RESUMEN
In this issue, Versluis et al.1 use a highly sensitive live-cell imaging system to examine transcription dynamics and functions of various key transcription elongation regulators at the Hsp70 loci.
Asunto(s)
Cromosomas Politénicos , Elongación de la Transcripción Genética , Animales , Cromosomas Politénicos/genética , Cromosomas Politénicos/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Transcripción Genética , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genéticaRESUMEN
Bacterial chromosomal DNA is structured and compacted by proteins known as bacterial chromatin proteins (i.e., nucleoid-associated proteins or NAPs). DNA-dependent RNA polymerase (RNAP) must frequently interact with bacterial chromatin proteins because they often bind DNA genome-wide. In some cases, RNAP must overcome barriers bacterial chromatin proteins impose on transcription. One key bacterial chromatin protein in Escherichia coli that influences transcription is the histone-like nucleoid structuring protein, H-NS. H-NS binds to DNA and forms nucleoprotein filaments. To investigate the effect of H-NS filaments on RNAP elongation, we developed an in vitro transcription assay to monitor RNAP progression on a DNA template bound by H-NS. In this method, initiation and elongation by RNAP are uncoupled by first initiating transcription in the presence of only three ribonucleoside triphosphates (rNTPs) to halt elongation just downstream of the promoter. Before elongation is restarted by addition of the fourth NTP, an H-NS filament is formed on the DNA so that transcript elongation occurs on an H-NS nucleoprotein filament template. Here, we provide detailed protocols for performing in vitro transcription through H-NS filaments, analysis of the transcription products, and visualization of H-NS filament formation on DNA by electrophoretic mobility shift assay (EMSA). These methods enable insight into how H-NS affects RNAP transcript elongation and provide a starting point to determine effects of other bacterial chromatin proteins on RNAP elongation.
Asunto(s)
ARN Polimerasas Dirigidas por ADN , Proteínas de Escherichia coli , Escherichia coli , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Transcripción Genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Elongación de la Transcripción Genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Proteínas Fimbrias/metabolismo , Proteínas Fimbrias/genéticaRESUMEN
Signal-induced transcriptional programs regulate critical biological processes through the precise spatiotemporal activation of Immediate Early Genes (IEGs); however, the mechanisms of transcription induction remain poorly understood. By combining an acute depletion system with several genomics approaches to interrogate synchronized, temporal transcription, we reveal that KAP1/TRIM28 is a first responder that fulfills the temporal and heightened transcriptional demand of IEGs. Acute KAP1 loss triggers an increase in RNA polymerase II elongation kinetics during early stimulation time points. This elongation defect derails the normal progression through the transcriptional cycle during late stimulation time points, ultimately leading to decreased recruitment of the transcription apparatus for re-initiation thereby dampening IEGs transcriptional output. Collectively, KAP1 plays a counterintuitive role by negatively regulating transcription elongation to support full activation across multiple transcription cycles of genes critical for cell physiology and organismal functions.
Asunto(s)
ARN Polimerasa II , Proteína 28 que Contiene Motivos Tripartito , Proteína 28 que Contiene Motivos Tripartito/metabolismo , Proteína 28 que Contiene Motivos Tripartito/genética , ARN Polimerasa II/metabolismo , Humanos , Cinética , Elongación de la Transcripción Genética , Genes Inmediatos-Precoces , Transcripción Genética , Transducción de Señal , Activación Transcripcional , AnimalesRESUMEN
Mutations in the Cockayne Syndrome group B (CSB) gene cause cancer in mice, but premature aging and severe neurodevelopmental defects in humans. CSB, a member of the SWI/SNF family of chromatin remodelers, plays diverse roles in regulating gene expression and transcription-coupled nucleotide excision repair (TC-NER); however, these functions do not explain the distinct phenotypic differences observed between CSB-deficient mice and humans. During investigating Cockayne Syndrome-associated genome instability, we uncover an intrinsic mechanism that involves elongating RNA polymerase II (RNAPII) undergoing transient pauses at internal T-runs where CSB is required to propel RNAPII forward. Consequently, CSB deficiency retards RNAPII elongation in these regions, and when coupled with G-rich sequences upstream, exacerbates genome instability by promoting R-loop formation. These R-loop prone motifs are notably abundant in relatively long genes related to neuronal functions in the human genome, but less prevalent in the mouse genome. These findings provide mechanistic insights into differential impacts of CSB deficiency on mice versus humans and suggest that the manifestation of the Cockayne Syndrome phenotype in humans results from the progressive evolution of mammalian genomes.
Asunto(s)
Síndrome de Cockayne , ADN Helicasas , Enzimas Reparadoras del ADN , Inestabilidad Genómica , Proteínas de Unión a Poli-ADP-Ribosa , Estructuras R-Loop , ARN Polimerasa II , Síndrome de Cockayne/genética , Síndrome de Cockayne/patología , Síndrome de Cockayne/metabolismo , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Animales , Humanos , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Ratones , ADN Helicasas/metabolismo , ADN Helicasas/genética , Estructuras R-Loop/genética , Reparación del ADN , Elongación de la Transcripción Genética , Ratones NoqueadosRESUMEN
RNA polymerase II drives mRNA gene expression, yet our understanding of Pol II degradation is limited. Using auxin-inducible degron, we degraded Pol II's RPB1 subunit, resulting in global repression. Surprisingly, certain genes exhibited increased RNA levels post-degradation. These genes are associated with GPCR ligand binding and are characterized by being less paused and comprising polycomb-bound short genes. RPB1 degradation globally increased KDM6B binding, which was insufficient to explain specific gene activation. In contrast, RPB2 degradation repressed nearly all genes, accompanied by decreased H3K9me3 and SUV39H1 occupancy. We observed a specific increase in serine 2 phosphorylated Pol II and RNA stability for RPB1 degradation-upregulated genes. Additionally, α-amanitin or UV treatment resulted in RPB1 degradation and global gene repression, unveiling subsets of upregulated genes. Our findings highlight the activated transcription elongation and increased RNA stability of signaling genes as potential mechanisms for mammalian cells to counter RPB1 degradation during stress.
Asunto(s)
ARN Polimerasa II , Estabilidad del ARN , Elongación de la Transcripción Genética , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Humanos , Ligandos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Alfa-Amanitina/farmacología , Fosforilación , ARN Mensajero/metabolismo , ARN Mensajero/genética , Histonas/metabolismo , ProteolisisRESUMEN
The RNA polymerase II (Pol II) elongation rate influences poly(A) site selection, with slow and fast Pol II derivatives causing upstream and downstream shifts, respectively, in poly(A) site utilization. In yeast, depletion of either of the histone chaperones FACT or Spt6 causes an upstream shift of poly(A) site use that strongly resembles the poly(A) profiles of slow Pol II mutant strains. Like slow Pol II mutant strains, FACT- and Spt6-depleted cells exhibit Pol II processivity defects, indicating that both Spt6 and FACT stimulate the Pol II elongation rate. Poly(A) profiles of some genes show atypical downstream shifts; this subset of genes overlaps well for FACT- or Spt6-depleted strains but is different from the atypical genes in Pol II speed mutant strains. In contrast, depletion of histone H3 or H4 causes a downstream shift of poly(A) sites for most genes, indicating that nucleosomes inhibit the Pol II elongation rate in vivo. Thus, chromatin-based control of the Pol II elongation rate is a potential mechanism, distinct from direct effects on the cleavage/polyadenylation machinery, to regulate alternative polyadenylation in response to genetic or environmental changes.
Asunto(s)
Cromatina , Histonas , Poliadenilación , ARN Polimerasa II , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Factores de Elongación Transcripcional , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Cromatina/metabolismo , Cromatina/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Histonas/metabolismo , Factores de Elongación Transcripcional/metabolismo , Factores de Elongación Transcripcional/genética , Nucleosomas/metabolismo , Nucleosomas/genética , Elongación de la Transcripción Genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Chaperonas de Histonas/metabolismo , Chaperonas de Histonas/genética , Poli A/metabolismoRESUMEN
RNA Polymerase II (Pol II) transcriptional elongation pausing is an integral part of the dynamic regulation of gene transcription in the genome of metazoans. It plays a pivotal role in many vital biological processes and disease progression. However, experimentally measuring genome-wide Pol II pausing is technically challenging and the precise governing mechanism underlying this process is not fully understood. Here, we develop RP3 (RNA Polymerase II Pausing Prediction), a network regularized logistic regression machine learning method, to predict Pol II pausing events by integrating genome sequence, histone modification, gene expression, chromatin accessibility, and protein-protein interaction data. RP3 can accurately predict Pol II pausing in diverse cellular contexts and unveil the transcription factors that are associated with the Pol II pausing machinery. Furthermore, we utilize a forward feature selection framework to systematically identify the combination of histone modification signals associated with Pol II pausing. RP3 is freely available at https://github.com/AMSSwanglab/RP3.
Asunto(s)
Código de Histonas , ARN Polimerasa II , ARN Polimerasa II/metabolismo , Humanos , Elongación de la Transcripción Genética , Cromatina/metabolismo , Cromatina/genética , Histonas/metabolismo , Aprendizaje Automático , AnimalesRESUMEN
Archaeal transcription is carried out by a multi-subunit RNA polymerase (RNAP) that is highly homologous in structure and function to eukaryotic RNAP II. Among the set of basal transcription factors, only Spt5 is found in all domains of life, but Spt5 has been shaped during evolution, which is also reflected in the heterodimerization of Spt5 with Spt4 in Archaea and Eukaryotes. To unravel the mechanistic basis of Spt4/5 function in Archaea, we performed structure-function analyses using the archaeal transcriptional machinery of Pyrococcus furiosus (Pfu). We report single-particle cryo-electron microscopy reconstructions of apo RNAP and the archaeal elongation complex (EC) in the absence and presence of Spt4/5. Surprisingly, Pfu Spt4/5 also binds the RNAP in the absence of nucleic acids in a distinct super-contracted conformation. We show that the RNAP clamp/stalk module exhibits conformational flexibility in the apo state of RNAP and that the enzyme contracts upon EC formation or Spt4/5 engagement. We furthermore identified a contact of the Spt5-NGN domain with the DNA duplex that stabilizes the upstream boundary of the transcription bubble and impacts Spt4/5 activity in vitro. This study, therefore, provides the structural basis for Spt4/5 function in archaeal transcription and reveals a potential role beyond the well-described support of elongation.
Asunto(s)
Proteínas Arqueales , ARN Polimerasas Dirigidas por ADN , Modelos Moleculares , Elongación de la Transcripción Genética , Factores de Elongación Transcripcional , Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Proteínas Arqueales/genética , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Microscopía por Crioelectrón , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Unión Proteica , Pyrococcus furiosus/enzimología , Pyrococcus furiosus/genética , Factores de Elongación Transcripcional/metabolismo , Factores de Elongación Transcripcional/química , Factores de Elongación Transcripcional/genéticaRESUMEN
Facilitates chromatin transcription (FACT) is a histone chaperone that supports transcription through chromatin in vitro, but its functional roles in vivo remain unclear. Here, we analyze the in vivo functions of FACT with the use of multi-omics analysis after rapid FACT depletion from human cells. We show that FACT depletion destabilizes chromatin and leads to transcriptional defects, including defective promoter-proximal pausing and elongation, and increased premature termination of RNA polymerase II. Unexpectedly, our analysis revealed that promoter-proximal pausing depends not only on the negative elongation factor (NELF) but also on the +1 nucleosome, which is maintained by FACT.
Asunto(s)
Cromatina , Proteínas del Grupo de Alta Movilidad , Nucleosomas , Regiones Promotoras Genéticas , ARN Polimerasa II , Transcripción Genética , Factores de Elongación Transcripcional , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Humanos , Factores de Elongación Transcripcional/metabolismo , Factores de Elongación Transcripcional/genética , Cromatina/metabolismo , Cromatina/genética , Nucleosomas/metabolismo , Nucleosomas/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas del Grupo de Alta Movilidad/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Células HeLa , Ensamble y Desensamble de Cromatina , Células HEK293 , Elongación de la Transcripción Genética , Terminación de la Transcripción GenéticaRESUMEN
The Prp19 complex (Prp19C), also named NineTeen Complex (NTC), is conserved from yeast to human and functions in many different processes such as genome stability, splicing, and transcription elongation. In the latter, Prp19C ensures TREX occupancy at transcribed genes. TREX, in turn, couples transcription to nuclear mRNA export by recruiting the mRNA exporter to transcribed genes and consequently to nascent mRNAs. Here, we assess the function of the nonessential Prp19C subunit Syf2 and the nonessential Prp19C-associated protein Cwc15 in the interaction of Prp19C and TREX with the transcription machinery, Prp19C and TREX occupancy, and transcription elongation. Whereas both proteins are important for Prp19C-TREX interaction, Syf2 is needed for full Prp19C occupancy, and Cwc15 is important for the interaction of Prp19C with RNA polymerase II and TREX occupancy. These partially overlapping functions are corroborated by a genetic interaction between Δcwc15 and Δsyf2 Finally, Cwc15 also interacts genetically with the transcription elongation factor Dst1 and functions in transcription elongation. In summary, we uncover novel roles of the Prp19C component Syf2 and the Prp19C-associated protein Cwc15 in Prp19C's function in transcription elongation.
Asunto(s)
ARN Polimerasa II , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Elongación de la Transcripción Genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Unión Proteica , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Factores de Empalme de ARNRESUMEN
The transition from transcription initiation to elongation is highly regulated in human cells but remains incompletely understood at the structural level. In particular, it is unclear how interactions between RNA polymerase II (RNA Pol II) and initiation factors are broken to enable promoter escape. Here, we reconstitute RNA Pol II promoter escape in vitro and determine high-resolution structures of initially transcribing complexes containing 8-, 10-, and 12-nt ordered RNAs and two elongation complexes containing 14-nt RNAs. We suggest that promoter escape occurs in three major steps. First, the growing RNA displaces the B-reader element of the initiation factor TFIIB without evicting TFIIB. Second, the rewinding of the transcription bubble coincides with the eviction of TFIIA, TFIIB, and TBP. Third, the binding of DSIF and NELF facilitates TFIIE and TFIIH dissociation, establishing the paused elongation complex. This three-step model for promoter escape fills a gap in our understanding of the initiation-elongation transition of RNA Pol II transcription.
Asunto(s)
Fosfoproteínas , Regiones Promotoras Genéticas , ARN Polimerasa II , Proteína de Unión a TATA-Box , Factor de Transcripción TFIIB , Factores de Transcripción , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Humanos , Factor de Transcripción TFIIB/metabolismo , Factor de Transcripción TFIIB/genética , Proteína de Unión a TATA-Box/metabolismo , Proteína de Unión a TATA-Box/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Iniciación de la Transcripción Genética , Factor de Transcripción TFIIH/metabolismo , Factor de Transcripción TFIIH/genética , Factor de Transcripción TFIIH/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Unión Proteica , Factor de Transcripción TFIIA/metabolismo , Factor de Transcripción TFIIA/genética , Transcripción Genética , Elongación de la Transcripción Genética , ARN/metabolismo , ARN/genética , Factores de Transcripción TFII/metabolismo , Factores de Transcripción TFII/genéticaRESUMEN
During development, inflammation or tissue injury, macrophages may successively engulf and process multiple apoptotic corpses via efferocytosis to achieve tissue homeostasis1. How macrophages may rapidly adapt their transcription to achieve continuous corpse uptake is incompletely understood. Transcriptional pause/release is an evolutionarily conserved mechanism, in which RNA polymerase (Pol) II initiates transcription for 20-60 nucleotides, is paused for minutes to hours and is then released to make full-length mRNA2. Here we show that macrophages, within minutes of corpse encounter, use transcriptional pause/release to unleash a rapid transcriptional response. For human and mouse macrophages, the Pol II pause/release was required for continuous efferocytosis in vitro and in vivo. Interestingly, blocking Pol II pause/release did not impede Fc receptor-mediated phagocytosis, yeast uptake or bacterial phagocytosis. Integration of data from three genomic approaches-precision nuclear run-on sequencing, RNA sequencing, and assay for transposase-accessible chromatin using sequencing (ATAC-seq)-on efferocytic macrophages at different time points revealed that Pol II pause/release controls expression of select transcription factors and downstream target genes. Mechanistic studies on transcription factor EGR3, prominently regulated by pause/release, uncovered EGR3-related reprogramming of other macrophage genes involved in cytoskeleton and corpse processing. Using lysosomal probes and a new genetic fluorescent reporter, we identify a role for pause/release in phagosome acidification during efferocytosis. Furthermore, microglia from egr3-deficient zebrafish embryos displayed reduced phagocytosis of apoptotic neurons and fewer maturing phagosomes, supporting defective corpse processing. Collectively, these data indicate that macrophages use Pol II pause/release as a mechanism to rapidly alter their transcriptional programs for efficient processing of the ingested apoptotic corpses and for successive efferocytosis.
Asunto(s)
Eferocitosis , Macrófagos , ARN Polimerasa II , Elongación de la Transcripción Genética , Animales , Humanos , Masculino , Ratones , Apoptosis , Citoesqueleto/metabolismo , Proteína 3 de la Respuesta de Crecimiento Precoz/deficiencia , Proteína 3 de la Respuesta de Crecimiento Precoz/genética , Eferocitosis/genética , Concentración de Iones de Hidrógeno , Macrófagos/inmunología , Macrófagos/metabolismo , Neuronas/metabolismo , Fagosomas/metabolismo , ARN Polimerasa II/metabolismo , Factores de Transcripción/genética , Pez Cebra/embriología , Pez Cebra/genética , Factores de TiempoRESUMEN
Direct tubular injury caused by several medications, especially chemotherapeutic drugs, is a common cause of AKI. Inhibition or loss of cyclin-dependent kinase 12 (CDK12) triggers a transcriptional elongation defect that results in deficiencies in DNA damage repair, producing genomic instability in a variety of cancers. Notably, 10-25% of individuals developed AKI after treatment with a CDK12 inhibitor, and the potential mechanism is not well understood. Here, we found that CDK12 was downregulated in the renal tubular epithelial cells in both patients with AKI and murine AKI models. Moreover, tubular cell-specific knockdown of CDK12 in mice enhanced cisplatin-induced AKI through promotion of genome instability, apoptosis, and proliferative inhibition, whereas CDK12 overexpression protected against AKI. Using the single molecule real-time (SMRT) platform on the kidneys of CDK12RTEC+/- mice, we found that CDK12 knockdown targeted Fgf1 and Cast through transcriptional elongation defects, thereby enhancing genome instability and apoptosis. Overall, these data demonstrated that CDK12 knockdown could potentiate the development of AKI by altering the transcriptional elongation defect of the Fgf1 and Cast genes, and more attention should be given to patients treated with CDK12 inhibitors to prevent AKI.
Asunto(s)
Lesión Renal Aguda , Quinasas Ciclina-Dependientes , Factor 1 de Crecimiento de Fibroblastos , Elongación de la Transcripción Genética , Animales , Humanos , Ratones , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/genética , Quinasas Ciclina-Dependientes/genética , Factor 1 de Crecimiento de Fibroblastos/genética , Inestabilidad Genómica , RiñónRESUMEN
Transcription is a tightly regulated, complex, and essential cellular process in all living organisms. Transcription is comprised of three steps, transcription initiation, elongation, and termination. The distinct transcription initiation and termination mechanisms of eukaryotic RNA polymerases I, II, and III (Pols I, II, and III) have long been appreciated. Recent methodological advances have empowered high-resolution investigations of the Pols' transcription elongation mechanisms. Here, we review the kinetic similarities and differences in the individual steps of Pol I-, II-, and III-catalyzed transcription elongation, including NTP binding, bond formation, pyrophosphate release, and translocation. This review serves as an important summation of Saccharomyces cerevisiae (yeast) Pol I, II, and III kinetic investigations which reveal that transcription elongation by the Pols is governed by distinct mechanisms. Further, these studies illustrate how basic, biochemical investigations of the Pols can empower the development of chemotherapeutic compounds.
Asunto(s)
Quimioterapia , ARN Polimerasa III , ARN Polimerasa II , ARN Polimerasa I , Saccharomyces cerevisiae , Elongación de la Transcripción Genética , Biocatálisis/efectos de los fármacos , Cinética , ARN Polimerasa I/metabolismo , ARN Polimerasa II/metabolismo , ARN Polimerasa III/metabolismo , Saccharomyces cerevisiae/enzimología , Elongación de la Transcripción Genética/efectos de los fármacosRESUMEN
Transcription elongation stalls at lesions in the DNA template1. For the DNA lesion to be repaired, the stalled transcription elongation complex (EC) has to be removed from the damaged site2. Here we show that translation, which is coupled to transcription in bacteria, actively dislodges stalled ECs from the damaged DNA template. By contrast, paused, but otherwise elongation-competent, ECs are not dislodged by the ribosome. Instead, they are helped back into processive elongation. We also show that the ribosome slows down when approaching paused, but not stalled, ECs. Our results indicate that coupled ribosomes functionally and kinetically discriminate between paused ECs and stalled ECs, ensuring the selective destruction of only the latter. This functional discrimination is controlled by the RNA polymerase's catalytic domain, the Trigger Loop. We show that the transcription-coupled DNA repair helicase UvrD, proposed to cause backtracking of stalled ECs3, does not interfere with ribosome-mediated dislodging. By contrast, the transcription-coupled DNA repair translocase Mfd4 acts synergistically with translation, and dislodges stalled ECs that were not destroyed by the ribosome. We also show that a coupled ribosome efficiently destroys misincorporated ECs that can cause conflicts with replication5. We propose that coupling to translation is an ancient and one of the main mechanisms of clearing non-functional ECs from the genome.
Asunto(s)
ARN Polimerasas Dirigidas por ADN , Escherichia coli , Biosíntesis de Proteínas , Transcripción Genética , Dominio Catalítico , ADN Helicasas/metabolismo , Reparación del ADN , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Cinética , Ribosomas/metabolismo , Moldes Genéticos , Elongación de la Transcripción Genética , Genoma BacterianoRESUMEN
Despite advances in defining diverse somatic mutations that cause myeloid malignancies, a significant heritable component for these cancers remains largely unexplained. Here, we perform rare variant association studies in a large population cohort to identify inherited predisposition genes for these blood cancers. CTR9, which encodes a key component of the PAF1 transcription elongation complex, is among the significant genes identified. The risk variants found in the cases cause loss of function and result in a â¼10-fold increased odds of acquiring a myeloid malignancy. Partial CTR9 loss of function expands human hematopoietic stem cells (HSCs) by increased super elongation complex-mediated transcriptional activity, which thereby increases the expression of key regulators of HSC self-renewal. By following up on insights from a human genetic study examining inherited predisposition to the myeloid malignancies, we define a previously unknown antagonistic interaction between the PAF1 and super elongation complexes. These insights could enable targeted approaches for blood cancer prevention.
Asunto(s)
Neoplasias Hematológicas , Fosfoproteínas , Elongación de la Transcripción Genética , Factores de Transcripción , Humanos , Neoplasias Hematológicas/genética , Células Madre Hematopoyéticas/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Fosfoproteínas/genéticaRESUMEN
Various transcript elongation factors (TEFs) including modulators of RNA polymerase II (RNAPII) activity and histone chaperones tune the efficiency of transcription in the chromatin context. TEFs are involved in establishing gene expression patterns during growth and development in Arabidopsis, while little is known about the genomic distribution of the TEFs and the way they facilitate transcription. We have mapped the genome-wide occupancy of the elongation factors SPT4-SPT5, PAF1C and FACT, relative to that of elongating RNAPII phosphorylated at residues S2/S5 within the carboxyterminal domain. The distribution of SPT4-SPT5 along transcribed regions closely resembles that of RNAPII-S2P, while the occupancy of FACT and PAF1C is rather related to that of RNAPII-S5P. Under transcriptionally challenging heat stress conditions, mutant plants lacking the corresponding TEFs are differentially impaired in transcript synthesis. Strikingly, in plants deficient in PAF1C, defects in transcription across intron/exon borders are observed that are cumulative along transcribed regions. Upstream of transcriptional start sites, the presence of FACT correlates with nucleosomal occupancy. Under stress conditions FACT is particularly required for transcriptional upregulation and to promote RNAPII transcription through +1 nucleosomes. Thus, Arabidopsis TEFs are differently distributed along transcribed regions, and are distinctly required during transcript elongation especially upon transcriptional reprogramming.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Factores de Elongación de Péptidos , ARN Polimerasa II , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Nucleosomas/genética , Nucleosomas/metabolismo , Factores de Elongación de Péptidos/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Elongación de la Transcripción Genética , Transcripción Genética , Factores de Elongación Transcripcional/metabolismoRESUMEN
IMPORTANCE: Infection with herpes simplex virus 1 (HSV-1) leads to lifelong infection due to the virus's remarkable ability to control transcription of its own genome, resulting in two transcriptional programs: lytic (highly active) and latent (restricted). The lytic program requires immediate early (IE) proteins to first repress transcription of late viral genes, which then undergo sequential de-repression, leading to a specific sequence of gene expression. Here, we show that the IE ICP4 functions to regulate the cascade by limiting RNA polymerase initiation at immediate early times. However, late viral genes that initiate too early in the absence of ICP4 do not yield mRNA as transcription stalls within gene bodies. It follows that other regulatory steps intercede to prevent elongation of genes at the incorrect time, demonstrating the precise control HSV-1 exerts over its own transcription.
Asunto(s)
Regulación Viral de la Expresión Génica , Herpesvirus Humano 1 , Proteínas Inmediatas-Precoces , Transcripción Genética , Humanos , Genes Virales/genética , Herpes Simple/virología , Herpesvirus Humano 1/genética , Proteínas Inmediatas-Precoces/deficiencia , Proteínas Inmediatas-Precoces/metabolismo , Iniciación de la Transcripción Genética , Elongación de la Transcripción Genética , Terminación de la Transcripción GenéticaRESUMEN
Physiological homeostasis becomes compromised during ageing, as a result of impairment of cellular processes, including transcription and RNA splicing1-4. However, the molecular mechanisms leading to the loss of transcriptional fidelity are so far elusive, as are ways of preventing it. Here we profiled and analysed genome-wide, ageing-related changes in transcriptional processes across different organisms: nematodes, fruitflies, mice, rats and humans. The average transcriptional elongation speed (RNA polymerase II speed) increased with age in all five species. Along with these changes in elongation speed, we observed changes in splicing, including a reduction of unspliced transcripts and the formation of more circular RNAs. Two lifespan-extending interventions, dietary restriction and lowered insulin-IGF signalling, both reversed most of these ageing-related changes. Genetic variants in RNA polymerase II that reduced its speed in worms5 and flies6 increased their lifespan. Similarly, reducing the speed of RNA polymerase II by overexpressing histone components, to counter age-associated changes in nucleosome positioning, also extended lifespan in flies and the division potential of human cells. Our findings uncover fundamental molecular mechanisms underlying animal ageing and lifespan-extending interventions, and point to possible preventive measures.
Asunto(s)
Envejecimiento , Longevidad , Elongación de la Transcripción Genética , Animales , Humanos , Ratones , Ratas , Envejecimiento/genética , Insulina/metabolismo , Longevidad/genética , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Transducción de Señal , Drosophila melanogaster/genética , Caenorhabditis elegans/genética , ARN Circular , Somatomedinas , Nucleosomas , Histonas , División Celular , Restricción CalóricaRESUMEN
Trimethylation of histone H3 lysine 4 (H3K4me3) is associated with transcriptional start sites and has been proposed to regulate transcription initiation1,2. However, redundant functions of the H3K4 SET1/COMPASS methyltransferase complexes complicate the elucidation of the specific role of H3K4me3 in transcriptional regulation3,4. Here, using mouse embryonic stem cells as a model system, we show that acute ablation of shared subunits of the SET1/COMPASS complexes leads to a complete loss of all H3K4 methylation. Turnover of H3K4me3 occurs more rapidly than that of H3K4me1 and H3K4me2 and is dependent on KDM5 demethylases. Notably, acute loss of H3K4me3 does not have detectable effects on transcriptional initiation but leads to a widespread decrease in transcriptional output, an increase in RNA polymerase II (RNAPII) pausing and slower elongation. We show that H3K4me3 is required for the recruitment of the integrator complex subunit 11 (INTS11), which is essential for the eviction of paused RNAPII and transcriptional elongation. Thus, our study demonstrates a distinct role for H3K4me3 in transcriptional pause-release and elongation rather than transcriptional initiation.