression. We show that ZFP-1 mediates this regulation by negatively influencing the recruitment of DAF-16/FOXO and PHA-4/FOXA to their target promoters. Consequently, zfp-1 is required for the enhanced longevity observed during DR and on knockdown of IIS. Our data reveal how two distinct sensor pathways control an overlapping set of genes, using different downstream transcription factors, integrating potentially diverse and temporally distinct nutritional situations.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Restrição Calórica , Cromatina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Insulina/metabolismo , Longevidade/fisiologia , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Caenorhabditis elegans/genética , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Isoformas de Proteínas/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Lowering insulin-IGF-1-like signalling (IIS) activates FOXO transcription factors (TF) to extend life span across species. To study the dynamics of FOXO chromatin occupancy under this condition in C. elegans, we report the first recruitment profile of endogenous DAF-16 and show that the response is conserved. DAF-16 predominantly acts as a transcriptional activator and binding within the 0.5 kb promoter-proximal region results in maximum induction of downstream targets that code for proteins involved in detoxification and longevity. Interestingly, genes that are activated under low IIS already have higher DAF-16 recruited to their promoters in WT. DAF-16 binds to variants of the FOXO consensus sequence in the promoter proximal regions of genes that are exclusively targeted during low IIS. We also define a set of 'core' direct targets, after comparing multiple studies, which tend to co-express and contribute robustly towards IIS-associated phenotypes. Additionally, we show that nuclear hormone receptor DAF-12 as well as zinc-finger TF EOR-1 may bind DNA in close proximity to DAF-16 and distinct TF classes that are direct targets of DAF-16 may be instrumental in regulating its indirect targets. Together, our study provides fundamental insights into the transcriptional biology of FOXO/DAF-16 and gene regulation downstream of the IIS pathway.