RESUMEN
Cyclin-dependent kinase 7 (CDK7), part of the general transcription factor TFIIH, promotes gene transcription by phosphorylating the C-terminal domain of RNA polymerase II (RNA Pol II). Here, we combine rapid CDK7 kinase inhibition with multi-omics analysis to unravel the direct functions of CDK7 in human cells. CDK7 inhibition causes RNA Pol II retention at promoters, leading to decreased RNA Pol II initiation and immediate global downregulation of transcript synthesis. Elongation, termination, and recruitment of co-transcriptional factors are not directly affected. Although RNA Pol II, initiation factors, and Mediator accumulate at promoters, RNA Pol II complexes can also proceed into gene bodies without promoter-proximal pausing while retaining initiation factors and Mediator. Further downstream, RNA Pol II phosphorylation increases and initiation factors and Mediator are released, allowing recruitment of elongation factors and an increase in RNA Pol II elongation velocity. Collectively, CDK7 kinase activity promotes the release of initiation factors and Mediator from RNA Pol II, facilitating RNA Pol II escape from the promoter.
Asunto(s)
Quinasa Activadora de Quinasas Ciclina-Dependientes , Quinasas Ciclina-Dependientes , Regiones Promotoras Genéticas , ARN Polimerasa II , Iniciación de la Transcripción Genética , Humanos , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/genética , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Complejo Mediador/metabolismo , Complejo Mediador/genética , Células HeLa , Factor de Transcripción TFIIH/metabolismo , Factor de Transcripción TFIIH/genética , Células HEK293RESUMEN
At active human genes, the +1 nucleosome is located downstream of the RNA polymerase II (RNA Pol II) pre-initiation complex (PIC). However, at inactive genes, the +1 nucleosome is found further upstream, at a promoter-proximal location. Here, we establish a model system to show that a promoter-proximal +1 nucleosome can reduce RNA synthesis in vivo and in vitro, and we analyze its structural basis. We find that the PIC assembles normally when the edge of the +1 nucleosome is located 18 base pairs (bp) downstream of the transcription start site (TSS). However, when the nucleosome edge is located further upstream, only 10 bp downstream of the TSS, the PIC adopts an inhibited state. The transcription factor IIH (TFIIH) shows a closed conformation and its subunit XPB contacts DNA with only one of its two ATPase lobes, inconsistent with DNA opening. These results provide a mechanism for nucleosome-dependent regulation of transcription initiation.
Asunto(s)
Nucleosomas , ARN Polimerasa II , Humanos , Nucleosomas/genética , ARN Polimerasa II/metabolismo , Regiones Promotoras Genéticas , Factor de Transcripción TFIIH/metabolismo , ADN/genética , ADN/química , Transcripción Genética , Sitio de Iniciación de la TranscripciónRESUMEN
Transcription by RNA polymerase II (Pol II) is coupled to pre-mRNA splicing, but the underlying mechanisms remain poorly understood. Co-transcriptional splicing requires assembly of a functional spliceosome on nascent pre-mRNA, but whether and how this influences Pol II transcription remains unclear. Here we show that inhibition of pre-mRNA branch site recognition by the spliceosome component U2 snRNP leads to a widespread and strong decrease in new RNA synthesis from human genes. Multiomics analysis reveals that inhibition of U2 snRNP function increases the duration of Pol II pausing in the promoter-proximal region, impairs recruitment of the pause release factor P-TEFb, and reduces Pol II elongation velocity at the beginning of genes. Our results indicate that efficient release of paused Pol II into active transcription elongation requires the formation of functional spliceosomes and that eukaryotic mRNA biogenesis relies on positive feedback from the splicing machinery to the transcription machinery.
Asunto(s)
ARN Polimerasa II/metabolismo , ARN Mensajero/biosíntesis , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Empalmosomas/enzimología , Elongación de la Transcripción Genética , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Retroalimentación Fisiológica , Regulación de la Expresión Génica , Células HeLa , Humanos , Células K562 , Factor B de Elongación Transcripcional Positiva/genética , Factor B de Elongación Transcripcional Positiva/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa II/genética , Precursores del ARN/genética , Precursores del ARN/metabolismo , Empalme del ARN , ARN Mensajero/genética , Ribonucleoproteína Nuclear Pequeña U2/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Empalmosomas/genética , Factores de TiempoRESUMEN
Eukaryotic RNA polymerase II (Pol II) contains a tail-like, intrinsically disordered carboxy-terminal domain (CTD) comprised of heptad-repeats, that functions in coordination of the transcription cycle and in coupling transcription to co-transcriptional processes. The CTD repeat number varies between species and generally increases with genome size, but the reasons for this are unclear. Here, we show that shortening the CTD in human cells to half of its length does not generally change pre-mRNA synthesis or processing in cells. However, CTD shortening decreases the duration of promoter-proximal Pol II pausing, alters transcription of putative enhancer elements, and delays transcription activation after stimulation of the MAP kinase pathway. We suggest that a long CTD is required for efficient enhancer-dependent recruitment of Pol II to target genes for their rapid activation.
Asunto(s)
ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , Eliminación de Secuencia , Activación Transcripcional , Elementos de Facilitación Genéticos , Perfilación de la Expresión Génica , Humanos , Sistema de Señalización de MAP Quinasas , Regiones Promotoras Genéticas , Dominios Proteicos , ARN Polimerasa II/genéticaRESUMEN
At the end of protein-coding genes, RNA polymerase (Pol) II undergoes a concerted transition that involves 3'-processing of the pre-mRNA and transcription termination. Here, we present a genome-wide analysis of the 3'-transition in budding yeast. We find that the 3'-transition globally requires the Pol II elongation factor Spt5 and factors involved in the recognition of the polyadenylation (pA) site and in endonucleolytic RNA cleavage. Pol II release from DNA occurs in a narrow termination window downstream of the pA site and requires the "torpedo" exonuclease Rat1 (XRN2 in human). The Rat1-interacting factor Rai1 contributes to RNA degradation downstream of the pA site. Defects in the 3'-transition can result in increased transcription at downstream genes.
Asunto(s)
ADN de Hongos/metabolismo , Procesamiento de Término de ARN 3' , ARN Polimerasa II/metabolismo , Precursores del ARN/biosíntesis , ARN de Hongos/biosíntesis , ARN Mensajero/biosíntesis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Sitios de Unión , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , ADN de Hongos/genética , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Modelos Genéticos , Unión Proteica , ARN Polimerasa II/genética , Precursores del ARN/genética , ARN de Hongos/genética , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
Stabilization of messenger RNA is an important step in posttranscriptional gene regulation. In the nucleus and cytoplasm of eukaryotic cells it is generally achieved by 5' capping and 3' polyadenylation, whereas additional mechanisms exist in bacteria and organelles. The mitochondrial mRNAs in the yeast Saccharomyces cerevisiae comprise a dodecamer sequence element that confers RNA stability and 3'-end processing via an unknown mechanism. Here, we isolated the protein that binds the dodecamer and identified it as Rmd9, a factor that is known to stabilize yeast mitochondrial RNA. We show that Rmd9 associates with mRNA around dodecamer elements in vivo and that recombinant Rmd9 specifically binds the element in vitro. The crystal structure of Rmd9 bound to its dodecamer target reveals that Rmd9 belongs to the family of pentatricopeptide (PPR) proteins and uses a previously unobserved mode of specific RNA recognition. Rmd9 protects RNA from degradation by the mitochondrial 3'-exoribonuclease complex mtEXO in vitro, indicating that recognition and binding of the dodecamer element by Rmd9 confers stability to yeast mitochondrial mRNAs.
Asunto(s)
Proteínas de la Membrana/metabolismo , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Regiones no Traducidas 3' , Genes Mitocondriales , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Motivos de Nucleótidos , Unión Proteica , Dominios Proteicos , ARN Mensajero/química , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Mouse embryonic stem cells (mESCs) can adopt naïve, ground, and paused pluripotent states that give rise to unique transcriptomes. Here, we use transient transcriptome sequencing (TT-seq) to define both coding and non-coding transcription units (TUs) in these three pluripotent states and combine TT-seq with RNA polymerase II occupancy profiling to unravel the kinetics of RNA metabolism genome-wide. Compared to the naïve state (serum), RNA synthesis and turnover rates are globally reduced in the ground state (2i) and the paused state (mTORi). The global reduction in RNA synthesis goes along with a genome-wide decrease of polymerase elongation velocity, which is related to epigenomic features and alterations in the Pol II termination window. Our data suggest that transcription activity is the main determinant of steady state mRNA levels in the naïve state and that genome-wide changes in transcription kinetics invoke ground and paused pluripotent states.
Asunto(s)
ARN Polimerasa II , Transcriptoma , Animales , Cinética , Ratones , Células Madre Embrionarias de Ratones/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , Transcriptoma/genéticaRESUMEN
The Cdk8 kinase module (CKM) is a dissociable part of the coactivator complex mediator, which regulates gene transcription by RNA polymerase II. The CKM has both negative and positive functions in gene transcription that remain poorly understood at the mechanistic level. In order to reconstitute the role of the CKM in transcription initiation, we prepared recombinant CKM from the yeast Saccharomyces cerevisiae. We showed that CKM bound to the core mediator (cMed) complex, sterically inhibiting cMed from binding to the polymerase II preinitiation complex (PIC) in vitro. We further showed that the Cdk8 kinase activity of the CKM weakened CKM-cMed interaction, thereby facilitating dissociation of the CKM and enabling mediator to bind the PIC in order to stimulate transcription initiation. Finally, we report that the kinase activity of Cdk8 is required for gene activation during the stressful condition of heat shock in vivo but not under steady-state growth conditions. Based on these results, we propose a model in which the CKM negatively regulates mediator function at upstream-activating sequences by preventing mediator binding to the PIC at the gene promoter. However, during gene activation in response to stress, the Cdk8 kinase activity of the CKM may release mediator and allow its binding to the PIC, thereby accounting for the positive function of CKM. This may impart improved adaptability to stress by allowing a rapid transcriptional response to environmental changes, and we speculate that a similar mechanism in metazoans may allow the precise timing of developmental transcription programs.
Asunto(s)
Quinasa 8 Dependiente de Ciclina/metabolismo , Complejo Mediador/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Modelos Moleculares , Unión Proteica , Mapas de Interacción de ProteínasRESUMEN
The growth of human cancer cells is driven by aberrant enhancer and gene transcription activity. Here, we use transient transcriptome sequencing (TT-seq) to map thousands of transcriptionally active putative enhancers in fourteen human cancer cell lines covering seven types of cancer. These enhancers were associated with cell type-specific gene expression, enriched for genetic variants that predispose to cancer, and included functionally verified enhancers. Enhancer-promoter (E-P) pairing by correlation of transcription activity revealed ~ 40,000 putative E-P pairs, which were depleted for housekeeping genes and enriched for transcription factors, cancer-associated genes, and 3D conformational proximity. The cell type specificity and transcription activity of target genes increased with the number of paired putative enhancers. Our results represent a rich resource for future studies of gene regulation by enhancers and their role in driving cancerous cell growth.
Asunto(s)
Elementos de Facilitación Genéticos , Perfilación de la Expresión Génica/métodos , Neoplasias/genética , Análisis de Secuencia de ADN/métodos , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Células HCT116 , Humanos , Mutación , Especificidad de Órganos , Análisis de Secuencia de ARN , Activación TranscripcionalRESUMEN
The Nrd1-Nab3-Sen1 (NNS) complex is essential for controlling pervasive transcription and generating sn/snoRNAs in S. cerevisiae. The NNS complex terminates transcription of noncoding RNA genes and promotes exosome-dependent processing/degradation of the released transcripts. The Trf4-Air2-Mtr4 (TRAMP) complex polyadenylates NNS target RNAs and favors their degradation. NNS-dependent termination and degradation are coupled, but the mechanism underlying this coupling remains enigmatic. Here we provide structural and functional evidence demonstrating that the same domain of Nrd1p interacts with RNA polymerase II and Trf4p in a mutually exclusive manner, thus defining two alternative forms of the NNS complex, one involved in termination and the other in degradation. We show that the Nrd1-Trf4 interaction is required for optimal exosome activity in vivo and for the stimulation of polyadenylation of NNS targets by TRAMP in vitro. We propose that transcription termination and RNA degradation are coordinated by switching between two alternative partners of the NNS complex.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , ARN Polimerasa II/metabolismo , ARN de Hongos/metabolismo , ARN no Traducido/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Terminación de la Transcripción Genética , Sitios de Unión , ADN Polimerasa Dirigida por ADN/química , Exosomas/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación de Ácido Nucleico , Poliadenilación , Estabilidad del ARN , Proteínas de Unión al ARN/metabolismoRESUMEN
The 3'-ends of eukaryotic pre-mRNAs are processed in the nucleus by a large multiprotein complex, the cleavage and polyadenylation factor (CPF). CPF cleaves RNA, adds a poly(A) tail and signals transcription termination. CPF harbors four enzymatic activities essential for these processes, but how these are coordinated remains poorly understood. Several subunits of CPF, including two protein phosphatases, are also found in the related 'associated with Pta1' (APT) complex, but the relationship between CPF and APT is unclear. Here, we show that the APT complex is physically distinct from CPF. The 21 kDa Syc1 protein is associated only with APT, and not with CPF, and is therefore the defining subunit of APT. Using ChIP-seq, PAR-CLIP and RNA-seq, we show that Syc1/APT has distinct, but possibly overlapping, functions from those of CPF. Syc1/APT plays a more important role in sn/snoRNA production whereas CPF processes the 3'-ends of protein-coding pre-mRNAs. These results define distinct protein machineries for synthesis of mature eukaryotic protein-coding and non-coding RNAs.
Asunto(s)
Complejos Multiproteicos/metabolismo , ARN no Traducido/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcripción Genética , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Inmunoprecipitación de Cromatina , Complejos Multiproteicos/genética , Subunidades de Proteína , ARN Nucleolar Pequeño/genética , ARN Nucleolar Pequeño/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
Metabolic control of gene expression coordinates the levels of specific gene products to meet cellular demand for their activities. This control can be exerted by metabolites acting as regulatory signals and/or a class of metabolic enzymes with dual functions as regulators of gene expression. However, little is known about how metabolic signals affect the balance between enzymatic and regulatory roles of these dual functional proteins. We previously described the RNA binding activity of a 63 kDa chloroplast protein from Chlamydomonas reinhardtii, which has been implicated in expression of the psbA mRNA, encoding the D1 protein of photosystem II. Here, we identify this factor as dihydrolipoamide acetyltransferase (DLA2), a subunit of the chloroplast pyruvate dehydrogenase complex (cpPDC), which is known to provide acetyl-CoA for fatty acid synthesis. Analyses of RNAi lines revealed that DLA2 is involved in the synthesis of both D1 and acetyl-CoA. Gel filtration analyses demonstrated an RNP complex containing DLA2 and the chloroplast psbA mRNA specifically in cells metabolizing acetate. An intrinsic RNA binding activity of DLA2 was confirmed by in vitro RNA binding assays. Results of fluorescence microscopy and subcellular fractionation experiments support a role of DLA2 in acetate-dependent localization of the psbA mRNA to a translation zone within the chloroplast. Reciprocally, the activity of the cpPDC was specifically affected by binding of psbA mRNA. Beyond that, in silico analysis and in vitro RNA binding studies using recombinant proteins support the possibility that RNA binding is an ancient feature of dihydrolipoamide acetyltransferases. Our results suggest a regulatory function of DLA2 in response to growth on reduced carbon energy sources. This raises the intriguing possibility that this regulation functions to coordinate the synthesis of lipids and proteins for the biogenesis of photosynthetic membranes.
Asunto(s)
Carbono/metabolismo , Tilacoides/metabolismo , Chlamydomonas reinhardtii/metabolismo , Microscopía Fluorescente , Biogénesis de Organelos , Complejo de Proteína del Fotosistema II/metabolismo , Proteínas de Plantas/biosíntesis , Biosíntesis de ProteínasRESUMEN
Bypass of Ess1 (Bye1) is a nuclear protein with a domain resembling the central domain in the transcription elongation factor TFIIS. Here we show that Bye1 binds with its TFIIS-like domain (TLD) to RNA polymerase (Pol) II, and report crystal structures of the Bye1 TLD bound to Pol II and three different Pol II-nucleic acid complexes. Like TFIIS, Bye1 binds with its TLD to the Pol II jaw and funnel. In contrast to TFIIS, however, it neither alters the conformation nor the in vitro functions of Pol II. In vivo, Bye1 is recruited to chromatin via its TLD and occupies the 5'-region of active genes. A plant homeo domain (PHD) in Bye1 binds histone H3 tails with trimethylated lysine 4, and this interaction is enhanced by the presence of neighboring posttranslational modifications (PTMs) that mark active transcription and conversely is impaired by repressive PTMs. We identify putative human homologs of Bye1, the proteins PHD finger protein 3 and death-inducer obliterator, which are both implicated in cancer. These results establish Bye1 as the founding member of a unique family of chromatin transcription factors that link histones with active PTMs to transcribing Pol II.
Asunto(s)
Cromatina/metabolismo , Modelos Moleculares , Complejos Multiproteicos/química , ARN Polimerasa II/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/fisiología , Transcripción Genética/fisiología , Factores de Elongación Transcripcional/química , Escherichia coli , Análisis por Micromatrices , Complejos Multiproteicos/metabolismo , Peptidilprolil Isomerasa de Interacción con NIMA , Isomerasa de Peptidilprolil/metabolismo , Conformación Proteica , ARN Polimerasa II/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Resonancia por Plasmón de Superficie , Factores de Elongación Transcripcional/metabolismoRESUMEN
DNA replication, transcription and repair involve the recruitment of protein complexes that change their composition as they progress along the genome in a directed or strand-specific manner. Chromatin immunoprecipitation in conjunction with hidden Markov models (HMMs) has been instrumental in understanding these processes, as they segment the genome into discrete states that can be related to DNA-associated protein complexes. However, current HMM-based approaches are not able to assign forward or reverse direction to states or properly integrate strand-specific (e.g., RNA expression) with non-strand-specific (e.g., ChIP) data, which is indispensable to accurately characterize directed processes. To overcome these limitations, we introduce bidirectional HMMs which infer directed genomic states from occupancy profiles de novo. Application to RNA polymerase II-associated factors in yeast and chromatin modifications in human T cells recovers the majority of transcribed loci, reveals gene-specific variations in the yeast transcription cycle and indicates the existence of directed chromatin state patterns at transcribed, but not at repressed, regions in the human genome. In yeast, we identify 32 new transcribed loci, a regulated initiation-elongation transition, the absence of elongation factors Ctk1 and Paf1 from a class of genes, a distinct transcription mechanism for highly expressed genes and novel DNA sequence motifs associated with transcription termination. We anticipate bidirectional HMMs to significantly improve the analyses of genome-associated directed processes.
Asunto(s)
Variación Genética , Genómica/métodos , Cadenas de Markov , ARN Polimerasa II/metabolismo , Transcripción Genética , Inmunoprecipitación de Cromatina , Bases de Datos Genéticas , Regulación de la Expresión Génica , Sitios Genéticos , Genoma Fúngico , Genoma Humano , Humanos , Modelos Teóricos , Regiones Promotoras Genéticas , ARN Polimerasa II/genética , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADN , Linfocitos T/metabolismoRESUMEN
The RNA polymerase II (RNApII) C-terminal domain (CTD)-interacting domain (CID) proteins are involved in two distinct RNApII termination pathways and recognize different phosphorylated forms of CTD. To investigate the role of differential CTD-CID interactions in the choice of termination pathway, we altered the CTD-binding specificity of Nrd1 by domain swapping. Nrd1 with the CID from Rtt103 (Nrd1(CID(Rtt103))) causes read-through transcription at many genes, but can also trigger termination where multiple Nrd1/Nab3-binding sites and the Ser(P)-2 CTD co-exist. Therefore, CTD-CID interactions target specific termination complexes to help choose an RNApII termination pathway. Interactions of Nrd1 with both CTD and nascent transcripts contribute to efficient termination by the Nrd1 complex. Surprisingly, replacing the Nrd1 CID with that from Rtt103 reduces binding to Rrp6/Trf4, and RNA transcripts terminated by Nrd1(CID(Rtt103)) are predominantly processed by core exosome. Thus, the Nrd1 CID couples Ser(P)-5 CTD not only to termination, but also to RNA processing by the nuclear exosome.
Asunto(s)
Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , ARN Polimerasa II/metabolismo , Procesamiento Postranscripcional del ARN , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Terminación de la Transcripción Genética , Alcohol Deshidrogenasa/genética , Alcohol Deshidrogenasa/metabolismo , Secuencia de Bases , Sitios de Unión , Núcleo Celular/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estructura Terciaria de Proteína , ARN Polimerasa II/química , ARN Polimerasa II/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
During viral infections cellular gene expression is subject to rapid alterations induced by both viral and antiviral mechanisms. In this study, we applied metabolic labeling of newly transcribed RNA with 4-thiouridine (4sU-tagging) to dissect the real-time kinetics of cellular and viral transcriptional activity during lytic murine cytomegalovirus (MCMV) infection. Microarray profiling on newly transcribed RNA obtained at different times during the first six hours of MCMV infection revealed discrete functional clusters of cellular genes regulated with distinct kinetics at surprising temporal resolution. Immediately upon virus entry, a cluster of NF-κB- and interferon-regulated genes was induced. Rapid viral counter-regulation of this coincided with a very transient DNA-damage response, followed by a delayed ER-stress response. Rapid counter-regulation of all three clusters indicated the involvement of novel viral regulators targeting these pathways. In addition, down-regulation of two clusters involved in cell-differentiation (rapid repression) and cell-cycle (delayed repression) was observed. Promoter analysis revealed all five clusters to be associated with distinct transcription factors, of which NF-κB and c-Myc were validated to precisely match the respective transcriptional changes observed in newly transcribed RNA. 4sU-tagging also allowed us to study the real-time kinetics of viral gene expression in the absence of any interfering virion-associated-RNA. Both qRT-PCR and next-generation sequencing demonstrated a sharp peak of viral gene expression during the first two hours of infection including transcription of immediate-early, early and even well characterized late genes. Interestingly, this was subject to rapid gene silencing by 5-6 hours post infection. Despite the rapid increase in viral DNA load during viral DNA replication, transcriptional activity of some viral genes remained remarkably constant until late-stage infection, or was subject to further continuous decline. In summary, this study pioneers real-time transcriptional analysis during a lytic herpesvirus infection and highlights numerous novel regulatory aspects of virus-host-cell interaction.
Asunto(s)
Regulación Viral de la Expresión Génica , Infecciones por Herpesviridae/genética , Interacciones Huésped-Patógeno/genética , Muromegalovirus/genética , Animales , Perfilación de la Expresión Génica/métodos , Genes Virales/genética , Infecciones por Herpesviridae/virología , Ratones , Análisis por Micromatrices , Familia de Multigenes/genética , Muromegalovirus/patogenicidad , Células 3T3 NIH , Regiones Promotoras Genéticas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción/genética , Transcripción Genética/genéticaRESUMEN
The Mediator is a highly conserved, large multiprotein complex that is involved essentially in the regulation of eukaryotic mRNA transcription. It acts as a general transcription factor by integrating regulatory signals from gene-specific activators or repressors to the RNA Polymerase II. The internal network of interactions between Mediator subunits that conveys these signals is largely unknown. Here, we introduce MC EMiNEM, a novel method for the retrieval of functional dependencies between proteins that have pleiotropic effects on mRNA transcription. MC EMiNEM is based on Nested Effects Models (NEMs), a class of probabilistic graphical models that extends the idea of hierarchical clustering. It combines mode-hopping Monte Carlo (MC) sampling with an Expectation-Maximization (EM) algorithm for NEMs to increase sensitivity compared to existing methods. A meta-analysis of four Mediator perturbation studies in Saccharomyces cerevisiae, three of which are unpublished, provides new insight into the Mediator signaling network. In addition to the known modular organization of the Mediator subunits, MC EMiNEM reveals a hierarchical ordering of its internal information flow, which is putatively transmitted through structural changes within the complex. We identify the N-terminus of Med7 as a peripheral entity, entailing only local structural changes upon perturbation, while the C-terminus of Med7 and Med19 appear to play a central role. MC EMiNEM associates Mediator subunits to most directly affected genes, which, in conjunction with gene set enrichment analysis, allows us to construct an interaction map of Mediator subunits and transcription factors.
Asunto(s)
Algoritmos , Complejo Mediador/química , Mapeo de Interacción de Proteínas/estadística & datos numéricos , Teorema de Bayes , Biología Computacional , Simulación por Computador , Perfilación de la Expresión Génica/estadística & datos numéricos , Complejo Mediador/genética , Modelos Biológicos , Modelos Estadísticos , Método de Montecarlo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Enhancer-mediated gene activation generally requires physical proximity between enhancers and their target gene promoters. However, the molecular mechanisms by which interactions between enhancers and promoters are formed are not well understood. Here, we investigate the function of the Mediator complex in the regulation of enhancer-promoter interactions, by combining rapid protein depletion and high-resolution MNase-based chromosome conformation capture approaches. We show that depletion of Mediator leads to reduced enhancer-promoter interaction frequencies, which are associated with a strong decrease in gene expression. In addition, we find increased interactions between CTCF-binding sites upon Mediator depletion. These changes in chromatin architecture are associated with a redistribution of the Cohesin complex on chromatin and a reduction in Cohesin occupancy at enhancers. Together, our results indicate that the Mediator and Cohesin complexes contribute to enhancer-promoter interactions and provide insights into the molecular mechanisms by which communication between enhancers and promoters is regulated.
Asunto(s)
Elementos de Facilitación Genéticos , Complejo Mediador , Complejo Mediador/genética , Complejo Mediador/metabolismo , Elementos de Facilitación Genéticos/genética , Cromatina , Regiones Promotoras Genéticas , Sitios de Unión , Proteínas de Ciclo Celular/metabolismo , Factor de Unión a CCCTC/genética , Factor de Unión a CCCTC/metabolismoRESUMEN
Isocitrate dehydrogenase (IDH) mutations are found in 20% of acute myeloid leukemia (AML) patients. However, only 30-40% of the patients respond to IDH inhibitors (IDHi). We aimed to identify a molecular vulnerability to tailor novel therapies for AML patients with IDH mutations. We characterized the transcriptional and epigenetic landscape with the IDH2i AG-221, using an IDH2 mutated AML cell line model and AML patient cohorts, and discovered a perturbed transcriptional regulatory network involving myeloid transcription factors that were partly restored after AG-221 treatment. In addition, hypermethylation of the HLA cluster caused a down-regulation of HLA class I genes, triggering an enhanced natural killer (NK) cell activation and an increased susceptibility to NK cell-mediated responses. Finally, analyses of DNA methylation data from IDHi-treated patients showed that non-responders still harbored hypermethylation in HLA class I genes. In conclusion, this study provides new insights suggesting that IDH mutated AML is particularly sensitive to NK cell-based personalized immunotherapy.
Asunto(s)
Isocitrato Deshidrogenasa , Leucemia Mieloide Aguda , Humanos , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Epigénesis Genética , Mutación , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Células Asesinas Naturales/metabolismoRESUMEN
DNA can determine where and when genes are expressed, but the full set of sequence determinants that control gene expression is unknown. Here, we measured the transcriptional activity of DNA sequences that represent an ~100 times larger sequence space than the human genome using massively parallel reporter assays (MPRAs). Machine learning models revealed that transcription factors (TFs) generally act in an additive manner with weak grammar and that most enhancers increase expression from a promoter by a mechanism that does not appear to involve specific TF-TF interactions. The enhancers themselves can be classified into three types: classical, closed chromatin and chromatin dependent. We also show that few TFs are strongly active in a cell, with most activities being similar between cell types. Individual TFs can have multiple gene regulatory activities, including chromatin opening and enhancing, promoting and determining transcription start site (TSS) activity, consistent with the view that the TF binding motif is the key atomic unit of gene expression.