RESUMO
Cell size varies greatly between cell types, yet within a specific cell type and growth condition, cell size is narrowly distributed. Why maintenance of a cell-type specific cell size is important remains poorly understood. Here we show that growing budding yeast and primary mammalian cells beyond a certain size impairs gene induction, cell-cycle progression, and cell signaling. These defects are due to the inability of large cells to scale nucleic acid and protein biosynthesis in accordance with cell volume increase, which effectively leads to cytoplasm dilution. We further show that loss of scaling beyond a certain critical size is due to DNA becoming limiting. Based on the observation that senescent cells are large and exhibit many of the phenotypes of large cells, we propose that the range of DNA:cytoplasm ratio that supports optimal cell function is limited and that ratios outside these bounds contribute to aging.
Assuntos
Crescimento Celular , Senescência Celular/fisiologia , Citoplasma/metabolismo , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Ciclo Celular , Proliferação de Células , Tamanho Celular , Senescência Celular/genética , Fibroblastos/metabolismo , Células HEK293 , Humanos , Cultura Primária de Células , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomycetales/genética , Saccharomycetales/crescimento & desenvolvimento , Saccharomycetales/metabolismo , Transdução de SinaisRESUMO
In addition to their annotated transcript, many eukaryotic mRNA promoters produce divergent noncoding transcripts. To define determinants of divergent promoter directionality, we used genomic replacement experiments. Sequences within noncoding transcripts specified their degradation pathways, and functional protein-coding transcripts could be produced in the divergent direction. To screen for mutants affecting the ratio of transcription in each direction, a bidirectional fluorescent protein reporter construct was introduced into the yeast nonessential gene deletion collection. We identified chromatin assembly as an important regulator of divergent transcription. Mutations in the CAF-I complex caused genome-wide derepression of nascent divergent noncoding transcription. In opposition to the CAF-I chromatin assembly pathway, H3K56 hyperacetylation, together with the nucleosome remodeler SWI/SNF, facilitated divergent transcription by promoting rapid nucleosome turnover. We propose that these chromatin-mediated effects control divergent transcription initiation, complementing downstream pathways linked to early termination and degradation of the noncoding RNAs.
Assuntos
Fator 1 de Modelagem da Cromatina/metabolismo , Cromatina/metabolismo , Regulação Fúngica da Expressão Gênica , RNA Fúngico/genética , RNA não Traduzido/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Montagem e Desmontagem da Cromatina , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Estabilidade de RNA , RNA Fúngico/metabolismo , RNA não Traduzido/metabolismo , Terminação da Transcrição Genética , Transcrição GênicaRESUMO
RNA polymerase II (RNA Pol II) transcription reconstituted from purified factors suggests pre-initiation complexes (PICs) can assemble by sequential incorporation of factors at the TATA box. However, these basal transcription reactions are generally independent of activators and co-activators. To study PIC assembly under more realistic conditions, we used single-molecule microscopy to visualize factor dynamics during activator-dependent reactions in nuclear extracts. Surprisingly, RNA Pol II, TFIIF, and TFIIE can pre-assemble on enhancer-bound activators before loading into PICs, and multiple RNA Pol II complexes can bind simultaneously to create a localized cluster. Unlike TFIIF and TFIIE, TFIIH binding is singular and dependent on the basal promoter. Activator-tethered factors exhibit dwell times on the order of seconds. In contrast, PICs can persist on the order of minutes in the absence of nucleotide triphosphates, although TFIIE remains unexpectedly dynamic even after TFIIH incorporation. Our kinetic measurements lead to a new branched model for activator-dependent PIC assembly.
Assuntos
Complexo Mediador/metabolismo , RNA Polimerase II/metabolismo , Iniciação da Transcrição Genética/fisiologia , Núcleo Celular/metabolismo , Complexo Mediador/genética , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula , TATA Box/genética , Proteína de Ligação a TATA-Box/genética , Fator de Transcrição TFIIH/metabolismo , Fatores de Transcrição TFII/metabolismo , Transcrição Gênica/genéticaRESUMO
The Set3 histone deacetylase complex (Set3C) binds histone H3 dimethylated at lysine 4 (H3K4me2) to mediate deacetylation of histones in 5'-transcribed regions. To discern how Set3C affects gene expression, genome-wide transcription was analyzed in yeast undergoing a series of carbon source shifts. Deleting SET3 primarily caused changes during transition periods, as genes were induced or repressed. Surprisingly, a majority of Set3-affected genes are overlapped by noncoding RNA (ncRNA) transcription. Many Set3-repressed genes have H3K4me2 instead of me3 over promoter regions, due to either reduced H3K4me3 or ncRNA transcription from distal or antisense promoters. Set3C also represses internal cryptic promoters, but in different regions of genes than the Set2/Rpd3S pathway. Finally, Set3C stimulates some genes by repressing an overlapping antagonistic antisense transcript. These results show that overlapping noncoding transcription can fine-tune gene expression, not via the ncRNA but by depositing H3K4me2 to recruit the Set3C deacetylase.
Assuntos
Regulação Fúngica da Expressão Gênica , Histona Desacetilases/metabolismo , RNA Antissenso/genética , RNA Fúngico/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Histonas/metabolismo , Cinética , Metilação , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
The cell-fate decision leading to gametogenesis is essential for sexual reproduction. In S. cerevisiae, only diploid MATa/α but not haploid MATa or MATα cells undergo gametogenesis, known as sporulation. We find that transcription of two long noncoding RNAs (lncRNAs) mediates mating-type control of sporulation. In MATa or MATα haploids, expression of IME1, the central inducer of gametogenesis, is inhibited in cis by transcription of the lncRNA IRT1, located in the IME1 promoter. IRT1 transcription recruits the Set2 histone methyltransferase and the Set3 histone deacetylase complex to establish repressive chromatin at the IME1 promoter. Inhibiting expression of IRT1 and an antisense transcript that antagonizes the expression of the meiotic regulator IME4 allows cells expressing the haploid mating type to sporulate with kinetics that are indistinguishable from that of MATa/α diploids. Conversely, expression of the two lncRNAs abolishes sporulation in MATa/α diploids. Thus, transcription of two lncRNAs governs mating-type control of gametogenesis in yeast.
Assuntos
Regulação Fúngica da Expressão Gênica , Genes Fúngicos Tipo Acasalamento , RNA Fúngico/genética , RNA Longo não Codificante/genética , Saccharomyces cerevisiae/genética , Transcrição Gênica , Gametogênese , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Esporos Fúngicos , Fatores de Transcrição/genéticaRESUMO
RNA polymerase II elongation complexes (ECs) were assembled from nuclear extract on immobilized DNA templates and analyzed by quantitative mass spectrometry. Time-course experiments showed that initiation factor TFIIF can remain bound to early ECs, while levels of core elongation factors Spt4-Spt5, Paf1C, Spt6-Spn1, and Elf1 remain steady. Importantly, the dynamic phosphorylation patterns of the Rpb1 C-terminal domain (CTD) and the factors that recognize them change as a function of postinitiation time rather than distance elongated. Chemical inhibition of Kin28/Cdk7 in vitro blocks both Ser5 and Ser2 phosphorylation, affects initiation site choice, and inhibits elongation efficiency. EC components dependent on CTD phosphorylation include capping enzyme, cap-binding complex, Set2, and the polymerase-associated factor (PAF1) complex. By recapitulating many known features of in vivo elongation, this system reveals new details that clarify how EC-associated factors change at each step of transcription.
Assuntos
RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ativação Enzimática , Fatores de Alongamento de Peptídeos/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , RNA Polimerase II/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Many non-coding transcripts (ncRNA) generated by RNA polymerase II in S. cerevisiae are terminated by the Nrd1-Nab3-Sen1 complex. However, Sen1 helicase levels are surprisingly low compared with Nrd1 and Nab3, raising questions regarding how ncRNA can be terminated in an efficient and timely manner. We show that Sen1 levels increase during the S and G2 phases of the cell cycle, leading to increased termination activity of NNS. Overexpression of Sen1 or failure to modulate its abundance by ubiquitin-proteasome-mediated degradation greatly decreases cell fitness. Sen1 toxicity is suppressed by mutations in other termination factors, and NET-seq analysis shows that its overexpression leads to a decrease in ncRNA production and altered mRNA termination. We conclude that Sen1 levels are carefully regulated to prevent aberrant termination. We suggest that ncRNA levels and coding gene transcription termination are modulated by Sen1 to fulfill critical cell cycle-specific functions.
Assuntos
DNA Helicases/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular , Regulação Fúngica da Expressão Gênica , RNA Helicases/metabolismo , RNA Fúngico/biossíntese , RNA Mensageiro/biossíntese , RNA não Traduzido/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Terminação da Transcrição Genética , DNA Helicases/genética , Viabilidade Microbiana , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , RNA Helicases/genética , RNA Fúngico/genética , RNA Mensageiro/genética , RNA não Traduzido/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , UbiquitinaçãoRESUMO
Various factors differentially recognize trimethylated histone H3 lysine 4 (H3K4me3) near promoters, H3K4me2 just downstream, and promoter-distal H3K4me1 to modulate gene expression. This methylation "gradient" is thought to result from preferential binding of the H3K4 methyltransferase Set1/complex associated with Set1 (COMPASS) to promoter-proximal RNA polymerase II. However, other studies have suggested that location-specific cues allosterically activate Set1. Chromatin immunoprecipitation sequencing (ChIP-seq) experiments show that H3K4 methylation patterns on active genes are not universal or fixed and change in response to both transcription elongation rate and frequency as well as reduced COMPASS activity. Fusing Set1 to RNA polymerase II results in H3K4me2 throughout transcribed regions and similarly extended H3K4me3 on highly transcribed genes. Tethered Set1 still requires histone H2B ubiquitylation for activity. These results show that higher-level methylations reflect not only Set1/COMPASS recruitment but also multiple rounds of transcription. This model provides a simple explanation for non-canonical methylation patterns at some loci or in certain COMPASS mutants.
Assuntos
Histonas/metabolismo , Modelos Biológicos , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Metilação , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitinação/fisiologiaRESUMO
TFIID binds promoter DNA to recruit RNA polymerase II and other basal factors for transcription. Although the TATA-binding protein (TBP) subunit of TFIID is necessary and sufficient for in vitro transcription, the TBP-associated factor (TAF) subunits recognize downstream promoter elements, act as coactivators, and interact with nucleosomes. In yeast nuclear extracts, transcription induces stable TAF binding to downstream promoter DNA, promoting subsequent activator-independent transcription reinitiation. In vivo, promoter responses to TAF mutations correlate with the level of downstream, rather than overall, Taf1 cross-linking. We propose a new model in which TAFs function as reinitiation factors, accounting for the differential responses of promoters to various transcription factor mutations.
Assuntos
Regiões Promotoras Genéticas/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Transcrição Gênica/genética , Acetilação , Histonas/metabolismo , Mutação/genética , Ligação Proteica , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores de Transcrição/metabolismoRESUMO
Cotranscriptional histone methylations by Set1 and Set2 have been shown to affect histone acetylation at promoters and 3' regions of genes, respectively. While histone H3K4 trimethylation (H3K4me3) is thought to promote nucleosome acetylation and remodeling near promoters, we show here that H3K4 dimethylation (H3K4me2) by Set1 leads to reduced histone acetylation levels near 5' ends of genes. H3K4me2 recruits the Set3 complex via the Set3 PHD finger, localizing the Hos2 and Hst1 subunits to deacetylate histones in 5' transcribed regions. Cells lacking the Set1-Set3 complex pathway are sensitive to mycophenolic acid and have reduced polymerase levels at a Set3 target gene, suggesting a positive role in transcription. We propose that Set1 establishes two distinct chromatin zones on genes: H3K4me3 leads to high levels of acetylation and low nucleosome density at promoters, while H3K4me2 just downstream recruits the Set3 complex to suppress nucleosome acetylation and remodeling.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Histona Desacetilases/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Acetilação , Montagem e Desmontagem da Cromatina , Histona-Lisina N-Metiltransferase , Metilação , Nucleossomos/metabolismo , Telômero/metabolismoRESUMO
Dynamic interactions between RNA polymerase II and various mRNA-processing and chromatin-modifying enzymes are mediated by the changing phosphorylation pattern on the C-terminal domain (CTD) of polymerase subunit Rpb1 during different stages of transcription. Phosphorylations within the repetitive heptamer sequence (YSPTSPS) of CTD have primarily been defined using antibodies, but these do not distinguish different repeats or allow comparative quantitation. Using a CTD modified for mass spectrometry (msCTD), we show that Ser5-P and Ser2-P occur throughout the length of CTD and are far more abundant than other phosphorylation sites. msCTD extracted from cells mutated in several CTD kinases or phosphatases showed the expected changes in phosphorylation. Furthermore, msCTD associated with capping enzyme was enriched for Ser5-P while that bound to the transcription termination factor Rtt103 had higher levels of Ser2-P. These results suggest a relatively sparse and simple "CTD code."
Assuntos
RNA Polimerase II/química , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Proliferação de Células , Espectrometria de Massas , Dados de Sequência Molecular , Mutação , Fosforilação , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Transcrição GênicaRESUMO
In eukaryotes, RNA polymerase II (RNApII) transcribes messenger RNA from template DNA. Decades of experiments have identified the proteins needed for transcription activation, initiation complex assembly, and productive elongation. However, the dynamics of recruitment of these proteins to transcription complexes, and of the transitions between these steps, are poorly understood. We used multiwavelength single-molecule fluorescence microscopy to directly image and quantitate these dynamics in a budding yeast nuclear extract that reconstitutes activator-dependent transcription in vitro. A strong activator (Gal4-VP16) greatly stimulated reversible binding of individual RNApII molecules to template DNA. Binding of labeled elongation factor Spt4/5 to DNA typically followed RNApII binding, was NTP dependent, and was correlated with association of mRNA binding protein Hek2, demonstrating specificity of Spt4/5 binding to elongation complexes. Quantitative kinetic modeling shows that only a fraction of RNApII binding events are productive and implies a rate-limiting step, probably associated with recruitment of general transcription factors, needed to assemble a transcription-competent preinitiation complex at the promoter. Spt4/5 association with transcription complexes was slowly reversible, with DNA-bound RNApII molecules sometimes binding and releasing Spt4/5 multiple times. The average Spt4/5 residence time was of similar magnitude to the time required to transcribe an average length yeast gene. These dynamics suggest that a single Spt4/5 molecule remains associated during a typical transcription event, yet can dissociate from RNApII to allow disassembly of abnormally long-lived (i.e., stalled) elongation complexes.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas Nucleares/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/metabolismo , Sítios de Ligação , Proteínas Cromossômicas não Histona/genética , Cinética , Modelos Teóricos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , RNA Polimerase II/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Imagem Individual de Molécula/métodos , Fatores de Elongação da Transcrição/genéticaRESUMO
Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is commonly overexpressed in human cancers, including pancreatic ductal adenocarcinoma (PDAC). While Pin1 is dispensable for viability in mice, it is required for activated Ras to induce tumorigenesis, suggesting a role for Pin1 inhibitors in Ras-driven tumors, such as PDAC. We report the development of rationally designed peptide inhibitors that covalently target Cys113, a highly conserved cysteine located in the Pin1 active site. The inhibitors were iteratively optimized for potency, selectivity and cell permeability to give BJP-06-005-3, a versatile tool compound with which to probe Pin1 biology and interrogate its role in cancer. In parallel to inhibitor development, we employed genetic and chemical-genetic strategies to assess the consequences of Pin1 loss in human PDAC cell lines. We demonstrate that Pin1 cooperates with mutant KRAS to promote transformation in PDAC, and that Pin1 inhibition impairs cell viability over time in PDAC cell lines.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Peptidilprolil Isomerase de Interação com NIMA/antagonistas & inibidores , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Animais , Antineoplásicos/química , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Cristalografia por Raios X , Cisteína/metabolismo , Desenho de Fármacos , Inibidores Enzimáticos/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Peptidilprolil Isomerase de Interação com NIMA/química , Peptidilprolil Isomerase de Interação com NIMA/genética , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Conformação Proteica , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismoRESUMO
Two new studies in this issue of Molecular Cell (Kim et al., 2013 and Wu et al., 2013) provide new insights and reignite debate over how histone H2B ubiquitination promotes methylation of histone H3 lysine 4.
RESUMO
The C-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II (RNApII), coordinates recruitment of RNA- and chromatin-modifying factors to transcription complexes. It is unclear whether the CTD communicates with the catalytic core region of Rpb1 and thus must be physically connected, or instead can function as an independent domain. To address this question, CTD was transferred to other RNApII subunits. Fusions to Rpb4 or Rpb6, two RNApII subunits located near the original position of CTD, support viability in a strain carrying a truncated Rpb1. In contrast, CTD fusion to Rpb9 on the other side of RNApII does not. Rpb4-CTD and Rpb6-CTD proteins are functional for phosphorylation and recruitment of various factors, albeit with some restrictions and minor defects. Normal CTD functions are not transferred to RNApI or RNApIII by Rbp6-CTD. These results show that, with some spatial constraints, CTD can function even when disconnected from Rpb1.
Assuntos
Cromatina/genética , Estrutura Terciária de Proteína/genética , RNA Polimerase II/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , RNA Polimerases Dirigidas por DNA/biossíntese , RNA Polimerases Dirigidas por DNA/genética , Fosforilação , RNA , RNA Polimerase II/química , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/química , Serina/metabolismoRESUMO
The essential helicase-like protein Sen1 mediates termination of RNA Polymerase II (Pol II) transcription at snoRNAs and other noncoding RNAs in yeast. A mutation in the Pol II subunit Rpb1 that increases the elongation rate increases read-through transcription at Sen1-mediated terminators. Termination and growth defects in sen1 mutant cells are partially suppressed by a slowly transcribing Pol II mutant and are exacerbated by a faster-transcribing Pol II mutant. Deletion of the nuclear exosome subunit Rrp6 allows visualization of noncoding RNA intermediates that are terminated but not yet processed. Sen1 mutants or faster-transcribing Pol II increase the average lengths of preprocessed snoRNA, CUT, and SUT transcripts, while slowed Pol II transcription produces shorter transcripts. These connections between transcription rate and Sen1 activity support a model whereby kinetic competition between elongating Pol II and Sen1 helicase establishes the temporal and spatial window for early Pol II termination.
Assuntos
DNA Helicases/metabolismo , RNA Helicases/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Terminação da Transcrição Genética , Alelos , Motivos de Aminoácidos , Substituição de Aminoácidos , Mapeamento Cromossômico , DNA Helicases/genética , Cinética , RNA Helicases/genética , RNA Polimerase II/genética , RNA Polimerase II/fisiologia , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Monoubiquitination of histone H2B (to H2Bub1) is required for downstream events including histone H3 methylation, transcription, and mRNA export. The mechanisms and players regulating these events have not yet been completely delineated. Here, we show that the conserved Ran-binding protein Mog1 is required to sustain normal levels of H2Bub1 and H3K4me3 in Saccharomyces cerevisiae Mog1 is needed for gene body recruitment of Rad6, Bre1, and Rtf1 that are involved in H2B ubiquitination and genetically interacts with these factors. We provide evidence that the absence of MOG1 impacts on cellular processes such as transcription, DNA replication, and mRNA export, which are linked to H2Bub1. Importantly, the mRNA export defect in mog1Δ strains is exacerbated by the absence of factors that decrease H2Bub1 levels. Consistent with a role in sustaining H2Bub and H3K4me3 levels, Mog1 co-precipitates with components that participate in these modifications such as Bre1, Rtf1, and the COMPASS-associated factors Shg1 and Sdc1. These results reveal a novel role for Mog1 in H2B ubiquitination, transcription, and mRNA biogenesis.
Assuntos
Histonas/metabolismo , RNA Polimerase II/genética , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteína ran de Ligação ao GTP/metabolismo , Imunoprecipitação da Cromatina , Repressão Epigenética , Regulação Fúngica da Expressão Gênica , Histonas/genética , RNA Polimerase II/metabolismo , Transporte de RNA , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Transcrição Gênica , Ubiquitinação , Proteína ran de Ligação ao GTP/genéticaRESUMO
The RNA polymerase II (RNApII) transcription cycle consists of multiple steps involving dozens of protein factors. Here we describe a useful approach to study the dynamics of initiation and early elongation, comprising an in vitro transcription system in which complexes are assembled on immobilized DNA templates and analyzed by quantitative mass spectrometry. This unbiased screening system allows quantitation of RNApII complex components on either naked DNA or chromatin templates. In addition to transcription, the system reproduces co-transcriptional mRNA capping and multiple transcription-related histone modifications. In combination with other biochemical and genetic methods, this approach can provide insights into the mechanistic details of gene expression by RNApII.
Assuntos
Proteômica , RNA Polimerase II/metabolismo , Transcrição Gênica , Espectrometria de Massas , Complexos Multiproteicos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Transcriptional memory is critical for the faster reactivation of necessary genes upon environmental changes and requires that the genes were previously in an active state. However, whether transcriptional repression also displays 'memory' of the prior transcriptionally inactive state remains unknown. In this study, we show that transcriptional repression of â¼540 genes in yeast occurs much more rapidly if the genes have been previously repressed during carbon source shifts. This novel transcriptional response has been termed transcriptional repression memory (TREM). Interestingly, Rpd3L histone deacetylase (HDAC), targeted to active promoters induces TREM. Mutants for Rpd3L exhibit increased acetylation at active promoters and delay TREM significantly. Surprisingly, the interaction between H3K4me3 and Rpd3L via the Pho23 PHD finger is critical to promote histone deacetylation and TREM by Rpd3L. Therefore, we propose that an active mark, H3K4me3 enriched at active promoters, instructs Rpd3L HDAC to induce histone deacetylation and TREM.