RESUMEN
Genome introgressions drive evolution across the animal1, plant2 and fungal3 kingdoms. Introgressions initiate from archaic admixtures followed by repeated backcrossing to one parental species. However, how introgressions arise in reproductively isolated species, such as yeast4, has remained unclear. Here we identify a clonal descendant of the ancestral yeast hybrid that founded the extant Saccharomyces cerevisiae Alpechin lineage5, which carries abundant Saccharomyces paradoxus introgressions. We show that this clonal descendant, hereafter defined as a 'living ancestor', retained the ancestral genome structure of the first-generation hybrid with contiguous S. cerevisiae and S. paradoxus subgenomes. The ancestral first-generation hybrid underwent catastrophic genomic instability through more than a hundred mitotic recombination events, mainly manifesting as homozygous genome blocks generated by loss of heterozygosity. These homozygous sequence blocks rescue hybrid fertility by restoring meiotic recombination and are the direct origins of the introgressions present in the Alpechin lineage. We suggest a plausible route for introgression evolution through the reconstruction of extinct stages and propose that genome instability allows hybrids to overcome reproductive isolation and enables introgressions to emerge.
Asunto(s)
Evolución Molecular , Introgresión Genética/genética , Genoma Fúngico/genética , Genómica , Filogenia , Saccharomyces cerevisiae/genética , Saccharomyces/genética , Cruzamientos Genéticos , Fertilidad/genética , Aptitud Genética/genética , Inestabilidad Genómica/genética , Recombinación Homóloga/genética , Pérdida de Heterocigocidad/genética , Meiosis/genética , Mitosis/genética , Reproducción Asexuada/genética , Saccharomyces/clasificación , Saccharomyces/citología , Saccharomyces cerevisiae/clasificación , Saccharomyces cerevisiae/citologíaRESUMEN
Large-scale population genomic surveys are essential to explore the phenotypic diversity of natural populations. Here we report the whole-genome sequencing and phenotyping of 1,011 Saccharomyces cerevisiae isolates, which together provide an accurate evolutionary picture of the genomic variants that shape the species-wide phenotypic landscape of this yeast. Genomic analyses support a single 'out-of-China' origin for this species, followed by several independent domestication events. Although domesticated isolates exhibit high variation in ploidy, aneuploidy and genome content, genome evolution in wild isolates is mainly driven by the accumulation of single nucleotide polymorphisms. A common feature is the extensive loss of heterozygosity, which represents an essential source of inter-individual variation in this mainly asexual species. Most of the single nucleotide polymorphisms, including experimentally identified functional polymorphisms, are present at very low frequencies. The largest numbers of variants identified by genome-wide association are copy-number changes, which have a greater phenotypic effect than do single nucleotide polymorphisms. This resource will guide future population genomics and genotype-phenotype studies in this classic model system.
Asunto(s)
Evolución Molecular , Variación Genética , Genoma Fúngico/genética , Saccharomyces cerevisiae/clasificación , Saccharomyces cerevisiae/genética , Alelos , Aneuploidia , China , Variaciones en el Número de Copia de ADN , Estudios de Asociación Genética , Estudio de Asociación del Genoma Completo , Genómica , Pérdida de Heterocigocidad , Fenotipo , Filogenia , Filogeografía , Ploidias , Polimorfismo de Nucleótido Simple , Saccharomyces cerevisiae/aislamiento & purificación , Análisis de Secuencia de ADNRESUMEN
Aging varies among individuals due to both genetics and environment, but the underlying molecular mechanisms remain largely unknown. Using a highly recombined Saccharomyces cerevisiae population, we found 30 distinct quantitative trait loci (QTLs) that control chronological life span (CLS) in calorie-rich and calorie-restricted environments and under rapamycin exposure. Calorie restriction and rapamycin extended life span in virtually all genotypes but through different genetic variants. We tracked the two major QTLs to the cell wall glycoprotein genes FLO11 and HPF1 We found that massive expansion of intragenic tandem repeats within the N-terminal domain of HPF1 was sufficient to cause pronounced life span shortening. Life span impairment by HPF1 was buffered by rapamycin but not by calorie restriction. The HPF1 repeat expansion shifted yeast cells from a sedentary to a buoyant state, thereby increasing their exposure to surrounding oxygen. The higher oxygenation altered methionine, lipid, and purine metabolism, and inhibited quiescence, which explains the life span shortening. We conclude that fast-evolving intragenic repeat expansions can fundamentally change the relationship between cells and their environment with profound effects on cellular lifestyle and longevity.
Asunto(s)
Expansión de las Repeticiones de ADN , Proteínas de Saccharomyces cerevisiae/genética , Pared Celular , Genes Fúngicos , Metabolismo de los Lípidos , Glicoproteínas de Membrana/genética , Metionina/metabolismo , Purinas/metabolismo , Sitios de Carácter Cuantitativo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sirolimus/farmacologíaRESUMEN
BACKGROUND: Mitochondria are essential organelles partially regulated by their own genomes. The mitochondrial genome maintenance and inheritance differ from the nuclear genome, potentially uncoupling their evolutionary trajectories. Here, we analysed mitochondrial sequences obtained from the 1011 Saccharomyces cerevisiae strain collection and identified pronounced differences with their nuclear genome counterparts. RESULTS: In contrast with pre-whole genome duplication fungal species, S. cerevisiae mitochondrial genomes show higher genetic diversity compared to the nuclear genomes. Strikingly, mitochondrial genomes appear to be highly admixed, resulting in a complex interconnected phylogeny with a weak grouping of isolates, whereas interspecies introgressions are very rare. Complete genome assemblies revealed that structural rearrangements are nearly absent with rare inversions detected. We tracked intron variation in COX1 and COB to infer gain and loss events throughout the species evolutionary history. Mitochondrial genome copy number is connected with the nuclear genome and linearly scale up with ploidy. We observed rare cases of naturally occurring mitochondrial DNA loss, petite, with a subset of them that do not suffer the expected growth defect in fermentable rich media. CONCLUSIONS: Overall, our results illustrate how differences in the biology of two genomes coexisting in the same cells can lead to discordant evolutionary histories.
Asunto(s)
Núcleo Celular/genética , Evolución Molecular , Variación Genética , Genoma Fúngico , Genoma Mitocondrial , Saccharomyces cerevisiae/genética , FilogeniaRESUMEN
Pre-existing and de novo genetic variants can both drive adaptation to environmental changes, but their relative contributions and interplay remain poorly understood. Here we investigated the evolutionary dynamics in drug-treated yeast populations with different levels of pre-existing variation by experimental evolution coupled with time-resolved sequencing and phenotyping. We found a doubling of pre-existing variation alone boosts the adaptation by 64.1% and 51.5% in hydroxyurea and rapamycin, respectively. The causative pre-existing and de novo variants were selected on shared targets: RNR4 in hydroxyurea and TOR1, TOR2 in rapamycin. Interestingly, the pre-existing and de novo TOR variants map to different functional domains and act via distinct mechanisms. The pre-existing TOR variants from two domesticated strains exhibited opposite rapamycin resistance effects, reflecting lineage-specific functional divergence. This study provides a dynamic view on how pre-existing and de novo variants interactively drive adaptation and deepens our understanding of clonally evolving populations.
Asunto(s)
Evolución Biológica , Farmacorresistencia Fúngica/genética , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Hidroxiurea , Mutación , Fosfatidilinositol 3-Quinasas/genética , Sitios de Carácter Cuantitativo , Proteínas de Saccharomyces cerevisiae/genética , Selección Genética , SirolimusRESUMEN
NF-kappaB and p53 are important regulators of the cellular response to stress. Here, we identify the Skp2 gene as being both an NF-kappaB and p53 target after DNA damage. However, Skp2 expression can be either induced or repressed in a manner requiring both the p52 NF-kappaB subunit and p53, with subsequent effects on autophagy, apoptosis, and p53 function. This process is regulated by the Akt(PKB)/GSK3beta pathway. When Akt is active, GSK3beta is repressed, allowing p52 and p53 to cooperatively induce Skp2 expression. However, if Akt is inactive, GSK3beta phosphorylates p52 at Ser 222. This modification disrupts p52 homodimer/Bcl-3 complexes and facilitates transcriptional repression by p52/-c-Rel. The Skp2 promoter therefore integrates signaling through the NF-kappaB, p53, and Akt/GSK3beta pathways to regulate cell fate in response to DNA damage.
Asunto(s)
Apoptosis , Autofagia , Glucógeno Sintasa Quinasa 3/metabolismo , FN-kappa B/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Quinasas Asociadas a Fase-S/genética , Proteína p53 Supresora de Tumor/metabolismo , Daño del ADN , Glucógeno Sintasa Quinasa 3 beta , Humanos , Transducción de SeñalRESUMEN
The question of how genetic variation in a population influences phenotypic variation and evolution is of major importance in modern biology. Yet much is still unknown about the relative functional importance of different forms of genome variation and how they are shaped by evolutionary processes. Here we address these questions by population level sequencing of 42 strains from the budding yeast Saccharomyces cerevisiae and its closest relative S. paradoxus. We find that genome content variation, in the form of presence or absence as well as copy number of genetic material, is higher within S. cerevisiae than within S. paradoxus, despite genetic distances as measured in single-nucleotide polymorphisms being vastly smaller within the former species. This genome content variation, as well as loss-of-function variation in the form of premature stop codons and frameshifting indels, is heavily enriched in the subtelomeres, strongly reinforcing the relevance of these regions to functional evolution. Genes affected by these likely functional forms of variation are enriched for functions mediating interaction with the external environment (sugar transport and metabolism, flocculation, metal transport, and metabolism). Our results and analyses provide a comprehensive view of genomic diversity in budding yeast and expose surprising and pronounced differences between the variation within S. cerevisiae and that within S. paradoxus. We also believe that the sequence data and de novo assemblies will constitute a useful resource for further evolutionary and population genomics studies.
Asunto(s)
Genes Fúngicos , Saccharomyces cerevisiae/genética , Arsenitos/farmacología , Variaciones en el Número de Copia de ADN , Farmacorresistencia Fúngica/genética , Evolución Molecular , Ligamiento Genético , Especiación Genética , Genoma Fúngico , Anotación de Secuencia Molecular , Familia de Multigenes , Filogenia , Polimorfismo de Nucleótido Simple , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrollo , Análisis de Secuencia de ADN , Compuestos de Sodio/farmacologíaRESUMEN
Interleukin-26 (IL-26), a member of the IL-10 cytokine family, induces the production of proinflammatory cytokines by epithelial cells. IL-26 has been also reported overexpressed in Crohn's disease, suggesting that it may be involved in the physiopathology of chronic inflammatory disorders. Here, we have analyzed the expression and role of IL-26 in rheumatoid arthritis (RA), a chronic inflammatory disorder characterized by joint synovial inflammation. We report that the concentrations of IL-26 are higher in the serums of RA patients than of healthy subjects and dramatically elevated in RA synovial fluids compared to RA serums. Immunohistochemistry reveals that synoviolin(+) fibroblast-like synoviocytes and CD68(+) macrophage-like synoviocytes are the main IL-26-producing cells in RA joints. Fibroblast-like synoviocytes from RA patients constitutively produce IL-26 and this production is upregulated by IL-1-beta and IL-17A. We have therefore investigated the role of IL-26 in the inflammatory process. Results show that IL-26 induces the production of the proinflammatory cytokines IL-1-beta, IL-6, and tumor necrosis factor (TNF)-alpha by human monocytes and also upregulates the expression of numerous chemokines (mainly CCL20). Interestingly, IL-26-stimulated monocytes selectively promote the generation of RORgamma t(+) Th17 cells, through IL-1-beta secretion by monocytes. More precisely, IL-26-stimulated monocytes switch non-Th17 committed (IL-23R(-) or CCR6(-) CD161(-)) CD4(+) memory T cells into Th17 cells. Finally, synovial fluids from RA patients also induce Th17 cell generation and this effect is reduced after IL-26 depletion. These findings show that IL-26 is constitutively produced by RA synoviocytes, induces proinflammatory cytokine secretion by myeloid cells, and favors Th17 cell generation. IL-26 thereby appears as a novel proinflammatory cytokine, located upstream of the proinflammatory cascade, that may constitute a promising target to treat RA and chronic inflammatory disorders.
Asunto(s)
Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Citocinas/biosíntesis , Mediadores de Inflamación/metabolismo , Interleucinas/metabolismo , Células Th17/inmunología , Artritis Reumatoide/sangre , Citocinas/metabolismo , Demografía , Femenino , Fibroblastos/inmunología , Fibroblastos/patología , Humanos , Inmunohistoquímica , Memoria Inmunológica , Interleucina-17/metabolismo , Interleucina-1beta/metabolismo , Interleucinas/sangre , Articulaciones/inmunología , Articulaciones/patología , Masculino , Persona de Mediana Edad , Modelos Inmunológicos , Monocitos/metabolismo , Células Mieloides/metabolismo , Líquido Sinovial/metabolismo , Membrana Sinovial/metabolismo , Membrana Sinovial/patologíaRESUMEN
Expression profiles represent new molecular tools that are useful to characterize the successive steps of tumor progression and the prediction of recurrence or chemotherapy response. In this study, we have used quantitative proteomic analysis to compare different stages of colorectal cancer. A combination of laser microdissection, OFFGEL separation, iTRAQ labeling, and MALDI-TOF/TOF MS was used to explore the proteome of 28 colorectal cancer tissues. Two software packages were used for identification and quantification of differentially expressed proteins: Protein Pilot and iQuantitator. Based on â¼1,190,702 MS/MS spectra, a total of 3138 proteins were identified, which represents the largest database of colorectal cancer realized to date and demonstrates the value of our quantitative proteomic approach. In this way, individual protein expression and variation have been identified for each patient and for each colorectal dysplasia and cancer stage (stages I-IV). A total of 555 proteins presenting a significant fold change were quantified in the different stages, and this differential expression correlated with immunohistochemistry results reported in the Human Protein Atlas database. To identify a candidate biomarker of the early stages of colorectal cancer, we focused our study on secreted proteins. In this way, we identified olfactomedin-4, which was overexpressed in adenomas and in early stages of colorectal tumors. This early stage overexpression was confirmed by immunohistochemistry in 126 paraffin-embedded tissues. Our results also indicate that OLFM4 is regulated by the Ras-NF-κB2 pathway, one of the main oncogenic pathways deregulated in colorectal tumors.
Asunto(s)
Adenocarcinoma/patología , Adenoma/patología , Biomarcadores de Tumor/metabolismo , Carcinoma/patología , Neoplasias Colorrectales/patología , Factor Estimulante de Colonias de Granulocitos/metabolismo , Adenocarcinoma/metabolismo , Adenoma/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Carcinoma/metabolismo , Neoplasias Colorrectales/metabolismo , Femenino , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glicosilación , Factor Estimulante de Colonias de Granulocitos/genética , Células HT29 , Humanos , Captura por Microdisección con Láser , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Proteoma/metabolismo , Proteómica , Reproducibilidad de los Resultados , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Telomeres are ribonucleoproteins that cap chromosome-ends and their DNA length is controlled by counteracting elongation and shortening processes. The budding yeast Saccharomyces cerevisiae has been a leading model to study telomere DNA length control and dynamics. Its telomeric DNA is maintained at a length that slightly varies between laboratory strains, but little is known about its variation at the species level. The recent publication of the genomes of over 1,000 S. cerevisiae strains enabled us to explore telomere DNA length variation at an unprecedented scale. Here, we developed a bioinformatic pipeline (YeaISTY) to estimate telomere DNA length from whole-genome sequences and applied it to the sequenced S. cerevisiae collection. Our results revealed broad natural telomere DNA length variation among the isolates. Notably, telomere DNA length is shorter in those derived from wild rather than domesticated environments. Moreover, telomere DNA length variation is associated with mitochondrial metabolism, and this association is driven by wild strains. Overall, these findings reveal broad variation in budding yeast's telomere DNA length regulation, which might be shaped by its different ecological life-styles.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Telómero/genética , Telómero/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Unión a Telómeros/genética , Secuencia de BasesRESUMEN
Oncogene-induced senescence (OIS) is a tumor suppressor response that induces permanent cell cycle arrest in response to oncogenic signaling. Through the combined activation of the p53-p21 and p16-Rb suppressor pathways, OIS leads to the transcriptional repression of proliferative genes. Although this protective mechanism has been essentially described in primary cells, we surprisingly observed in this study that the OIS program is conserved in established colorectal cell lines. In response to the RAS oncogene and despite the inactivation of p53 and p16(INK4), HT29 cells enter senescence, up-regulate p21(WAF1), and induce senescence-associated heterochromatin foci formation. The same effect was observed in response to B-RAF(v600E) in LS174T cells. We also observed that p21(WAF1) prevents the expression of the CDC25A and PLK1 genes to induce cell cycle arrest. Using ChIP and luciferase experiments, we have observed that p21(WAF1) binds to the PLK1 promoter to induce its down-regulation during OIS induction. Following 4-5 weeks, several clones were able to resume proliferation and escape this tumor suppressor pathway. Tumor progression was associated with p21(WAF1) down-regulation and CDC25A and PLK1 reexpression. In addition, OIS and p21(WAF1) escape was associated with an increase in DNA damage, an induction of the epithelial-mesenchymal transition program, and an increase in the proportion of cells expressing the CD24(low)/CD44(high) phenotype. Results also indicate that malignant cells having escaped OIS rely on survival pathways induced by Bcl-xL/MCL1 signaling. In light of these observations, it appears that the transcriptional functions of p21(WAF1) are active during OIS and that the inactivation of this protein is associated with cell dedifferentiation and enhanced survival.
Asunto(s)
Desdiferenciación Celular , Senescencia Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína bcl-X/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/genética , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/genética , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , Mutación Missense , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteína Oncogénica p21(ras)/genética , Proteína Oncogénica p21(ras)/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Transducción de Señal/genética , Factores de Tiempo , Transcripción Genética/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/genética , Proteína bcl-X/genética , Fosfatasas cdc25/genética , Fosfatasas cdc25/metabolismo , Quinasa Tipo Polo 1RESUMEN
Domestication of plants and animals is the foundation for feeding the world human population but can profoundly alter the biology of the domesticated species. Here we investigated the effect of domestication on one of our prime model organisms, the yeast Saccharomyces cerevisiae, at a species-wide level. We tracked the capacity for sexual and asexual reproduction and the chronological life span across a global collection of 1,011 genome-sequenced yeast isolates and found a remarkable dichotomy between domesticated and wild strains. Domestication had systematically enhanced fermentative and reduced respiratory asexual growth, altered the tolerance to many stresses and abolished or impaired the sexual life cycle. The chronological life span remained largely unaffected by domestication and was instead dictated by clade-specific evolution. We traced the genetic origins of the yeast domestication syndrome using genome-wide association analysis and genetic engineering and disclosed causative effects of aneuploidy, gene presence/absence variations, copy number variations and single-nucleotide polymorphisms. Overall, we propose domestication to be the most dramatic event in budding yeast evolution, raising questions about how much domestication has distorted our understanding of the natural biology of this key model species.
Asunto(s)
Domesticación , Saccharomycetales , Animales , Variaciones en el Número de Copia de ADN , Estudio de Asociación del Genoma Completo , Estadios del Ciclo de Vida , Saccharomyces cerevisiae/genética , Saccharomycetales/genéticaRESUMEN
The STAT3 transcription factors are cytoplasmic proteins that induce gene activation in response to growth factor stimulation. Following tyrosine phosphorylation, STAT3 proteins dimerize, translocate to the nucleus, and activate specific target genes involved in cell-cycle progression. Despite its importance in cancer cells, the molecular mechanisms by which this protein is regulated in response to DNA damage remain to be characterized. In this study, we show that STAT3 is activated in response to topoisomerase I inhibition. Following treatment, STAT3 is phosphorylated on its C-terminal serine 727 residue but not on its tyrosine 705 site. We also show that topoisomerase I inhibition induced the up-regulation of the cdk5 kinase, a protein initially described in neuronal stress responses. In co-immunoprecipitations, cdk5 was found to associate with STAT3, and pulldown experiments indicated that it associates with the C-terminal activation domain of STAT3 upon DNA damage. Importantly, the cdk5-STAT3 pathway reduced DNA damage in response to topoisomerase I inhibition through the up-regulation of Eme1, an endonuclease involved in DNA repair. ChIP experiments indicated that STAT3 can be found associated with the Eme1 promoter when phosphorylated only on its serine 727 residue and not on tyrosine 705. We therefore propose that the cdk5-STAT3 oncogenic pathway plays an important role in the expression of DNA repair genes and that these proteins could be used as predictive markers of tumors that will fail to respond to chemotherapy.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Quinasa 5 Dependiente de la Ciclina/metabolismo , Daño del ADN/efectos de los fármacos , ADN-Topoisomerasas de Tipo I/metabolismo , Inhibidores Enzimáticos/farmacología , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Factor de Transcripción STAT3/metabolismo , Inhibidores de Topoisomerasa I , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Quinasa 5 Dependiente de la Ciclina/genética , ADN-Topoisomerasas de Tipo I/genética , Endodesoxirribonucleasas/biosíntesis , Endodesoxirribonucleasas/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Proteínas de Neoplasias/genética , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Fosforilación/efectos de los fármacos , Fosforilación/genética , Regiones Promotoras Genéticas/genética , Multimerización de Proteína/efectos de los fármacos , Multimerización de Proteína/genética , Estructura Terciaria de Proteína , Factor de Transcripción STAT3/genéticaRESUMEN
BACKGROUND: Anti-apoptotic signals induced downstream of HER2 are known to contribute to the resistance to current treatments of breast cancer cells that overexpress this member of the EGFR family. Whether or not some of these signals are also involved in tumor maintenance by counteracting constitutive death signals is much less understood. To address this, we investigated what role anti- and pro-apoptotic Bcl-2 family members, key regulators of cancer cell survival, might play in the viability of HER2 overexpressing breast cancer cells. METHODS: We used cell lines as an in vitro model of HER2-overexpressing cells in order to evaluate how anti-apoptotic Bcl-2, Bcl-xL and Mcl-1, and pro-apoptotic Puma and Bim impact on their survival, and to investigate how the constitutive expression of these proteins is regulated. Expression of the proteins of interest was confirmed using lysates from HER2-overexpressing tumors and through analysis of publicly available RNA expression data. RESULTS: We show that the depletion of Mcl-1 is sufficient to induce apoptosis in HER2-overexpressing breast cancer cells. This Mcl-1 dependence is due to Bim expression and it directly results from oncogenic signaling, as depletion of the oncoprotein c-Myc, which occupies regions of the Bim promoter as evaluated in ChIP assays, decreases Bim levels and mitigates Mcl-1 dependence. Consistently, a reduction of c-Myc expression by inhibition of mTORC1 activity abrogates occupancy of the Bim promoter by c-Myc, decreases Bim expression and promotes tolerance to Mcl-1 depletion. Western blot analysis confirms that naïve HER2-overexpressing tumors constitutively express detectable levels of Mcl-1 and Bim, while expression data hint on enrichment for Mcl-1 transcripts in these tumors. CONCLUSIONS: This work establishes that, in HER2-overexpressing tumors, it is necessary, and maybe sufficient, to therapeutically impact on the Mcl-1/Bim balance for efficient induction of cancer cell death.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptor ErbB-2/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Neoplasias de la Mama , Agregación Celular , Línea Celular Tumoral , Supervivencia Celular , Everolimus , Femenino , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Proteínas de la Membrana/genética , Complejos Multiproteicos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Regiones Promotoras Genéticas , Proteínas/antagonistas & inhibidores , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Interferencia de ARN , Transducción de Señal , Sirolimus/análogos & derivados , Sirolimus/farmacología , Serina-Treonina Quinasas TOR , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
Aberrantly active NF-kappaB complexes can contribute to tumorigenesis by regulating genes that promote the growth and survival of cancer cells. We have investigated NF-kappaB during the cell cycle and find that its ability to regulate the G1-phase expression of key proto-oncogenes is subject to regulation by the integrated activity of IkappaB kinase (IKK)alpha, IKKbeta, Akt and Chk1. The coordinated binding of NF-kappaB subunits to the Cyclin D1, c-Myc and Skp2 promoters is dynamic with distinct changes in promoter occupancy and RelA(p65) phosphorylation occurring through G1, S and G2 phases, concomitant with a switch from coactivator to corepressor recruitment. Akt activity is required for IKK-dependent phosphorylation of NF-kappaB subunits in G1 and G2 phases, where Chk1 is inactive. However, in S-phase, Akt is inactivated, while Chk1 phosphorylates RelA and associates with IKKalpha, inhibiting the processing of the p100 (NF-kappaB2) subunit, which also plays a critical role in the regulation of these genes. These data reveal a complex regulatory network integrating NF-kappaB with the DNA-replication checkpoint and the expression of critical regulators of cell proliferation.
Asunto(s)
Regulación de la Expresión Génica , Quinasa I-kappa B/biosíntesis , FN-kappa B/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Humanos , Quinasa I-kappa B/metabolismo , Ratones , Transcripción GenéticaRESUMEN
To understand the genetic basis and selective forces acting on longevity, it is useful to examine lifespan variation among closely related species, or ecologically diverse isolates of the same species, within a controlled environment. In particular, this approach may lead to understanding mechanisms underlying natural variation in lifespan. Here, we analyzed 76 ecologically diverse wild yeast isolates and discovered a wide diversity of replicative lifespan (RLS). Phylogenetic analyses pointed to genes and environmental factors that strongly interact to modulate the observed aging patterns. We then identified genetic networks causally associated with natural variation in RLS across wild yeast isolates, as well as genes, metabolites, and pathways, many of which have never been associated with yeast lifespan in laboratory settings. In addition, a combined analysis of lifespan-associated metabolic and transcriptomic changes revealed unique adaptations to interconnected amino acid biosynthesis, glutamate metabolism, and mitochondrial function in long-lived strains. Overall, our multiomic and lifespan analyses across diverse isolates of the same species shows how gene-environment interactions shape cellular processes involved in phenotypic variation such as lifespan.
Asunto(s)
Redes Reguladoras de Genes , Genes Fúngicos , Saccharomyces cerevisiae/fisiología , Saccharomyces/fisiología , Saccharomyces/genética , Saccharomyces cerevisiae/genéticaRESUMEN
Hybrids between diverged lineages contain novel genetic combinations but an impaired meiosis often makes them evolutionary dead ends. Here, we explore to what extent an aborted meiosis followed by a return-to-growth (RTG) promotes recombination across a panel of 20 Saccharomyces cerevisiae and S. paradoxus diploid hybrids with different genomic structures and levels of sterility. Genome analyses of 275 clones reveal that RTG promotes recombination and generates extensive regions of loss-of-heterozygosity in sterile hybrids with either a defective meiosis or a heavily rearranged karyotype, whereas RTG recombination is reduced by high sequence divergence between parental subgenomes. The RTG recombination preferentially arises in regions with low local heterozygosity and near meiotic recombination hotspots. The loss-of-heterozygosity has a profound impact on sexual and asexual fitness, and enables genetic mapping of phenotypic differences in sterile lineages where linkage analysis would fail. We propose that RTG gives sterile yeast hybrids access to a natural route for genome recombination and adaptation.
Asunto(s)
Diploidia , Hibridación Genética , Infertilidad/genética , Meiosis , Saccharomyces cerevisiae/genética , Mapeo Cromosómico , Evolución Molecular , Genoma Fúngico , Recombinación Homóloga , Fenotipo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
During the G2 phase of the cell cycle, the Aurora-A kinase plays an important role in centrosome maturation and progression to mitosis. In this study, we show in colorectal cell lines that Aurora-A expression is downregulated in response to topoisomerase I inhibition. Using chromatin immunoprecipitation assays, we have observed that the Myc transcription factor and its Max binding partner are associated with the Aurora-A promoter during the G2 phase of the cell cycle. RNA interference experiments indicated that Myc is involved in the regulation of the Aurora-A gene. Following topoisomerase I inhibition, the expression of Myc decreased whereas Mad was upregulated, and the association of Myc and Max with the promoter of the kinase was inhibited. In parallel, an increased association of Mad and Miz-1 was detected on DNA, associated with an inhibition of the recruitment of transcriptional coactivators. Interestingly, a gain of H3K9 trimethylation and HP1gamma recruitment was observed on the Aurora-A promoter following sn38 treatment, suggesting that this promoter is located within SAHF foci following genotoxic treatment. Since Aurora-A is involved in centrosome maturation, we observed as expected that topoisomerase I inhibition prevented centrosome separation but did not affect their duplication. As a consequence, this led to G2 arrest and senescence induction.These results suggest a model by which the Aurora-A gene is inactivated by the G2 checkpoint following topoisomerase I inhibition. We therefore propose the hypothesis that the coordinated overexpression of Myc and Aurora-A, together with a downregulation of Mad and Miz-1 should be tested as a prognosis signature of poor responses to topoisomerase I inhibitors.
Asunto(s)
ADN-Topoisomerasas de Tipo I/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-myc/fisiología , Aurora Quinasas , Secuencia de Bases , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Cartilla de ADN , Humanos , Regiones Promotoras GenéticasRESUMEN
Constitutive activation of STAT3 (signal transducer and activator of transcription) has been reported in several primary cancers and tumor cell lines where it induces cell transformation through a combined inhibition of apoptosis and cell-cycle activation. Several studies have suggested that STAT3 prevents cell-cycle arrest and cell death through upregulation of survival proteins and downregulation of tumor suppressors. As a consequence of anti-apoptotic and proliferative lesions, we propose that this oncogenic pathway is also involved in intrinsic drug resistance and that STAT3-expressing tumors are resistant to chemotherapeutic agents. If this hypothesis is correct, the detection of the activated form of this protein should help to define subsets of tumors that fail to respond to chemotherapy. Furthermore, interfering with the STAT3 oncogenic pathway might restore the sensitivity to anticancer drugs.
Asunto(s)
Biomarcadores/análisis , Transformación Celular Neoplásica/genética , Resistencia a Antineoplásicos/genética , Neoplasias/tratamiento farmacológico , Factor de Transcripción STAT3/genética , Transducción de Señal/genética , Apoptosis/genética , Humanos , Neoplasias/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
Besides its function as a cell cycle regulator, cyclin D1 interacts with transcription factors to regulate gene activation. In this study, we show that cyclin D1 is recruited to the p21waf1 promoter by a STAT3-NcoA complex. The association of cyclin D1 with DNA prevented the recruitment of the CBP histone acetylase and RNA polymerase II, leading to an inhibition of the p21waf1 gene. Confirming the transcriptional function of the protein, the expression of the p21waf1 gene was enhanced in cyclin D1-/- fibroblasts or upon siRNA-mediated down-regulation of the cyclin. Moreover, the STAT3-mediated activation of p21waf1 was also inhibited in breast cancer cells containing elevated levels of cyclin D1. Altogether, these results suggest that the transcriptional activities of cyclin D1 might play an important role in the regulation of cell-cycle regulatory genes and that these functions are probably involved in cell transformation.