RESUMEN
Messenger RNA precursors (pre-mRNA) generally undergo 3' end processing by cleavage and polyadenylation (CPA), which is specified by a polyadenylation site (PAS) and adjacent RNA sequences and regulated by a large variety of core and auxiliary CPA factors. To date, most of the human CPA factors have been discovered through biochemical and proteomic studies. However, genetic identification of the human CPA factors has been hampered by the lack of a reliable genome-wide screening method. We describe here a dual fluorescence readthrough reporter system with a PAS inserted between two fluorescent reporters. This system enables measurement of the efficiency of 3' end processing in living cells. Using this system in combination with a human genome-wide CRISPR/Cas9 library, we conducted a screen for CPA factors. The screens identified most components of the known core CPA complexes and other known CPA factors. The screens also identified CCNK/CDK12 as a potential core CPA factor, and RPRD1B as a CPA factor that binds RNA and regulates the release of RNA polymerase II at the 3' ends of genes. Thus, this dual fluorescence reporter coupled with CRISPR/Cas9 screens reliably identifies bona fide CPA factors and provides a platform for investigating the requirements for CPA in various contexts.
Asunto(s)
Sistemas CRISPR-Cas , Genes Reporteros , Precursores del ARN , Factores de Escisión y Poliadenilación de ARNm , Humanos , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/genética , Genoma Humano , Células HEK293 , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética , Poliadenilación , División del ARN , ARN Polimerasa II/metabolismo , Precursores del ARN/metabolismo , Precursores del ARN/genéticaRESUMEN
Survival of motor neuron (SMN) functions in diverse biological pathways via recognition of symmetric dimethylarginine (Rme2s) on proteins by its Tudor domain, and deficiency of SMN leads to spinal muscular atrophy. Here we report a potent and selective antagonist with a 4-iminopyridine scaffold targeting the Tudor domain of SMN. Our structural and mutagenesis studies indicate that both the aromatic ring and imino groups of compound 1 contribute to its selective binding to SMN. Various on-target engagement assays support that compound 1 specifically recognizes SMN in a cellular context and prevents the interaction of SMN with the R1810me2s of RNA polymerase II subunit POLR2A, resulting in transcription termination and R-loop accumulation mimicking SMN depletion. Thus, in addition to the antisense, RNAi and CRISPR/Cas9 techniques, potent SMN antagonists could be used as an efficient tool to understand the biological functions of SMN.
Asunto(s)
ARN Polimerasa II , Proteínas del Complejo SMN , Humanos , Neuronas Motoras/metabolismo , Atrofia Muscular Espinal/metabolismo , ARN Polimerasa II/efectos de los fármacos , ARN Polimerasa II/metabolismo , Proteínas del Complejo SMN/antagonistas & inhibidores , Proteínas del Complejo SMN/efectos de los fármacos , Proteínas del Complejo SMN/metabolismoRESUMEN
Alternative polyadenylation (APA) enhances gene regulatory potential by increasing the diversity of mRNA transcripts. 3' UTR shortening through APA correlates with enhanced cellular proliferation and is a widespread phenomenon in tumor cells. Here, we show that the ubiquitously expressed transcription factor Sp1 binds RNA in vivo and is a common repressor of distal poly(A) site usage. RNA sequencing identified 2,344 genes (36% of the total mapped mRNA transcripts) with lengthened 3' UTRs upon Sp1 depletion. Sp1 preferentially binds the 3' UTRs of such lengthened transcripts and inhibits cleavage at distal sites by interacting with the subunits of the core cleavage and polyadenylation (CPA) machinery. The 3' UTR lengths of Sp1 target genes in breast cancer patient RNA-seq data correlate with Sp1 expression levels, implicating Sp1-mediated APA regulation in modulating tumorigenic properties. Taken together, our findings provide insights into the mechanism for dynamic APA regulation by unraveling a previously unknown function of the DNA-binding transcription factor Sp1.
Asunto(s)
Poli A , Poliadenilación , Regiones no Traducidas 3' , Humanos , Poli A/metabolismo , ARN Mensajero/metabolismo , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Zinc/metabolismoRESUMEN
Long-term exposure to high levels of arsenic (As) will result in damage to organs. Compared with free arsenic, protein-bound arsenic are more difficult to be excreted from the bodies due to their complexation with biological macromolecules. We developed a method of size exclusion chromatography (SEC) and ion exchange chromatography (IEC) combined with inductively coupled plasma-mass spectrometry (ICP-MS) and multiple reaction monitoring (MRM) mode, which was used to determine bound-arsenic species. DMAV was identified as bound arsenic species in rat livers after As4S4 overexposure. Subsequent proteomics analysis showed the potential binding partners included hemoglobin, glutathione S-transferases, superoxide dismutase [Cu-Zn] & [Mn], thiosulfate sulfurtransferase, and metallothionein-2. The method developed here was sensitive, repeatable, and conducive to arsenic analysis, especially for toxicity evaluation of arsenic-containing substances in vivo.
Asunto(s)
Arsénico , Arsenicales , Animales , Arsénico/toxicidad , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Intercambio Iónico , Hígado , RatasRESUMEN
Post-translational modifications of the RNA polymerase II C-terminal domain (CTD) coordinate the transcription cycle. Crosstalk between different modifications is poorly understood. Here, we show how acetylation of lysine residues at position 7 of characteristic heptad repeats (K7ac)-only found in higher eukaryotes-regulates phosphorylation of serines at position 5 (S5p), a conserved mark of polymerases initiating transcription. We identified the regulator of pre-mRNA-domain-containing (RPRD) proteins as reader proteins of K7ac. K7ac enhanced CTD peptide binding to the CTD-interacting domain (CID) of RPRD1A and RPRD1B proteins in isothermal calorimetry and molecular modeling experiments. Deacetylase inhibitors increased K7ac- and decreased S5-phosphorylated polymerases, consistent with acetylation-dependent S5 dephosphorylation by an RPRD-associated S5 phosphatase. Consistent with this model, RPRD1B knockdown increased S5p but enhanced K7ac, indicating that RPRD proteins recruit K7 deacetylases, including HDAC1. We also report autoregulatory crosstalk between K7ac and S5p via RPRD proteins and their interactions with acetyl- and phospho-eraser proteins.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Neoplasias/metabolismo , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , ARN Polimerasa II/metabolismo , Acetilación , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Células HEK293 , Humanos , Ratones , Modelos Moleculares , Células 3T3 NIH , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fosforilación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , ARN Polimerasa II/química , ARN Polimerasa II/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , TermodinámicaRESUMEN
The P-element-induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway plays a central role in transposon silencing and genome protection in the animal germline. A family of Tudor domain proteins regulates the piRNA pathway through direct Tudor domain-PIWI interactions. Tudor domains are known to fulfill this function by binding to methylated PIWI proteins in an arginine methylation-dependent manner. Here, we report a mechanism of methylation-independent Tudor domain-PIWI interaction. Unlike most other Tudor domains, the extended Tudor domain of mammalian Tudor domain-containing protein 2 (TDRD2) preferentially recognizes an unmethylated arginine-rich sequence from PIWI-like protein 1 (PIWIL1). Structural studies reveal an unexpected Tudor domain-binding mode for the PIWIL1 sequence in which the interface of Tudor and staphylococcal nuclease domains is primarily responsible for PIWIL1 peptide recognition. Mutations disrupting the TDRD2-PIWIL1 interaction compromise piRNA maturation via 3'-end trimming in vitro. Our work presented here reveals the molecular divergence of the interactions between different Tudor domain proteins and PIWI proteins.
Asunto(s)
Arginina/metabolismo , Proteínas Argonautas/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Proteínas Argonautas/química , Proteínas Argonautas/genética , Cristalografía por Rayos X , Epigénesis Genética , Células HEK293 , Humanos , Masculino , Metilación , Modelos Moleculares , Mutación , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Transducción de Señal , Relación Estructura-ActividadRESUMEN
BACKGROUND: STAT1 and IRF1 collaborate to induce interferon-γ (IFNγ) stimulated genes (ISGs), but the extent to which they act alone or together is unclear. The effect of single nucleotide polymorphisms (SNPs) on in vivo binding is also largely unknown. RESULTS: We show that IRF1 binds at proximal or distant ISG sites twice as often as STAT1, increasing to sixfold at the MHC class I locus. STAT1 almost always bound with IRF1, while most IRF1 binding events were isolated. Dual binding sites at remote or proximal enhancers distinguished ISGs that were responsive to IFNγ versus cell-specific resistant ISGs, which showed fewer and mainly single binding events. Surprisingly, inducibility in one cell type predicted ISG-responsiveness in other cells. Several dbSNPs overlapped with STAT1 and IRF1 binding motifs, and we developed methodology to rapidly assess their effects. We show that in silico prediction of SNP effects accurately reflects altered binding both in vitro and in vivo. CONCLUSIONS: These data reveal broad cooperation between STAT1 and IRF1, explain cell type specific differences in ISG-responsiveness, and identify genetic variants that may participate in the pathogenesis of immune disorders.
Asunto(s)
Factor 1 Regulador del Interferón/genética , Interferón gamma/inmunología , Polimorfismo de Nucleótido Simple , Factor de Transcripción STAT1/genética , Elementos de Facilitación Genéticos , Genes MHC Clase I , Células HeLa , Humanos , Factor 1 Regulador del Interferón/inmunología , Factor 1 Regulador del Interferón/metabolismo , Sitios Menores de Histocompatibilidad , Unión Proteica , Factor de Transcripción STAT1/inmunología , Factor de Transcripción STAT1/metabolismo , Regulación hacia ArribaRESUMEN
Determination of the physical interactions of environmental chemicals with cellular proteins is important for characterizing biological and toxic mechanism of action. Yet despite the discovery of numerous bioactive natural brominated compounds, such as hydroxylated polybrominated diphenyl ethers (OH-PBDEs), their corresponding protein targets remain largely unclear. Here, we reported a systematic and unbiased chemical proteomics assay (Target Identification by Ligand Stabilization, TILS) for target identification of bioactive molecules based on monitoring ligand-induced thermal stabilization. We first validated the broad applicability of this approach by identifying both known and unexpected proteins bound by diverse compounds (anticancer drugs, antibiotics). We then applied TILS to identify the bacterial target of 6-OH-BDE-47 as enoyl-acyl carrier protein reductase (FabI), an essential and widely conserved enzyme. Using affinity pull-down and in vitro enzymatic assays, we confirmed the potent antibacterial activity of 6-OH-BDE-47 occurs via direct binding and inhibition of FabI. Conversely, overexpression of FabI rescued the growth inhibition of Escherichia coli by 6-OH-BDE-47, validating it as the primary in vivo target. This study documents a chemical proteomics strategy for identifying the physical and functional targets of small molecules, and its potential high-throughput application to investigate the modes-of-action of environmental compounds.
RESUMEN
Glucolipotoxicity is one of the critical causal factors of diabetic complications. Whether mesenchymal stem cells (MSCs) have effects on glucolipotoxicity in human umbilical vein endothelial cells (HUVECs) and mechanisms involved are unclear. Thirty mM glucose plus 100 µM palmitic acid was used to induce glucolipotoxicity in HUVECs. MSCs and HUVECs were co-cultured at the ratio of 1:5 via Transwell system. The mRNA expressions of inflammatory factors were detected by RT-qPCR. The productions of reactive oxygen species (ROS), cell cycle and apoptosis were analyzed by flow cytometry. The tumor necrosis factor-α stimulated protein 6 (TSG-6) was knockdown in MSCs by RNA interference. High glucose and palmitic acid remarkably impaired cell viability and tube formation capacity, as well as increased the mRNA expression of inflammatory factors, ROS levels, and cell apoptosis in HUVECs. MSC co-cultivation ameliorated these detrimental effects in HUVECs, but no effect on ROS production. Moreover, TSG-6 was dramatically up-regulated by high glucose and fatty acid stimulation in both MSCs and HUVECs. TSG-6 knockdown partially abolished the protection mediated by MSCs. MSCs had protective effects on high glucose and palmitic acid induced glucolipotoxicity in HUVECs, and TSG-6 secreted by MSCs was likely to play an important role in this process.
Asunto(s)
Apoptosis/efectos de los fármacos , Glucosa/toxicidad , Células Madre Mesenquimatosas/citología , Ácido Palmítico/toxicidad , Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Quimiocinas/genética , Quimiocinas/metabolismo , Técnicas de Cocultivo , Citocinas/genética , Citocinas/metabolismo , Citometría de Flujo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Células Madre Mesenquimatosas/metabolismo , Microscopía Fluorescente , FN-kappa B/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Our analysis examines the conservation of multiprotein complexes among metazoa through use of high resolution biochemical fractionation and precision mass spectrometry applied to soluble cell extracts from 5 representative model organisms Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, Strongylocentrotus purpuratus, and Homo sapiens. The interaction network obtained from the data was validated globally in 4 distant species (Xenopus laevis, Nematostella vectensis, Dictyostelium discoideum, Saccharomyces cerevisiae) and locally by targeted affinity-purification experiments. Here we provide details of our massive set of supporting biochemical fractionation data available via ProteomeXchange (PXD002319-PXD002328), PPIs via BioGRID (185267); and interaction network projections via (http://metazoa.med.utoronto.ca) made fully accessible to allow further exploration. The datasets here are related to the research article on metazoan macromolecular complexes in Nature [1].
RESUMEN
The carboxy-terminal domain (CTD) of the RNA polymerase II (RNAP II) subunit POLR2A is a platform for modifications specifying the recruitment of factors that regulate transcription, mRNA processing, and chromatin remodelling. Here we show that a CTD arginine residue (R1810 in human) that is conserved across vertebrates is symmetrically dimethylated (me2s). This R1810me2s modification requires protein arginine methyltransferase 5 (PRMT5) and recruits the Tudor domain of the survival of motor neuron (SMN, also known as GEMIN1) protein, which is mutated in spinal muscular atrophy. SMN interacts with senataxin, which is sometimes mutated in ataxia oculomotor apraxia type 2 and amyotrophic lateral sclerosis. Because POLR2A R1810me2s and SMN, like senataxin, are required for resolving RNA-DNA hybrids created by RNA polymerase II that form R-loops in transcription termination regions, we propose that R1810me2s, SMN, and senataxin are components of an R-loop resolution pathway. Defects in this pathway can influence transcription termination and may contribute to neurodegenerative disorders.
Asunto(s)
Arginina/metabolismo , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Terminación de la Transcripción Genética , Línea Celular , Daño del ADN , ADN Helicasas , Humanos , Metilación , Enzimas Multifuncionales , Enfermedades Neurodegenerativas/genética , Unión Proteica , Estructura Terciaria de Proteína , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Elongación de la Transcripción GenéticaRESUMEN
An in vitro cell metabolomics study was performed on human L02 liver cells to investigate the toxic biomarkers of pekinenal from the herb Euphorbia pekinensis Rupr. Pekinenal significantly induced L02 cell damage, which was characterised by necrosis and apoptosis. Metabolomics combined with data pattern recognition showed that pekinenal significantly altered the profiles of more than 1299 endogenous metabolites with variable importance in the projection (VIP) > 1. Further, screening correlation coefficients between the intensities of all metabolites and the extent of L02 cell damage (MTT) identified 12 biomarker hits: ten were downregulated and two were upregulated. Among these hits, LysoPC(18:1(9Z)/(11Z)), PC(22:0/15:0) and PC(20:1(11Z)/14:1(9Z)) were disordered, implying the initiation of inflammation and cell damage. Several fatty acids (FAs) (3-hydroxytetradecanedioic acid, pivaloylcarnitine and eicosapentaenoyl ethanolamide) decreased due to fatty acid oxidation. Dihydroceramide and Cer(d18:0/14:0) were also altered and are associated with apoptosis. Additional examination of the levels of intracellular reactive oxygen species (ROS) and two eicosanoids (PGE2, PGF2α) in the cell supernatant confirmed the fatty acid oxidation and arachidonic acid metabolism pathways, respectively. In summary, cell metabolomics is a highly efficient approach for identifying toxic biomarkers and helping understand toxicity mechanisms and predict herb-induced liver injury.
Asunto(s)
Apoptosis/efectos de los fármacos , Biomarcadores/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Diterpenos/efectos adversos , Hígado/patología , Metabolómica/métodos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Euphorbia/química , Ácidos Grasos/metabolismo , Humanos , Técnicas In Vitro , Metabolismo de los Lípidos , Hígado/efectos de los fármacos , Hígado/metabolismo , Necrosis , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Macromolecular complexes are essential to conserved biological processes, but their prevalence across animals is unclear. By combining extensive biochemical fractionation with quantitative mass spectrometry, here we directly examined the composition of soluble multiprotein complexes among diverse metazoan models. Using an integrative approach, we generated a draft conservation map consisting of more than one million putative high-confidence co-complex interactions for species with fully sequenced genomes that encompasses functional modules present broadly across all extant animals. Clustering reveals a spectrum of conservation, ranging from ancient eukaryotic assemblies that have probably served cellular housekeeping roles for at least one billion years, ancestral complexes that have accrued contemporary components, and rarer metazoan innovations linked to multicellularity. We validated these projections by independent co-fractionation experiments in evolutionarily distant species, affinity purification and functional analyses. The comprehensiveness, centrality and modularity of these reconstructed interactomes reflect their fundamental mechanistic importance and adaptive value to animal cell systems.
Asunto(s)
Evolución Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Mapas de Interacción de Proteínas , Animales , Conjuntos de Datos como Asunto , Humanos , Mapeo de Interacción de Proteínas , Reproducibilidad de los Resultados , Biología de Sistemas , Espectrometría de Masas en TándemRESUMEN
The RNA polymerase II (RNAPII) C-terminal domain (CTD) heptapeptide repeats (1-YSPTSPS-7) undergo dynamic phosphorylation and dephosphorylation during the transcription cycle to recruit factors that regulate transcription, RNA processing and chromatin modification. We show here that RPRD1A and RPRD1B form homodimers and heterodimers through their coiled-coil domains and interact preferentially via CTD-interaction domains (CIDs) with RNAPII CTD repeats phosphorylated at S2 and S7. Crystal structures of the RPRD1A, RPRD1B and RPRD2 CIDs, alone and in complex with RNAPII CTD phosphoisoforms, elucidate the molecular basis of CTD recognition. In an example of cross-talk between different CTD modifications, our data also indicate that RPRD1A and RPRD1B associate directly with RPAP2 phosphatase and, by interacting with CTD repeats where phospho-S2 and/or phospho-S7 bracket a phospho-S5 residue, serve as CTD scaffolds to coordinate the dephosphorylation of phospho-S5 by RPAP2.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Neoplasias/química , Procesamiento Proteico-Postraduccional , ARN Polimerasa II/química , Proteínas Represoras/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cristalografía por Rayos X , Células HEK293 , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , ARN Polimerasa II/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Serina/químicaRESUMEN
Chromatin regulation is driven by multicomponent protein complexes, which form functional modules. Deciphering the components of these modules and their interactions is central to understanding the molecular pathways these proteins are regulating, their functions, and their relation to both normal development and disease. We describe the use of affinity purifications of tagged human proteins coupled with mass spectrometry to generate a protein-protein interaction map encompassing known and predicted chromatin-related proteins. On the basis of 1,394 successful purifications of 293 proteins, we report a high-confidence (85% precision) network involving 11,464 protein-protein interactions among 1,738 different human proteins, grouped into 164 often overlapping protein complexes with a particular focus on the family of JmjC-containing lysine demethylases, their partners, and their roles in chromatin remodeling. We show that RCCD1 is a partner of histone H3K36 demethylase KDM8 and demonstrate that both are important for cell-cycle-regulated transcriptional repression in centromeric regions and accurate mitotic division.
Asunto(s)
Proteínas Portadoras/metabolismo , Cromatina/metabolismo , Segregación Cromosómica , Histona Demetilasas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Portadoras/genética , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Unión ProteicaRESUMEN
Protein complexes and protein-protein interactions (PPIs) are fundamental for most biological functions. Deciphering the extensive protein interaction networks that occur within cellular contexts has become a logical extension to the human genome project. Proteome-scale interactome analysis of mammalian systems requires efficient methods for accurately detecting PPIs with specific considerations for the intrinsic technical challenges of mammalian genome manipulation. In this chapter, we outline in detail an innovative lentiviral-based functional proteomic approach that can be used to rapidly characterize protein complexes from a broad range of mammalian cell lines. This method integrates the following key features: (1) lentiviral elements for efficient delivery of tagged constructs into mammalian cell lines; (2) site-specific Gateway™ recombination sites for easy cloning; (3) versatile epitope-tagging system for flexible affinity purification strategies; and (4) LC-MS-based protein identification using tandem mass spectrometry.
Asunto(s)
Lentivirus/genética , Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas , Proteínas/análisis , Proteómica/métodos , Animales , Línea Celular , Cromatografía de Afinidad , Clonación Molecular , Células HEK293 , Humanos , Unión Proteica , Proteínas/metabolismo , Proteoma/análisis , Espectrometría de Masas en TándemRESUMEN
RNA polymerase II (RNAP II) C-terminal domain (CTD) phosphorylation is important for various transcription-related processes. Here, we identify by affinity purification and mass spectrometry three previously uncharacterized human CTD-interaction domain (CID)-containing proteins, RPRD1A, RPRD1B and RPRD2, which co-purify with RNAP II and three other RNAP II-associated proteins, RPAP2, GRINL1A and RECQL5, but not with the Mediator complex. RPRD1A and RPRD1B can accompany RNAP II from promoter regions to 3'-untranslated regions during transcription in vivo, predominantly interact with phosphorylated RNAP II, and can reduce CTD S5- and S7-phosphorylated RNAP II at target gene promoters. Thus, the RPRD proteins are likely to have multiple important roles in transcription.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Neoplasias/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Proteínas Represoras/metabolismo , Regiones no Traducidas 3' , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Células HEK293 , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fosforilación , Dominios y Motivos de Interacción de Proteínas , ARN Polimerasa II/química , ARN Polimerasa II/genética , RecQ Helicasas/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Transcripción GenéticaRESUMEN
Protein complexes and protein-protein interactions are essential for almost all cellular processes. Here, we establish a mammalian affinity purification and lentiviral expression (MAPLE) system for characterizing the subunit compositions of protein complexes. The system is flexible (i.e. multiple N- and C-terminal tags and multiple promoters), is compatible with Gateway cloning, and incorporates a reference peptide. Its major advantage is that it permits efficient and stable delivery of affinity-tagged open reading frames into most mammalian cell types. We benchmarked MAPLE with a number of human protein complexes involved in transcription, including the RNA polymerase II-associated factor, negative elongation factor, positive transcription elongation factor b, SWI/SNF, and mixed lineage leukemia complexes. In addition, MAPLE was used to identify an interaction between the reprogramming factor Klf4 and the Swi/Snf chromatin remodeling complex in mouse embryonic stem cells. We show that the SWI/SNF catalytic subunit Smarca2/Brm is up-regulated during the process of induced pluripotency and demonstrate a role for the catalytic subunits of the SWI/SNF complex during somatic cell reprogramming. Our data suggest that the transcription factor Klf4 facilitates chromatin remodeling during reprogramming.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Lentivirus/metabolismo , Células Madre Pluripotentes/metabolismo , Proteómica/métodos , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Reprogramación Celular/genética , Cromatografía de Afinidad , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Datos de Secuencia Molecular , Complejos Multiproteicos/metabolismo , Células Madre Pluripotentes/citología , Unión Proteica , Transcripción GenéticaRESUMEN
The chromatin-remodeling enzyme BRG1 is critical for interferon-gamma (IFN-gamma)-mediated gene induction. Promoter-proximal elements are sufficient to mediate BRG1 dependency at some IFN-gamma targets. In contrast, we show here that at CIITA, which encodes the 'master regulator' of induction of major histocompatibility complex class II, distal elements conferred BRG1 dependency. At the uninduced locus, many sites formed BRG1-independent loops. One loop juxtaposed a far downstream element adjacent to a far upstream site. Notably, BRG1 was recruited to the latter site, which triggered the appearance of a histone 'mark' linked to activation. This subtle change was crucial, as subsequent IFN-gamma-induced recruitment of the transcription factors STAT1, IRF1 and p300, as well as histone modifications, accessibility and additional loops, showed BRG1 dependency. Like BRG1, each remote element was critical for the induction of CIITA expression. Thus, BRG1 regulates CIITA through many interdependent remote enhancers, not through the promoter alone.
Asunto(s)
Cromatina/genética , ADN Helicasas/genética , Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica , Proteínas Nucleares/genética , Transactivadores/genética , Factores de Transcripción/genética , Expresión Génica , Genes Reguladores/genética , Células HeLa , Humanos , Interferón gamma/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Activación TranscripcionalRESUMEN
IL-6 is an immunoregulatory cytokine with multiple functions in hemopoiesis, proliferation, and tumorigenesis. IL-6 triggers phosphorylation, dimerization, and nuclear translocation of STAT3, which binds to target promoters and activates transcription. Brahma-related gene 1 (BRG1), the enzymatic engine of the yeast-mating type-switching and sucrose-nonfermenting chromatin-remodeling complex, is essential for recruitment of STAT1 or STAT1/STAT2-containing complexes to IFN targets. We hypothesized that BRG1 might also be required for STAT3 recruitment. In this study, we show that induction of a subset of human IL-6-responsive genes is BRG1 dependent. BRG1 is constitutively present at these targets and is required for STAT3 recruitment, downstream histone modifications, and IL-6-induced chromatin remodeling. IL-6-induced recruitment of STAT3 to the IFN regulatory factor 1 promoter and subsequent mRNA synthesis is BRG1 dependent, even though IFN-gamma-mediated STAT1 recruitment to this locus is BRG1 independent. BRG1 also increased basal expression of IFN-induced transmembrane protein 3 and IFN-gamma-induced protein 16, and the basal chromatin accessibility at the promoter of IFN regulatory factor 1. The effect on basal expression was STAT3 independent, as revealed by small interfering RNA knockdown. Together with prior observations, these data reveal that BRG1 has a broad role in mediating STAT accessibility at multiple cytokine-responsive promoters and exposes promoter specific differences in both the effect of BRG1 on basal chromatin accessibility and on access of different STAT proteins to the same target.