RESUMO
The packaging of the genetic material into chromatin imposes the remodeling of this barrier to allow efficient transcription. RNA polymerase II activity is coupled with several histone modification complexes that enforce remodeling. How RNA polymerase III (Pol III) counteracts the inhibitory effect of chromatin is unknown. We report here a mechanism where RNA Polymerase II (Pol II) transcription is required to prime and maintain nucleosome depletion at Pol III loci and contributes to efficient Pol III recruitment upon re-initiation of growth from stationary phase in Fission yeast. The Pcr1 transcription factor participates in the recruitment of Pol II, which affects local histone occupancy through the associated SAGA complex and a Pol II phospho-S2 CTD / Mst2 pathway. These data expand the central role of Pol II in gene expression beyond mRNA synthesis.
Assuntos
Montagem e Desmontagem da Cromatina , RNA Polimerase II , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromatina/genética , Cromatina/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Transcrição GênicaRESUMO
The epitranscriptome has emerged as a new fundamental layer of control of gene expression. Nevertheless, the determination of the transcriptome-wide occupancy and function of RNA modifications remains challenging. Here we have developed Rho-seq, an integrated pipeline detecting a range of modifications through differential modification-dependent rhodamine labeling. Using Rho-seq, we confirm that the reduction of uridine to dihydrouridine (D) by the Dus reductase enzymes targets tRNAs in E. coli and fission yeast. We find that the D modification is also present on fission yeast mRNAs, particularly those encoding cytoskeleton-related proteins, which is supported by large-scale proteome analyses and ribosome profiling. We show that the α-tubulin encoding mRNA nda2 undergoes Dus3-dependent dihydrouridylation, which affects its translation. The absence of the modification on nda2 mRNA strongly impacts meiotic chromosome segregation, resulting in low gamete viability. Applying Rho-seq to human cells revealed that tubulin mRNA dihydrouridylation is evolutionarily conserved.
Assuntos
Segregação de Cromossomos , Escherichia coli/genética , Meiose , Processamento Pós-Transcricional do RNA , RNA Bacteriano/genética , RNA Fúngico/genética , RNA Mensageiro/genética , Schizosaccharomyces/genética , Uridina/metabolismo , Cromossomos Bacterianos , Cromossomos Fúngicos , Cromossomos Humanos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Evolução Molecular , Células HCT116 , Humanos , Oxirredução , RNA Bacteriano/metabolismo , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Análise de Sequência de RNA , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
Nutrient availability has a profound impact on cell fate. Upon nitrogen starvation, wild-type fission yeast cells uncouple cell growth from cell division to generate small, round-shaped cells that are competent for sexual differentiation. The TORC1 (TOR complex 1) and TORC2 complexes exert opposite controls on cell growth and cell differentiation, but little is known about how their activity is coordinated. We show that transfer RNA (tRNA) modifications by Elongator are critical for this regulation by promoting the translation of both key components of TORC2 and repressors of TORC1. We further identified the TORC2 pathway as an activator of Elongator by down-regulating a Gsk3 (glycogen synthase kinase 3)-dependent inhibitory phosphorylation of Elongator. Therefore, a feedback control is operating between TOR complex (TORC) signaling and tRNA modification by Elongator to enforce the advancement of mitosis that precedes cell differentiation.
Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Nutrientes/genética , Elongação Traducional da Cadeia Peptídica/genética , RNA de Transferência/genética , Schizosaccharomyces/genética , Transdução de Sinais/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Regulação Fúngica da Expressão Gênica/genética , Quinase 3 da Glicogênio Sintase/genética , Mitose/genética , Fosforilação/genéticaRESUMO
Antisense (as)lncRNAs can regulate gene expression but the underlying mechanisms and the different cofactors involved remain unclear. Using Native Elongating Transcript sequencing, here we show that stabilization of antisense Exo2-sensitivite lncRNAs (XUTs) results in the attenuation, at the nascent transcription level, of a subset of highly expressed genes displaying prominent promoter-proximal nucleosome depletion and histone acetylation. Mechanistic investigations on the catalase gene ctt1 revealed that its induction following oxidative stress is impaired in Exo2-deficient cells, correlating with the accumulation of an asXUT. Interestingly, expression of this asXUT was also activated in wild-type cells upon oxidative stress, concomitant to ctt1 induction, indicating a potential attenuation feedback. This attenuation correlates with asXUT abundance, it is transcriptional, characterized by low RNAPII-ser5 phosphorylation, and it requires an histone deacetylase activity and the conserved Set2 histone methyltransferase. Finally, we identified Dicer as another RNA processing factor acting on ctt1 induction, but independently of Exo2. We propose that asXUTs could modulate the expression of their paired-sense genes when it exceeds a critical threshold, using a conserved mechanism independent of RNAi.
Assuntos
Regulação Fúngica da Expressão Gênica , RNA Antissenso/metabolismo , RNA Fúngico/metabolismo , RNA Longo não Codificante/metabolismo , Schizosaccharomyces/genética , Acetilação , Catalase/genética , Endorribonucleases/metabolismo , Exodesoxirribonucleases/genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Estresse Oxidativo/genética , Regiões Promotoras Genéticas , Interferência de RNA , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Transcrição Gênica/genéticaRESUMO
The distribution of modified histones within the fission yeast Schizosaccharomyces pombe genome is ultimately dependent upon the transcriptional activity and in turn influences the ability of the polymerases to bind and progress through the chromatin template. The Chromatin Immunoprecipitation-Polymerase Chain Reaction (ChIP-PCR) method currently provides the highest resolution, accuracy, and reproducibility to characterize histones modifications within a defined region of the genome. The following protocol details the method applied to S. pombe.
Assuntos
Imunoprecipitação da Cromatina/métodos , Metilação de DNA/fisiologia , Genoma Fúngico , Reação em Cadeia da Polimerase/métodos , Schizosaccharomyces , Ubiquitinação/fisiologia , Acetilação , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMO
The cell fate decision leading to gametogenesis requires the convergence of multiple signals on the promoter of a master regulator. In fission yeast, starvation-induced signaling leads to the transcriptional induction of the ste11 gene, which encodes the central inducer of mating and gametogenesis, known as sporulation. We find that the long intergenic non-coding (linc) RNA rse1 is transcribed divergently upstream of the ste11 gene. During vegetative growth, rse1 directly recruits a Mug187-Lid2-Set1 complex that mediates cis repression at the ste11 promoter through SET3C-dependent histone deacetylation. The absence of rse1 bypasses the starvation-induced signaling and induces gametogenesis in the presence of nutrients. Our data reveal that the remodeling of chromatin through ncRNA scaffolding of repressive complexes that is observed in higher eukaryotes is a conserved, likely very ancient mechanism for tight control of cell differentiation.
Assuntos
RNA Fúngico/metabolismo , RNA Longo não Codificante/metabolismo , Schizosaccharomyces/fisiologia , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Antisense transcription can regulate sense gene expression. However, previous annotations of antisense transcription units have been based on detection of mature antisense long noncoding (aslnc)RNAs by RNA-seq and/or microarrays, only giving a partial view of the antisense transcription landscape and incomplete molecular bases for antisense-mediated regulation. Here, we used native elongating transcript sequencing to map genome-wide nascent antisense transcription in fission yeast. Strikingly, antisense transcription was detected for most protein-coding genes, correlating with low sense transcription, especially when overlapping the mRNA start site. RNA profiling revealed that the resulting aslncRNAs mainly correspond to cryptic Xrn1/Exo2-sensitive transcripts (XUTs). ChIP-seq analyses showed that antisense (as)XUT's expression is associated with specific histone modification patterns. Finally, we showed that asXUTs are controlled by the histone chaperone Spt6 and respond to meiosis induction, in both cases anti-correlating with levels of the paired-sense mRNAs, supporting physiological significance to antisense-mediated gene attenuation. Our work highlights that antisense transcription is much more extended than anticipated and might constitute an additional nonpromoter determinant of gene regulation complexity.
Assuntos
Regulação Fúngica da Expressão Gênica , RNA Antissenso/biossíntese , Schizosaccharomyces/genética , Transcrição Gênica , Chaperonas de Histonas/metabolismo , Código das Histonas , Meiose/genética , Elongação Traducional da Cadeia Peptídica , Interferência de RNA , Estabilidade de RNA , RNA Antissenso/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Análise de Sequência de RNARESUMO
In fission yeast, the ste11 gene encodes the master regulator initiating the switch from vegetative growth to gametogenesis. In a previous paper, we showed that the methylation of H3K4 and consequent promoter nucleosome deacetylation repress ste11 induction and cell differentiation (Materne et al., 2015) but the regulatory steps remain poorly understood. Here we report a genetic screen that highlighted H2B deubiquitylation and the RSC remodeling complex as activators of ste11 expression. Mechanistic analyses revealed more complex, opposite roles of H2Bubi at the promoter where it represses expression, and over the transcribed region where it sustains it. By promoting H3K4 methylation at the promoter, H2Bubi initiates the deacetylation process, which decreases chromatin remodeling by RSC. Upon induction, this process is reversed and efficient NDR (nucleosome depleted region) formation leads to high expression. Therefore, H2Bubi represses gametogenesis by opposing the recruitment of RSC at the promoter of the master regulator ste11 gene.
Assuntos
Montagem e Desmontagem da Cromatina , Regulação Fúngica da Expressão Gênica , Histonas/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Fatores de Transcrição/antagonistas & inibidores , UbiquitinaçãoRESUMO
Tumor initiation in the intestine can rapidly occur from Lgr5(+) crypt columnar stem cells. Dclk1 is a marker of differentiated Tuft cells and, when coexpressed with Lgr5, also marks intestinal cancer stem cells. Here, we show that Elp3, the catalytic subunit of the Elongator complex, is required for Wnt-driven intestinal tumor initiation and radiation-induced regeneration by maintaining a subpool of Lgr5(+)/Dclk1(+)/Sox9(+) cells. Elp3 deficiency dramatically delayed tumor appearance in Apc-mutated intestinal epithelia and greatly prolonged mice survival without affecting the normal epithelium. Specific ablation of Elp3 in Lgr5(+) cells resulted in marked reduction of polyp formation upon Apc inactivation, in part due to a decreased number of Lgr5(+)/Dclk1(+)/Sox9(+) cells. Mechanistically, Elp3 is induced by Wnt signaling and promotes Sox9 translation, which is needed to maintain the subpool of Lgr5(+)/Dclk1(+) cancer stem cells. Consequently, Elp3 or Sox9 depletion led to similar defects in Dclk1(+) cancer stem cells in ex vivo organoids. Finally, Elp3 deficiency strongly impaired radiation-induced intestinal regeneration, in part because of decreased Sox9 protein levels. Together, our data demonstrate the crucial role of Elp3 in maintaining a subpopulation of Lgr5-derived and Sox9-expressing cells needed to trigger Wnt-driven tumor initiation in the intestine.
Assuntos
Histona Acetiltransferases/metabolismo , Intestinos/fisiopatologia , Neoplasias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Regeneração/fisiologia , Proteínas Wnt/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Quinases Semelhantes a Duplacortina , Expressão Gênica/efeitos da radiação , Células HCT116 , Células HEK293 , Células HT29 , Histona Acetiltransferases/genética , Humanos , Mucosa Intestinal/metabolismo , Intestinos/efeitos da radiação , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Neoplasias/genética , Neoplasias/patologia , Proteínas do Tecido Nervoso/genética , Técnicas de Cultura de Órgãos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Regeneração/genética , Regeneração/efeitos da radiação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Proteínas Wnt/genéticaRESUMO
The phosphorylation of the RNA polymerase II C-terminal domain (CTD) plays a key role in delineating transcribed regions within chromatin by recruiting histone methylases and deacetylases. Using genome-wide nucleosome mapping, we show that CTD S2 phosphorylation controls nucleosome dynamics in the promoter of a subset of 324 genes, including the regulators of cell differentiation ste11 and metabolic adaptation inv1. Mechanistic studies on these genes indicate that during gene activation a local increase of phospho-S2 CTD nearby the promoter impairs the phospho-S5 CTD-dependent recruitment of Set1 and the subsequent recruitment of specific HDACs, which leads to nucleosome depletion and efficient transcription. The early increase of phospho-S2 results from the phosphorylation of the CTD S2 kinase Lsk1 by MAP kinase in response to cellular signalling. The artificial tethering of the Lsk1 kinase at the ste11 promoter is sufficient to activate transcription. Therefore, signalling through the CTD code regulates promoter nucleosomes dynamics.
Assuntos
Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional , RNA Polimerase II/metabolismo , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Dados de Sequência Molecular , Fosforilação , Regiões Promotoras Genéticas , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Análise de Sequência de DNA , Transdução de Sinais , Transcrição Gênica , Ativação TranscricionalRESUMO
Cyclin-dependent kinase (Cdk) activation and RNA polymerase II transcription are linked by the Cdk7 kinase, which phosphorylates Cdks as a trimeric Cdk-activating kinase (CAK) complex, and serine 5 within the polymerase II (Pol II) C-terminal domain (CTD) as transcription factor TFIIH-bound CAK. However, the physiological importance of integrating these processes is not understood. Besides the Cdk7 ortholog Mcs6, fission yeast possesses a second CAK, Csk1. The two enzymes have been proposed to act redundantly to activate Cdc2. Using an improved analogue-sensitive Mcs6-as kinase, we show that Csk1 is not a relevant CAK for Cdc2. Further analyses revealed that Csk1 lacks a 20-amino-acid sequence required for its budding yeast counterpart, Cak1, to bind Cdc2. Transcriptome profiling of the Mcs6-as mutant in the presence or absence of the budding yeast Cak1 kinase, in order to uncouple the CTD kinase and CAK activities of Mcs6, revealed an unanticipated role of the CAK branch in the transcriptional control of the cluster of genes implicated in ribosome biogenesis and cell growth. The analysis of a Cdc2 CAK site mutant confirmed these data. Our data show that the Cdk7 kinase modulates transcription through its well-described RNA Pol II CTD kinase activity and also through the Cdc2-activating kinase activity.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Regulação Fúngica da Expressão Gênica , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Proteína Quinase CDC2/metabolismo , Quinases Ciclina-Dependentes/química , Ativação Enzimática , Fosforilação , Estrutura Terciária de Proteína , Schizosaccharomyces/metabolismo , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Sexual reproduction is a fundamental aspect of eukaryotic cells, and a conserved feature of gametogenesis is its dependency on a master regulator. The ste11 gene was isolated more than 20 years ago by the Yamamoto laboratory as a suppressor of the uncontrolled meiosis driven by a pat1 mutant. Numerous studies from this laboratory and others have established the role of the Ste11 transcription factor as the master regulator of the switch between proliferation and differentiation in fission yeast. The transcriptional and post-transcriptional controls of ste11 expression are intricate, but most are not redundant. Whereas the transcriptional controls ensure that the gene is transcribed at a high level only when nutrients are rare, the post-transcriptional controls restrict the ability of Ste11 to function as a transcription factor to the G1-phase of the cell cycle from where the differentiation programme is initiated. Several feedback loops ensure that the cell fate decision is irreversible. The complete panel of molecular mechanisms operating to warrant the timely expression of the ste11 gene and its encoded protein basically mirrors the advances in the understanding of the numerous ways by which gene expression can be modulated.
Assuntos
Gametogênese , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Fatores de Transcrição/metabolismo , Proliferação de Células , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Transcrição/genética , Transcrição Gênica/genéticaRESUMO
The large Mediator (L-Mediator) is a general coactivator of RNA polymerase II transcription and is formed by the reversible association of the small Mediator (S-Mediator) and the kinase-module-harboring Cdk8. It is not known how the kinase module association/dissociation is regulated. We describe the fission yeast Cdk11-L-type cyclin pombe (Lcp1) complex and show that its inactivation alters the global expression profile in a manner very similar to that of mutations of the kinase module. Cdk11 is broadly distributed onto chromatin and phosphorylates the Med27 and Med4 Mediator subunits on conserved residues. The association of the kinase module and the S-Mediator is strongly decreased by the inactivation of either Cdk11 or the mutation of its target residues on the Mediator. These results show that Cdk11-Lcp1 regulates the association of the kinase module and the S-Mediator to form the L-Mediator complex.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Complexo Mediador/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Sequência de Aminoácidos , Cromatina/metabolismo , Quinase 8 Dependente de Ciclina/metabolismo , Perfilação da Expressão Gênica , Complexo Mediador/genética , Dados de Sequência Molecular , Mutação , Fosforilação , Schizosaccharomyces/metabolismo , Alinhamento de Sequência , Transcrição GênicaRESUMO
The "CTD code" links the combinatorial potential of the modifications found on the Rpb1 C-terminal domain (CTD) to the growing group of CTD binding effectors. The genetic dissection of serine 2 and serine 7 function within the CTD in both budding and fission yeast reveals distinct in vivo requirement.
Assuntos
Estrutura Terciária de Proteína/fisiologia , RNA Polimerase II/fisiologia , Saccharomycetales/fisiologia , Schizosaccharomyces/fisiologia , Mutação , Fenótipo , Fosforilação , RNA Polimerase II/químicaRESUMO
We compared the DNA-binding activity of transcription factors and gene expression patterns in BJ human diploid fibroblasts (HDFs) expressing or not telomerase (hTERT) in stress-induced premature senescence (SIPS). Senescent BJ cells were also studied. Hydrogen peroxide (H2O2)-induced SIPS modulated gene expression in both BJ and hTERT-BJ1 cells. Increased p21(WAF-1) mRNA level was amongst the common gene expression changes in BJ and hTERT-BJ1 cells induced by SIPS. Telomerase expression markedly changed gene expression in non-stressful conditions. Expression patterns of senescent BJ cells partially overlapped those of BJ and hTERT-BJ1 cells in SIPS. The basal levels of DNA-binding activity of NF-kappaB and phosphorylated ATF-2 were different in BJ and hTERT-BJ1 cells. Both cell lines displayed a higher DNA-binding activity of p53 and HIF-1 72 h after H2O2 exposure. Our results indicate that similar mechanisms involving p21(WAF-1) and probably p53 are at work in BJ and hTERT-BJ1 HDFs under H2O2-induced SIPS, suggesting that generalized DNA damage rather than telomere length/telomerase plays a crucial role in H2O2induced SIPS. We propose that H2O2-induced SIPS involves a rearrangement of proliferative and apoptotic pathways. The marked changes in gene expression induced by telomerase suggest that apart from immortalization of HDFs, telomerase also alters the normal cellular functions but does not protect against SIPS.
Assuntos
Senescência Celular/genética , Fibroblastos/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Telomerase/metabolismo , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica/fisiologia , Humanos , Peróxido de Hidrogênio/farmacologia , Masculino , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Telomerase/genética , Fatores de Transcrição/metabolismoRESUMO
Normal human diploid fibroblasts (HDFs) exposed to a single H(2)O(2) subcytotoxic stress display features of premature senescence, termed stress-induced premature senescence (SIPS). In this work, our aim was to study SIPS in Werner syndrome (WS) fibroblasts, derived from a patient with WS, a disease resembling accelerated aging. The subcytotoxic dose for WS fibroblasts was found to be inferior to that of normal HDFs, indicating WS fibroblasts are more sensitive to hydrogen peroxide than normal HDFs. SA beta-gal activity has been shown to occur both in vitro and in vivo, and we studied the proportion of WS cells positive for SA beta-gal. Intriguingly, the percentage of positive cells did not increase with the dose of H(2)O(2) used. Contrary to other HDFs, the DNA-binding activity of p53 in WS fibroblasts did not increase in SIPS. We found, based on our results, that WS fibroblasts feature an altered stress response and do not reach SIPS from H(2)O(2). We suggest that the proportion of cells that in normal HDFs would enter SIPS instead die in WS fibroblasts. Last, we propose that aging derives from a loss of integrity of the chromatin structure, which occurs faster in WS patients.