RESUMO
The interactions of ultrasound with biological materials are exploited for diagnostic, interventional, and therapeutic applications in humans and can improve productivity in industrial-scale generation of organic molecules such as biofuels, vaccines, and antibodies. Accordingly, there is great interest in better understanding the biological effects of ultrasound. We studied the impact of low-intensity pulsed ultrasound (LIPUS) on RNA expression and metabolism of S. cerevisiae. Although the transcript expression signature of LIPUS-treated cells does not differ significantly from that of untreated cells after 5 days, metabolomic profiling by chemical-isotopic-labeling-liquid-chromatography-mass-spectrometry suggests that LIPUS has an impact on the pathways of pyrimidine, proline, alanine, aspartate, glutamate, and arginine metabolism. Therefore, LIPUS triggers metabolic effects beyond reprogramming of the core pathways of carbon metabolism. Further characterization of metabolism will likely be important for elucidation of the biological effects of LIPUS.
Assuntos
Metaboloma/efeitos da radiação , RNA Mensageiro/efeitos da radiação , Saccharomyces cerevisiae/efeitos da radiação , Ondas Ultrassônicas , Cromatografia Líquida , Espectrometria de Massas , RNA Mensageiro/análise , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
BACKGROUND: The maintenance of viability during periods when a glycolytic carbon source is limited (or absent) is a major obstacle for cells whose mitochondrial DNA (mtDNA) has been damaged or lost. METHODS: We utilized genome wide transcriptional profiling and in gel mobility analyses to examine the transcriptional response and characterize defects in the phosphorylation dependent signaling events that occur during acute glucose starvation in ρ(0) cells that lack mtDNA. Genetic and pharmacological interventions were employed to clarify the contribution of nutrient responsive kinases to regulation of the transcription factors that displayed abnormal phosphoregulation in ρ(0) cells. RESULTS: The transcriptional response to glucose deprivation is dampened but not blocked in ρ(0) cells. Genes regulated by the transcription factors Mig1, Msn2, Gat1, and Ume6 were noticeably affected and phosphorylation of these factors in response to nutrient depletion is abnormal in ρ(0) cells. Regulation of the nutrient responsive kinases PKA and Snf1 remains normal in ρ(0) cells. The phosphorylation defect results from ATP depletion and loss of the activity of kinases including GSK3ß, Rim15, and Yak1. Interventions which rescue phosphoregulation of transcription factors bolster maintenance of viability in ρ(0) cells during subsequent glucose deprivation. CONCLUSIONS: A subset of nutrient responsive kinases is especially sensitive to ATP levels and their misregulation may underlie regulatory defects presented by ρ(0) cells. GENERAL SIGNIFICANCE: Abnormal regulation of mitochondrial function is implicated in numerous human disorders. This work illustrates that some signaling pathways are more sensitive than others to metabolic defects caused by mitochondrial dysfunction.
Assuntos
DNA Mitocondrial/genética , Regulação Fúngica da Expressão Gênica , Glucose/deficiência , Saccharomyces cerevisiae/metabolismo , Divisão Celular , Glucose/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Ativação TranscricionalRESUMO
Information about how yeast metabolism is rewired in response to internal and external cues can inform the development of metabolic engineering strategies for food, fuel, and chemical production in this organism. We report a new metabolomics workflow for the characterization of such metabolic rewiring. The workflow combines efficient cell lysis without using chemicals that may interfere with downstream sample analysis and differential chemical isotope labeling liquid chromatography mass spectrometry (CIL LC-MS) for in-depth yeast metabolome profiling. Using (12)C- and (13)C-dansylation (Dns) labeling to analyze the amine/phenol submetabolome, we detected and quantified a total of 5719 peak pairs or metabolites. Among them, 120 metabolites were positively identified using a library of 275 Dns-metabolite standards, and 2980 metabolites were putatively identified based on accurate mass matches to metabolome databases. We also applied (12)C- and (13)C-dimethylaminophenacyl (DmPA) labeling to profile the carboxylic acid submetabolome and detected over 2286 peak pairs, from which 33 metabolites were positively identified using a library of 188 DmPA-metabolite standards, and 1595 metabolites were putatively identified. Using this workflow for metabolomic profiling of cells challenged by ammonium limitation revealed unexpected links between ammonium assimilation and pantothenate accumulation that might be amenable to engineering for better acetyl-CoA production in yeast. We anticipate that efforts to improve other schemes of metabolic engineering will benefit from application of this workflow to multiple cell types.
Assuntos
Compostos de Amônio/metabolismo , Engenharia Metabólica/métodos , Metabolômica/métodos , Saccharomyces cerevisiae/metabolismo , Acetilcoenzima A/biossíntese , Aminas/metabolismo , Ácidos Carboxílicos/metabolismo , Cromatografia Líquida/métodos , Mineração de Dados , Marcação por Isótopo , Espectrometria de Massas/métodos , Fenóis/metabolismoRESUMO
The promoter activity of yeast genes can depend on lysine 56 (K56) acetylation of histone H3. This modification of H3 is performed by lysine acetylase Rtt109 acting in concert with histone chaperone Asf1. We have examined the contributions of Rtt109, Asf1, and H3 K56 acetylation to nutrient regulation of a well-studied metabolic gene, ARG1. As expected, Rtt109, Asf1, and H3 K56 acetylation are required for maximal transcription of ARG1 under inducing conditions. However, Rtt109 and Asf1 also inhibit ARG1 under repressing conditions. This inhibition requires Asf1 binding to H3-H4 and Rtt109 KAT activity, but not tail acetylation of H3-H4 or K56 acetylation of H3. These observations suggest the existence of a unique mechanism of transcriptional regulation by Rtt109. Indeed, chromatin immunoprecipitation and genetic interaction studies support a model in which promoter-targeted Rtt109 represses ARG1 by silencing a pathway of transcriptional activation that depends on ASF1. Collectively, our results show that ARG1 transcription intensity at its induced and repressed set points is controlled by different mechanisms of functional interplay between Rtt109 and Asf1.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Histona Acetiltransferases/metabolismo , Chaperonas Moleculares/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Acetilação/efeitos dos fármacos , Arginase/genética , Proteínas de Ciclo Celular/genética , Imunoprecipitação da Cromatina , Meios de Cultura/farmacologia , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Histona Acetiltransferases/genética , Histonas/genética , Histonas/metabolismo , Lisina/metabolismo , Chaperonas Moleculares/genética , Mutação , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Some metabolic enzymes normally occur in the nucleus and cytoplasm. These compartments differ in molecular composition. Since post-translational modification and interaction with allosteric effectors can tune enzyme activity, it follows that the behavior of an enzyme as a catalyst may differ between the cytoplasm and nucleus. We explored this possibility for the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Homogenates of pristine nuclei and cytoplasms isolated from Xenopus laevis oocytes were used for whole compartment activity profiling in a near-physiological buffer. Titrations of NAD+ revealed similar whole compartment activity profiles for GAPDH in nuclear and cytoplasmic homogenates. Surprisingly however GAPDH in these compartments did not have the same behavior in assays of the dependence of initial velocity (v0) on G3P concentration. First, the peak v0 for nuclear GAPDH was up to 2.5-fold higher than the peak for cytoplasmic GAPDH. Second, while Michaelis Menten-like behavior was observed in all assays of cytoplasm, the v0 versus [G3P] plots for nuclear GAPDH typically exhibited a non-Michaelis Menten (sigmoidal) profile. Apparent Km and Vmax (G3P) values for nuclear GAPDH activity were highly variable, even between replicates of the same sample. Possible sources of this variability include in vitro processing of a metabolite that allosterically regulates GAPDH, turnover of a post-translational modification of the enzyme, and fluctuation of the state of interaction of GAPDH with other proteins. Collectively these findings are consistent with the hypothesis that the environment of the nucleus is distinct from the environment of the cytoplasm with regard to GAPDH activity and its modulation. This finding warrants further comparison of the regulation of nuclear and cytoplasmic GAPDH, as well as whole compartment activity profiling of other enzymes of metabolism with cytosolic and nuclear pools.
Assuntos
Núcleo Celular , Gliceraldeído-3-Fosfato Desidrogenases , Citoplasma , Citosol , BioensaioRESUMO
Asf1 is a conserved histone H3/H4 chaperone. We find that Asf1 in budding yeast promotes an essential cellular response to replication stress caused by the ribonucleotide reductase inhibitor hydroxyurea. That is, Asf1 stimulates derepression of DNA damage response (DDR) genes during the S phase. Derepression of DDR genes strongly correlates with Asf1 binding to their promoters. Having identified the C terminus and histone-binding domains of Asf1 as molecular determinants of its constitutive and inducible association with chromatin, we tested whether Asf1 binding to DDR genes is mechanistically important for their derepression. Our results provide little support for this hypothesis. Rather, the contribution of Asf1 to DDR gene derepression depends on its ability to stimulate H3K56 acetylation by lysine acetyltransferase Rtt109. The precise regulation of H3K56 acetylation in the promoters of DDR genes is unexpected: DDR gene promoters are occupied by H3K56-acetylated nucleosomes under repressing conditions, and the steady state level of H3K56 promoter acetylation does not change upon derepression. We propose that replication-coupled deposition of Lys(56)-acetylated H3 poises the DDR genes in newly synthesized daughter duplexes for derepression during the S phase. In this model, the presence of a histone mark that destabilizes nucleosomes is compatible with suppression of transcription because in the uninduced state, DDR gene promoters are constitutively occupied by a potent repressor-corepressor complex.
Assuntos
Proteínas de Ciclo Celular/genética , Dano ao DNA/fisiologia , Chaperonas Moleculares/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Estresse Fisiológico/fisiologia , Fosfatase Ácida/genética , Fosfatase Ácida/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/fisiologia , Regulação Fúngica da Expressão Gênica , Biblioteca Gênica , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Hidroxiureia/farmacologia , Chaperonas Moleculares/metabolismo , Inibidores da Síntese de Ácido Nucleico/farmacologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/fisiologia , Estresse Fisiológico/efeitos dos fármacosRESUMO
In solid organs, cells of the same "type" can vary in their molecular phenotype. The basis of this state variation is being revealed by characterizing cell features including the expression pattern of mRNAs and the internal distribution of proteins. Here, the variability of metabolic state between cells is probed by enzyme activity profiling. We study individual cells of types that can be identified during the post-mitotic phase of oogenesis in Xenopus laevis. Whole-cell homogenates of isolated oocytes are used for kinetic analysis of enzymes, with a focus on the initial reaction rate. For each oocyte type studied, the activity signatures of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and malate dehydrogenase 1 (MDH1) vary more between the homogenates of single oocytes than between repeat samplings of control homogenates. Unexpectedly, the activity signatures of GAPDH and MDH1 strongly co-vary between oocytes of each type and change in strength of correlation during oogenesis. Therefore, variability of the kinetic behavior of these housekeeping enzymes between "identical" cells is physiologically programmed. Based on these findings, we propose that single-cell profiling of enzyme kinetics will improve understanding of how metabolic state heterogeneity is related to heterogeneity revealed by omics methods including proteomics, epigenomics, and metabolomics.
RESUMO
Little is known about what enzyme complexes or mechanisms control global lysine acetylation in the amino-terminal tails of the histones. Here, we show that glucose induces overall acetylation of H3 K9, 18, 27 and H4 K5, 8, 12 in quiescent yeast cells mainly by stimulating two KATs, Gcn5 and Esa1. Genetic and pharmacological perturbation of carbon metabolism, combined with (1)H-NMR metabolic profiling, revealed that glucose induction of KAT activity directly depends on increased glucose catabolism. Glucose-inducible Esa1 and Gcn5 activities predominantly reside in the picNuA4 and SAGA complexes, respectively, and act on chromatin by an untargeted mechanism. We conclude that direct metabolic regulation of globally acting KATs can be a potent driving force for reconfiguration of overall histone acetylation in response to a physiological cue.
Assuntos
Glucose/metabolismo , Glicólise , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismo , Acetilação , Histonas/química , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismoRESUMO
Chromatin assembly in a crude DEAE (CD) fraction from budding yeast is ATP dependent and generates arrays of physiologically spaced nucleosomes which significantly protect constituent DNA from restriction endonuclease digestion. The CD fractions from mutants harboring deletions of the genes encoding histone-binding factors (NAP1, ASF1, and a subunit of CAF-I) and SNF2-like DEAD/H ATPases (SNF2, ISW1, ISW2, CHD1, SWR1, YFR038w, and SPT20) were screened for activity in this replication-independent system. ASF1 deletion substantially inhibits assembly, a finding consistent with published evidence that Asf1p is a chromatin assembly factor. Surprisingly, a strong assembly defect is also associated with deletion of CHD1, suggesting that like other SNF2-related groups of nucleic acid-stimulated ATPases, the chromodomain (CHD) group may contain a member involved in chromatin reconstitution. In contrast to the effects of disrupting ASF1 and CHD1, deletion of SNF2 is associated with increased resistance of chromatin to digestion by micrococcal nuclease. We discuss the possible implications of these findings for current understanding of the diversity of mechanisms by which chromatin reconstitution and remodeling can be achieved in vivo.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona , Proteínas Nucleares , Nucleossomos/genética , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Deleção de Genes , Inativação Gênica , Genes Fúngicos , Chaperonas Moleculares , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Acetylation and phosphorylation of the amino-terminal tails of the core histones fluctuate on a global scale in concert with other major events in chromosome metabolism. A ubiquitin ligase, the anaphase-promoting complex (APC), controls events in chromosome metabolism such as sister chromatid cohesion and may regulate H3 phosphorylation by targeting Aurora A, one of several S10-directed H3 kinases in vertebrate cells, for destruction by the proteasome. Our analysis of apc10Delta and apc11(ts) loss-of-function mutants reveals that the APC controls the global level of H3 S10 phosphorylation in cycling yeast cells. Surprisingly, it also regulates dephosphorylation of H3 and global deacetylation of H2B, H3, and H4 during exit from the cell cycle into G(0). Genetic, biochemical, and microarray analyses suggest that APC-dependent cell cycle control of H3 phosphorylation is exerted at the level of an Aurora H3 kinase, Ipl1p, while APC-dependent transcriptional induction of GLC7, an essential H3 phosphatase, contributes to sustained H3 dephosphorylation upon cell cycle withdrawal. Collectively, our results establish that core histone acetylation state and H3 phosphorylation are physiologically regulated by the APC and suggest a model in which global reconfiguration of H3 phosphorylation state involves APC-dependent control of both an H3 kinase and a conserved phosphatase.
Assuntos
Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Acetilação , Ciclossomo-Complexo Promotor de Anáfase , Aurora Quinases , Sequência de Bases , Ciclo Celular , Cromatina/metabolismo , DNA Fúngico/genética , Genes Fúngicos , Histonas/química , Peptídeos e Proteínas de Sinalização Intracelular , Mutação , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína Fosfatase 1 , Proteínas Serina-Treonina Quinases , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Complexos Ubiquitina-Proteína Ligase/genéticaRESUMO
The mechanisms of biological chromatin assembly and their regulation have been studied intensively using cellular extracts, particularly those from the embryonic cells of various metazoans. Here we describe how to prepare and use a crude chromatographic fraction from budding yeast, which also supports biological chromatin assembly. In this system, nucleosomes are assembled by a replication-independent mechanism into physiologically spaced arrays that significantly protect underlying DNA from restriction endonuclease digestion. The formation of correctly spaced nucleosome arrays absolutely requires ATP and exogenous core histones of yeast or Drosophila. We have explored how cell cycle and DNA damage signals affect assembly activity in this system.
Assuntos
Montagem e Desmontagem da Cromatina , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Fracionamento Celular/métodos , DNA Fúngico/metabolismo , Proteínas de Drosophila/metabolismo , Histonas/metabolismo , Técnicas In Vitro , Micologia/métodos , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoAssuntos
Células/enzimologia , RNA Polimerase III/metabolismo , RNA Polimerase I/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Núcleo Celular/genética , Montagem e Desmontagem da Cromatina , Epigênese Genética , RNA Polimerase I/genética , RNA Polimerase III/genética , Processamento Pós-Transcricional do RNA , Transdução de Sinais , Transcrição Gênica , Leveduras/enzimologia , Leveduras/genéticaRESUMO
Abnormal respiratory metabolism plays a role in numerous human disorders. We find that regulation of overall histone acetylation is perturbed in respiratory-incompetent (ρ(0)) yeast. Because histone acetylation is highly sensitive to acetyl-coenzyme A (acetyl-CoA) availability, we sought interventions that suppress this ρ(0) phenotype through reprogramming metabolism. Nutritional intervention studies led to the discovery that genetic coactivation of the mitochondrion-to-nucleus retrograde (RTG) response and the AMPK (Snf1) pathway prevents abnormal histone deacetylation in ρ(0) cells. Metabolic profiling of signaling mutants uncovered links between chromatin-dependent phenotypes of ρ(0) cells and metabolism of ATP, acetyl-CoA, glutathione, branched-chain amino acids, and the storage carbohydrate trehalose. Importantly, RTG/AMPK activation reprograms energy metabolism to increase the supply of acetyl-CoA to lysine acetyltransferases and extend the chronological lifespan of ρ(0) cells. Our results strengthen the framework for rational design of nutrient supplementation schemes and drug-discovery initiatives aimed at mimicking the therapeutic benefits of dietary interventions.
Assuntos
Respiração Celular , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilcoenzima A/metabolismo , Acetilação , Núcleo Celular/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transdução de Sinais , Fatores de TempoRESUMO
The histone chaperone Asf1 and the chromatin remodeler SWI/SNF have been separately implicated in derepression of the DNA damage response (DDR) genes in yeast cells treated with genotoxins that cause replication interference. Using genetic and biochemical approaches, we have tested if derepression of the DDR genes in budding yeast involves functional interplay between Asf1 and SWI/SNF. We find that Asf1 and SWI/SNF are both recruited to DDR genes under replication stress triggered by hydroxyurea, and have detected a soluble complex that contains Asf1 and the Snf2 subunit of SWI/SNF. SWI/SNF recruitment to DDR genes however does not require Asf1, and deletion of Snf2 does not affect Asf1 occupancy of DDR gene promoters. A checkpoint engagement defect is sufficient to explain the synthetic effect of deletion of ASF1 and SNF2 on derepression of the DDR genes in hydroxyurea-treated cells. Collectively, our results show that the DDR genes fall into a class in which Asf1 and SWI/SNF independently control transcriptional induction.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Dano ao DNA/fisiologia , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Imunoprecipitação da Cromatina , Dano ao DNA/genética , Replicação do DNA/genética , Replicação do DNA/fisiologia , Citometria de Fluxo , Immunoblotting , Ligação ProteicaRESUMO
The RNA polymerase III pre-initiation complex (PIC) assembled on yeast tRNA genes naturally causes replication fork pausing that contributes to genome instability. Mechanistic coupling of the fork pausing activity of tRNA genes to replication has long been considered likely, but only recently demonstrated. In contrast to the expectation that this coupling might occur by a passive mechanism such as direct disruption of transcription factor-DNA complexes by a component of the replisome, it turns out that disassembly of the RNA polymerase III PIC is actively controlled by the replication stress checkpoint signal transduction pathway. This advance supports a new model in which checkpoint-dependent disassembly of the transcription machinery at tRNA genes is a vital component of an overall system of genome stability control that also targets replication and DNA repair proteins.
RESUMO
Asf1 is a conserved histone H3/H4 chaperone that can assemble and disassemble nucleosomes and promote histone acetylation. Set2 is an H3 K36 methyltransferase. The functions of these proteins intersect in the context of transcription elongation by RNA polymerase II: both contribute to the establishment of repressive chromatin structures that inhibit spurious intragenic transcription. Here we characterize further interactions between budding yeast (Saccharomyces cerevisiae) Asf1 and Set2 using assays of intragenic transcription, H3/H4 posttranslational modification, coding region cross-linking of Asf1 and Set2, and cooccurrence of Asf1 and Set2 in protein complexes. We find that at some genes Asf1 and Set2 control chromatin metabolism as components of separate pathways. However, the existence of a low-abundance complex containing both proteins suggests that Asf1 and Set2 can more directly collaborate in chromatin regulation. Consistent with this possibility, we show that Asf1 stimulates Set2 occupancy of the coding region of a highly transcribed gene by a mechanism that depends on Asf1 binding to H3/H4. This function of Asf1 promotes the switch from di- to trimethylation of H3 K36 at that gene. These results support the view that Set2 function in chromatin metabolism can intimately involve histone chaperone Asf1.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Histonas/metabolismo , Metiltransferases/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/genética , Cromatina/genética , Cromatina/metabolismo , Dano ao DNA , DNA Fúngico/genética , DNA Fúngico/metabolismo , Deleção de Genes , Redes Reguladoras de Genes , Genes Fúngicos , Histonas/química , Metilação , Metiltransferases/genética , Modelos Biológicos , Chaperonas Moleculares/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transcrição GênicaRESUMO
In budding yeast, the transcriptional machinery at tRNA genes naturally interferes with replication in a way that can promote chromosome breakage. Here we show that a signaling module composed of core components of the replication stress checkpoint pathway represses this fork-pausing machinery in normally cycling and genotoxin-treated cells. Specifically, the sensor kinase Mec1, the signaling adaptor Mrc1 and the transducer kinase Rad53 relay signals that globally repress tRNA gene transcription during unchallenged proliferation and under conditions of replication stress. Repressive signaling in genotoxin-treated cells requires Rad53-dependent activation of a conserved repressor of tRNA gene transcription, Maf1. Cells lacking Maf1 are sensitive to replication stress under conditions of elevated tRNA gene transcription. We propose that checkpoint control of the fork-pausing activity of tRNA genes complements the repertoire of replisome-targeted mechanisms by which checkpoint proteins promote faithful DNA replication.