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1.
Nat Immunol ; 19(9): 932-941, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-30127433

RESUMEN

Cohesin is important for 3D genome organization. Nevertheless, even the complete removal of cohesin has surprisingly little impact on steady-state gene transcription and enhancer activity. Here we show that cohesin is required for the core transcriptional response of primary macrophages to microbial signals, and for inducible enhancer activity that underpins inflammatory gene expression. Consistent with a role for inflammatory signals in promoting myeloid differentiation of hematopoietic stem and progenitor cells (HPSCs), cohesin mutations in HSPCs led to reduced inflammatory gene expression and increased resistance to differentiation-inducing inflammatory stimuli. These findings uncover an unexpected dependence of inducible gene expression on cohesin, link cohesin with myeloid differentiation, and may help explain the prevalence of cohesin mutations in human acute myeloid leukemia.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular/genética , Autorrenovación de las Células/genética , Proteínas Cromosómicas no Histona/metabolismo , Células Madre Hematopoyéticas/fisiología , Leucemia Mieloide Aguda/genética , Macrófagos/fisiología , Proteínas Nucleares/genética , Fosfoproteínas/genética , Animales , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN , Regulación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inflamación/genética , Lipopolisacáridos/inmunología , Ratones , Ratones Noqueados , Mutación/genética , Cohesinas
2.
Mol Cell ; 81(3): 488-501.e9, 2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33338397

RESUMEN

Polycomb repressive complex 2 (PRC2) silences expression of developmental transcription factors in pluripotent stem cells by methylating lysine 27 on histone H3. Two mutually exclusive subcomplexes, PRC2.1 and PRC2.2, are defined by the set of accessory proteins bound to the core PRC2 subunits. Here we introduce separation-of-function mutations into the SUZ12 subunit of PRC2 to drive it into a PRC2.1 or 2.2 subcomplex in human induced pluripotent stem cells (iPSCs). We find that PRC2.2 occupies polycomb target genes at low levels and that homeobox transcription factors are upregulated when this complex is exclusively present. In contrast with previous studies, we find that chromatin occupancy of PRC2 increases drastically when it is forced to form PRC2.1. Additionally, several cancer-associated mutations also coerce formation of PRC2.1. We suggest that PRC2 chromatin occupancy can be altered in the context of disease or development by tuning the ratio of PRC2.1 to PRC2.2.


Asunto(s)
Cromatina/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas de Neoplasias/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Factores de Transcripción/metabolismo , Sitios de Unión , Unión Competitiva , Cromatina/genética , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Mutación , Proteínas de Neoplasias/genética , Complejo Represivo Polycomb 2/genética , Unión Proteica , Factores de Transcripción/genética
3.
Cell ; 153(6): 1327-39, 2013 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-23746844

RESUMEN

The transcription factor HIF1A is a key mediator of the cellular response to hypoxia. Despite the importance of HIF1A in homeostasis and various pathologies, little is known about how it regulates RNA polymerase II (RNAPII). We report here that HIF1A employs a specific variant of the Mediator complex to stimulate RNAPII elongation. The Mediator-associated kinase CDK8, but not the paralog CDK19, is required for induction of many HIF1A target genes. HIF1A induces binding of CDK8-Mediator and the super elongation complex (SEC), containing AFF4 and CDK9, to alleviate RNAPII pausing. CDK8 is dispensable for HIF1A chromatin binding and histone acetylation, but it is essential for binding of SEC and RNAPII elongation. Global analysis of active RNAPII reveals that hypoxia-inducible genes are paused and active prior to their induction. Our results provide a mechanistic link between HIF1A and CDK8, two potent oncogenes, in the cellular response to hypoxia.


Asunto(s)
Hipoxia de la Célula , Quinasa 8 Dependiente de Ciclina/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Complejo Mediador/metabolismo , Neoplasias/metabolismo , ARN Polimerasa II/metabolismo , Elongación de la Transcripción Genética , Acetilación , Línea Celular Tumoral , Quinasa 8 Dependiente de Ciclina/química , Quinasas Ciclina-Dependientes/metabolismo , Células HeLa , Histonas/metabolismo , Humanos
4.
Mol Cell ; 78(4): 785-793.e8, 2020 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-32229306

RESUMEN

RNA polymerase II (RNAPII) transcription is governed by the pre-initiation complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNAPII, and Mediator. After initiation, RNAPII enzymes pause after transcribing less than 100 bases; precisely how RNAPII pausing is enforced and regulated remains unclear. To address specific mechanistic questions, we reconstituted human RNAPII promoter-proximal pausing in vitro, entirely with purified factors (no extracts). As expected, NELF and DSIF increased pausing, and P-TEFb promoted pause release. Unexpectedly, the PIC alone was sufficient to reconstitute pausing, suggesting RNAPII pausing is an inherent PIC function. In agreement, pausing was lost upon replacement of the TFIID complex with TATA-binding protein (TBP), and PRO-seq experiments revealed widespread disruption of RNAPII pausing upon acute depletion (t = 60 min) of TFIID subunits in human or Drosophila cells. These results establish a TFIID requirement for RNAPII pausing and suggest pause regulatory factors may function directly or indirectly through TFIID.


Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa II/genética , Factor de Transcripción TFIID/metabolismo , Transcripción Genética , Animales , Drosophila/genética , Proteínas de Drosophila/genética , Células HCT116 , Humanos , Unión Proteica , ARN Polimerasa II/metabolismo , Factor de Transcripción TFIID/genética
5.
Genes Dev ; 34(21-22): 1452-1473, 2020 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-33060135

RESUMEN

CDK7 associates with the 10-subunit TFIIH complex and regulates transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Few additional CDK7 substrates are known. Here, using the covalent inhibitor SY-351 and quantitative phosphoproteomics, we identified CDK7 kinase substrates in human cells. Among hundreds of high-confidence targets, the vast majority are unique to CDK7 (i.e., distinct from other transcription-associated kinases), with a subset that suggest novel cellular functions. Transcription-associated factors were predominant CDK7 substrates, including SF3B1, U2AF2, and other splicing components. Accordingly, widespread and diverse splicing defects, such as alternative exon inclusion and intron retention, were characterized in CDK7-inhibited cells. Combined with biochemical assays, we establish that CDK7 directly activates other transcription-associated kinases CDK9, CDK12, and CDK13, invoking a "master regulator" role in transcription. We further demonstrate that TFIIH restricts CDK7 kinase function to the RNAPII CTD, whereas other substrates (e.g., SPT5 and SF3B1) are phosphorylated by the three-subunit CDK-activating kinase (CAK; CCNH, MAT1, and CDK7). These results suggest new models for CDK7 function in transcription and implicate CAK dissociation from TFIIH as essential for kinase activation. This straightforward regulatory strategy ensures CDK7 activation is spatially and temporally linked to transcription, and may apply toward other transcription-associated kinases.


Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Modelos Biológicos , Factor de Transcripción TFIIH/metabolismo , Transcripción Genética/genética , Empalme Alternativo/genética , Supervivencia Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/genética , Activación Enzimática/genética , Células HL-60 , Humanos , Quinasa Activadora de Quinasas Ciclina-Dependientes
6.
Mol Cell ; 76(3): 485-499.e8, 2019 11 07.
Artículo en Inglés | MEDLINE | ID: mdl-31495563

RESUMEN

Transcriptional responses to external stimuli remain poorly understood. Using global nuclear run-on followed by sequencing (GRO-seq) and precision nuclear run-on sequencing (PRO-seq), we show that CDK8 kinase activity promotes RNA polymerase II pause release in response to interferon-γ (IFN-γ), a universal cytokine involved in immunity and tumor surveillance. The Mediator kinase module contains CDK8 or CDK19, which are presumed to be functionally redundant. We implemented cortistatin A, chemical genetics, transcriptomics, and other methods to decouple their function while assessing enzymatic versus structural roles. Unexpectedly, CDK8 and CDK19 regulated different gene sets via distinct mechanisms. CDK8-dependent regulation required its kinase activity, whereas CDK19 governed IFN-γ responses through its scaffolding function (i.e., it was kinase independent). Accordingly, CDK8, not CDK19, phosphorylates the STAT1 transcription factor (TF) during IFN-γ stimulation, and CDK8 kinase inhibition blocked activation of JAK-STAT pathway TFs. Cytokines such as IFN-γ rapidly mobilize TFs to "reprogram" cellular transcription; our results implicate CDK8 and CDK19 as essential for this transcriptional reprogramming.


Asunto(s)
Quinasa 8 Dependiente de Ciclina/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Fibroblastos/efectos de los fármacos , Interferón gamma/farmacología , Transcripción Genética/efectos de los fármacos , Animales , Quinasa 8 Dependiente de Ciclina/genética , Quinasas Ciclina-Dependientes/genética , Fibroblastos/enzimología , Fibroblastos/virología , Células HCT116 , Interacciones Huésped-Patógeno , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , ARN Polimerasa II/metabolismo , Factor de Transcripción STAT1/metabolismo , Transducción de Señal , Vesiculovirus/patogenicidad
7.
J Virol ; 98(3): e0156323, 2024 Mar 19.
Artículo en Inglés | MEDLINE | ID: mdl-38323811

RESUMEN

Macrophages are important target cells for diverse viruses and thus represent a valuable system for studying virus biology. Isolation of primary human macrophages is done by culture of dissociated tissues or from differentiated blood monocytes, but these methods are both time consuming and result in low numbers of recovered macrophages. Here, we explore whether macrophages derived from human induced pluripotent stem cells (iPSCs)-which proliferate indefinitely and potentially provide unlimited starting material-could serve as a faithful model system for studying virus biology. Human iPSC-derived monocytes were differentiated into macrophages and then infected with HIV-1, dengue virus, or influenza virus as model human viruses. We show that iPSC-derived macrophages support the replication of these viruses with kinetics and phenotypes similar to human blood monocyte-derived macrophages. These iPSC-derived macrophages were virtually indistinguishable from human blood monocyte-derived macrophages based on surface marker expression (flow cytometry), transcriptomics (RNA sequencing), and chromatin accessibility profiling. iPSC lines were additionally generated from non-human primate (chimpanzee) fibroblasts. When challenged with dengue virus, human and chimpanzee iPSC-derived macrophages show differential susceptibility to infection, thus providing a valuable resource for studying the species-tropism of viruses. We also show that blood- and iPSC-derived macrophages both restrict influenza virus at a late stage of the virus lifecycle. Collectively, our results substantiate iPSC-derived macrophages as an alternative to blood monocyte-derived macrophages for the study of virus biology. IMPORTANCE: Macrophages have complex relationships with viruses: while macrophages aid in the removal of pathogenic viruses from the body, macrophages are also manipulated by some viruses to serve as vessels for viral replication, dissemination, and long-term persistence. Here, we show that iPSC-derived macrophages are an excellent model that can be exploited in virology.


Asunto(s)
Virus del Dengue , VIH-1 , Células Madre Pluripotentes Inducidas , Macrófagos , Modelos Biológicos , Orthomyxoviridae , Virología , Animales , Humanos , Diferenciación Celular/genética , VIH-1/crecimiento & desarrollo , VIH-1/fisiología , Células Madre Pluripotentes Inducidas/citología , Macrófagos/citología , Macrófagos/metabolismo , Macrófagos/virología , Orthomyxoviridae/crecimiento & desarrollo , Orthomyxoviridae/fisiología , Pan troglodytes , Virus del Dengue/crecimiento & desarrollo , Virus del Dengue/fisiología , Fibroblastos/citología , Monocitos/citología , Replicación Viral , Citometría de Flujo , Perfilación de la Expresión Génica , Ensamble y Desensamble de Cromatina , Tropismo Viral , Virología/métodos , Biomarcadores/análisis , Biomarcadores/metabolismo
8.
BMC Bioinformatics ; 25(1): 19, 2024 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-38216877

RESUMEN

In experiments with significant perturbations to transcription, nascent RNA sequencing protocols are dependent on external spike-ins for reliable normalization. Unlike in RNA-seq, these spike-ins are not standardized and, in many cases, depend on a run-on reaction that is assumed to have constant efficiency across samples. To assess the validity of this assumption, we analyze a large number of published nascent RNA spike-ins to quantify their variability across existing normalization methods. Furthermore, we develop a new biologically-informed Bayesian model to estimate the error in spike-in based normalization estimates, which we term Virtual Spike-In (VSI). We apply this method both to published external spike-ins as well as using reads at the [Formula: see text] end of long genes, building on prior work from Mahat (Mol Cell 62(1):63-78, 2016. https://doi.org/10.1016/j.molcel.2016.02.025 ) and Vihervaara (Nat Commun 8(1):255, 2017. https://doi.org/10.1038/s41467-017-00151-0 ). We find that spike-ins in existing nascent RNA experiments are typically under sequenced, with high variability between samples. Furthermore, we show that these high variability estimates can have significant downstream effects on analysis, complicating biological interpretations of results.


Asunto(s)
ARN , ARN/genética , Teorema de Bayes , Análisis de Secuencia de ARN , RNA-Seq
9.
PLoS Biol ; 19(8): e3001364, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-34351910

RESUMEN

The naturally occurring Δ40p53 isoform heterotetramerizes with wild-type p53 (WTp53) to regulate development, aging, and stress responses. How Δ40p53 alters WTp53 function remains enigmatic because their co-expression causes tetramer heterogeneity. We circumvented this issue with a well-tested strategy that expressed Δ40p53:WTp53 as a single transcript, ensuring a 2:2 tetramer stoichiometry. Human MCF10A cell lines expressing Δ40p53:WTp53, WTp53, or WTp53:WTp53 (as controls) from the native TP53 locus were examined with transcriptomics (precision nuclear run-on sequencing [PRO-seq] and RNA sequencing [RNA-seq]), metabolomics, and other methods. Δ40p53:WTp53 was transcriptionally active, and, although phenotypically similar to WTp53 under normal conditions, it failed to induce growth arrest upon Nutlin-induced p53 activation. This occurred via Δ40p53:WTp53-dependent inhibition of enhancer RNA (eRNA) transcription and subsequent failure to induce mRNA biogenesis, despite similar genomic occupancy to WTp53. A different stimulus (5-fluorouracil [5FU]) also showed Δ40p53:WTp53-specific changes in mRNA induction; however, other transcription factors (TFs; e.g., E2F2) could then drive the response, yielding similar outcomes vs. WTp53. Our results establish that Δ40p53 tempers WTp53 function to enable compensatory responses by other stimulus-specific TFs. Such modulation of WTp53 activity may be an essential physiological function for Δ40p53. Moreover, Δ40p53:WTp53 functional distinctions uncovered herein suggest an eRNA requirement for mRNA biogenesis and that human p53 evolved as a tetramer to support eRNA transcription.


Asunto(s)
Proteína p53 Supresora de Tumor/metabolismo , Línea Celular , Fluorouracilo , Genes p53 , Humanos , Imidazoles , Piperazinas , Isoformas de Proteínas , Estructura Cuaternaria de Proteína , Factores de Transcripción/metabolismo , Transcripción Genética , Transcriptoma
10.
Proc Natl Acad Sci U S A ; 118(21)2021 05 25.
Artículo en Inglés | MEDLINE | ID: mdl-33972412

RESUMEN

We analyze data from the fall 2020 pandemic response efforts at the University of Colorado Boulder, where more than 72,500 saliva samples were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using qRT-PCR. All samples were collected from individuals who reported no symptoms associated with COVID-19 on the day of collection. From these, 1,405 positive cases were identified. The distribution of viral loads within these asymptomatic individuals was indistinguishable from what has been previously observed in symptomatic individuals. Regardless of symptomatic status, ∼50% of individuals who test positive for SARS-CoV-2 seem to be in noninfectious phases of the disease, based on having low viral loads in a range from which live virus has rarely been isolated. We find that, at any given time, just 2% of individuals carry 90% of the virions circulating within communities, serving as viral "supercarriers" and possibly also superspreaders.


Asunto(s)
COVID-19/virología , Portador Sano/virología , SARS-CoV-2 , Infecciones Asintomáticas/epidemiología , COVID-19/diagnóstico , COVID-19/epidemiología , COVID-19/transmisión , Portador Sano/diagnóstico , Portador Sano/epidemiología , Portador Sano/transmisión , Colorado/epidemiología , Hospitalización/estadística & datos numéricos , Humanos , Tamizaje Masivo/estadística & datos numéricos , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Saliva/virología , Universidades , Carga Viral , Virión
11.
BMC Biol ; 21(1): 228, 2023 11 10.
Artículo en Inglés | MEDLINE | ID: mdl-37946204

RESUMEN

BACKGROUND: The increase in DNA copy number in Down syndrome (DS; caused by trisomy 21) has led to the DNA dosage hypothesis, which posits that the level of gene expression is proportional to the gene's DNA copy number. Yet many reports have suggested that a proportion of chromosome 21 genes are dosage compensated back towards typical expression levels (1.0×). In contrast, other reports suggest that dosage compensation is not a common mechanism of gene regulation in trisomy 21, providing support to the DNA dosage hypothesis. RESULTS: In our work, we use both simulated and real data to dissect the elements of differential expression analysis that can lead to the appearance of dosage compensation, even when compensation is demonstrably absent. Using lymphoblastoid cell lines derived from a family with an individual with Down syndrome, we demonstrate that dosage compensation is nearly absent at both nascent transcription (GRO-seq) and steady-state RNA (RNA-seq) levels. Furthermore, we link the limited apparent dosage compensation to expected allelic variation in transcription levels. CONCLUSIONS: Transcription dosage compensation does not occur in Down syndrome. Simulated data containing no dosage compensation can appear to have dosage compensation when analyzed via standard methods. Moreover, some chromosome 21 genes that appear to be dosage compensated are consistent with allele specific expression.


Asunto(s)
Síndrome de Down , Humanos , Síndrome de Down/genética , Cromosoma X , Compensación de Dosificación (Genética) , Regulación de la Expresión Génica , ADN
12.
Physiol Genomics ; 54(10): 389-401, 2022 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-36062885

RESUMEN

Military Deployment to Southwest Asia and Afghanistan and exposure to toxic airborne particulates have been associated with an increased risk of developing respiratory disease, collectively termed deployment-related respiratory diseases (DRRDs). Our knowledge about how particulates mediate respiratory disease is limited, precluding the appropriate recognition or management. Central to this limitation is the lack of understanding of how exposures translate into dysregulated cell identity with dysregulated transcriptional programs. The small airway epithelium is involved in both the pathobiology of DRRD and fine particulate matter deposition. To characterize small airway epithelial cell epigenetic and transcriptional responses to Afghan desert particulate matter (APM) and investigate the functional interactions of transcription factors that mediate these responses, we applied two genomics assays, the assay for transposase accessible chromatin with sequencing (ATAC-seq) and Precision Run-on sequencing (PRO-seq). We identified activity changes in a series of transcriptional pathways as candidate regulators of susceptibility to subsequent insults, including signal-dependent pathways, such as loss of cytochrome P450 or P53/P63, and lineage-determining transcription factors, such as GRHL2 loss or TEAD3 activation. We further demonstrated that TEAD3 activation was unique to APM exposure despite similar inflammatory responses when compared with wood smoke particle exposure and that P53/P63 program loss was uniquely positioned at the intersection of signal-dependent and lineage-determining transcriptional programs. Our results establish the utility of an integrated genomics approach in characterizing responses to exposures and identifying genomic targets for the advanced investigation of the pathogenesis of DRRD.


Asunto(s)
Células Epiteliales Alveolares , Material Particulado , Factores de Transcripción , Afganistán , Células Epiteliales Alveolares/metabolismo , Cromatina/metabolismo , Epigénesis Genética , Genómica/métodos , Despliegue Militar , Material Particulado/toxicidad , Enfermedades Respiratorias/epidemiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transposasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo
13.
J Biol Chem ; 297(4): 101147, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34520756

RESUMEN

The heterogeneity of respirable particulates and compounds complicates our understanding of transcriptional responses to air pollution. Here, we address this by applying precision nuclear run-on sequencing and the assay for transposase-accessible chromatin sequencing to measure nascent transcription and chromatin accessibility in airway epithelial cells after wood smoke particle (WSP) exposure. We used transcription factor enrichment analysis to identify temporally distinct roles for ternary response factor-serum response factor complexes, the aryl hydrocarbon receptor (AHR), and NFκB in regulating transcriptional changes induced by WSP. Transcription of canonical targets of the AHR, such as CYP1A1 and AHRR, was robustly increased after just 30 min of WSP exposure, and we discovered novel AHR-regulated pathways and targets including the DNA methyltransferase, DNMT3L. Transcription of these genes and associated enhancers rapidly returned to near baseline by 120 min after exposure. The kinetics of AHR- and NFκB-regulated responses to WSP were distinguishable based on the timing of both transcriptional responses and chromatin remodeling, with induction of several cytokines implicated in maintaining NFκB-mediated responses through 120 min of exposure. In aggregate, our data establish a direct and primary role for AHR in mediating airway epithelial responses to WSP and identify crosstalk between AHR and NFκB signaling in controlling proinflammatory gene expression. This work also defines an integrated genomics-based strategy for deconvoluting multiplexed transcriptional responses to heterogeneous environmental exposures.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal , Humo/efectos adversos , Transcripción Genética , Madera , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Línea Celular Transformada , Ensamble y Desensamble de Cromatina , Citocromo P-450 CYP1A1/biosíntesis , Citocromo P-450 CYP1A1/genética , ADN (Citosina-5-)-Metiltransferasas/biosíntesis , ADN (Citosina-5-)-Metiltransferasas/genética , Humanos , Ratones , FN-kappa B/genética , FN-kappa B/metabolismo , Células 3T3 NIH , Receptores de Hidrocarburo de Aril/genética , Proteínas Represoras/genética
14.
BMC Genomics ; 23(1): 187, 2022 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-35255806

RESUMEN

BACKGROUND: A variety of protocols exist for producing whole genome run-on transcription datasets. However, little is known about how differences between these protocols affect the signal within the resulting libraries. RESULTS: Using run-on transcription datasets generated from the same biological system, we show that a variety of GRO- and PRO-seq preparation methods leave identifiable signatures within each library. Specifically we show that the library preparation method results in differences in quality control metrics, as well as differences in the signal distribution at the 5 ' end of transcribed regions. These shifts lead to disparities in eRNA identification, but do not impact analyses aimed at inferring the key regulators involved in changes to transcription. CONCLUSIONS: Run-on sequencing protocol variations result in technical signatures that can be used to identify both the enrichment and library preparation method of a particular data set. These technical signatures are batch effects that limit detailed comparisons of pausing ratios and eRNAs identified across protocols. However, these batch effects have only limited impact on our ability to infer which regulators underlie the observed transcriptional changes.


Asunto(s)
Biblioteca Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Bases de Datos Genéticas , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Control de Calidad , Transcripción Genética
15.
Genome Res ; 29(11): 1753-1765, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31519741

RESUMEN

The glucocorticoid receptor (NR3C1, also known as GR) binds to specific DNA sequences and directly induces transcription of anti-inflammatory genes that contribute to cytokine repression, frequently in cooperation with NF-kB. Whether inflammatory repression also occurs through local interactions between GR and inflammatory gene regulatory elements has been controversial. Here, using global run-on sequencing (GRO-seq) in human airway epithelial cells, we show that glucocorticoid signaling represses transcription within 10 min. Many repressed regulatory regions reside within "hyper-ChIPable" genomic regions that are subject to dynamic, yet nonspecific, interactions with some antibodies. When this artifact was accounted for, we determined that transcriptional repression does not require local GR occupancy. Instead, widespread transcriptional induction through canonical GR binding sites is associated with reciprocal repression of distal TNF-regulated enhancers through a chromatin-dependent process, as evidenced by chromatin accessibility and motif displacement analysis. Simultaneously, transcriptional induction of key anti-inflammatory effectors is decoupled from primary repression through cooperation between GR and NF-kB at a subset of regulatory regions. Thus, glucocorticoids exert bimodal restraints on inflammation characterized by rapid primary transcriptional repression without local GR occupancy and secondary anti-inflammatory effects resulting from transcriptional cooperation between GR and NF-kB.


Asunto(s)
Dexametasona/farmacología , Inflamación/metabolismo , ARN Mensajero/genética , Receptores de Glucocorticoides/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Cromatina/metabolismo , Dexametasona/metabolismo , Elementos de Facilitación Genéticos , Células HEK293 , Humanos , FN-kappa B/metabolismo , Transducción de Señal
16.
Infect Immun ; 89(11): e0027321, 2021 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-34370511

RESUMEN

Nutritional immunity involves cellular and physiological responses to invading pathogens, such as limiting iron, increasing exposure to bactericidal copper, and altering zinc to restrict the growth of pathogens. Here, we examine infection of bone marrow-derived macrophages from 129S6/SvEvTac mice by Salmonella enterica serovar Typhimurium. The 129S6/SvEvTac mice possess a functional Slc11a1 (Nramp-1), a phagosomal transporter of divalent cations that plays an important role in modulating metal availability to the pathogen. We carried out global RNA sequencing upon treatment with live or heat-killed Salmonella at 2 h and 18 h postinfection and observed widespread changes in metal transport, metal-dependent genes, and metal homeostasis genes, suggesting significant remodeling of iron, copper, and zinc availability by host cells. Changes in host cell gene expression suggest infection increases cytosolic zinc while simultaneously limiting zinc within the phagosome. Using a genetically encoded sensor, we demonstrate that cytosolic labile zinc increases 45-fold at 12 h postinfection. Further, manipulation of zinc in the medium alters bacterial clearance and replication, with zinc depletion inhibiting both processes. Comparing the transcriptomic changes to published data on infection of C57BL/6 macrophages revealed notable differences in metal regulation and the global immune response. Our results reveal that 129S6 macrophages represent a distinct model system compared to C57BL/6 macrophages. Further, our results indicate that manipulation of zinc at the host-pathogen interface is more nuanced than that of iron or copper. The 129S6 macrophages leverage intricate means of manipulating zinc availability and distribution to limit the pathogen's access to zinc, while simultaneously ensuring sufficient zinc to support the immune response.


Asunto(s)
Macrófagos/inmunología , Metales/metabolismo , Salmonelosis Animal/inmunología , Animales , Proteínas del Sistema Complemento/inmunología , Femenino , Expresión Génica , Interacciones Huésped-Patógeno , Ratones , Ratones Endogámicos C57BL , Salmonella typhimurium , Zinc/metabolismo
17.
Genome Res ; 2018 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-29449408

RESUMEN

Transcription factors (TFs) exert their regulatory influence through the binding of enhancers, resulting in coordination of gene expression programs. Active enhancers are often characterized by the presence of short, unstable transcripts termed enhancer RNAs (eRNAs). While their function remains unclear, we demonstrate that eRNAs are a powerful readout of TF activity. We infer sites of eRNA origination across hundreds of publicly available nascent transcription data sets and show that eRNAs initiate from sites of TF binding. By quantifying the colocalization of TF binding motif instances and eRNA origins, we derive a simple statistic capable of inferring TF activity. In doing so, we uncover dozens of previously unexplored links between diverse stimuli and the TFs they affect.

18.
Nature ; 519(7543): 349-52, 2015 Mar 19.
Artículo en Inglés | MEDLINE | ID: mdl-25731168

RESUMEN

Polyploidy is observed across the tree of life, yet its influence on evolution remains incompletely understood. Polyploidy, usually whole-genome duplication, is proposed to alter the rate of evolutionary adaptation. This could occur through complex effects on the frequency or fitness of beneficial mutations. For example, in diverse cell types and organisms, immediately after a whole-genome duplication, newly formed polyploids missegregate chromosomes and undergo genetic instability. The instability following whole-genome duplications is thought to provide adaptive mutations in microorganisms and can promote tumorigenesis in mammalian cells. Polyploidy may also affect adaptation independently of beneficial mutations through ploidy-specific changes in cell physiology. Here we perform in vitro evolution experiments to test directly whether polyploidy can accelerate evolutionary adaptation. Compared with haploids and diploids, tetraploids undergo significantly faster adaptation. Mathematical modelling suggests that rapid adaptation of tetraploids is driven by higher rates of beneficial mutations with stronger fitness effects, which is supported by whole-genome sequencing and phenotypic analyses of evolved clones. Chromosome aneuploidy, concerted chromosome loss, and point mutations all provide large fitness gains. We identify several mutations whose beneficial effects are manifest specifically in the tetraploid strains. Together, these results provide direct quantitative evidence that in some environments polyploidy can accelerate evolutionary adaptation.


Asunto(s)
Adaptación Fisiológica/genética , Evolución Biológica , Poliploidía , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Aneuploidia , Cromosomas Fúngicos/genética , Células Clonales/citología , Células Clonales/metabolismo , Diploidia , Aptitud Genética/genética , Haploidia , Tasa de Mutación , Mutación Puntual/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Factores de Tiempo
19.
Nucleic Acids Res ; 46(4): 1756-1776, 2018 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-29240919

RESUMEN

Histone deacetylase inhibitors (HDACIs) are known to alter gene expression by both up- and down-regulation of protein-coding genes in normal and cancer cells. However, the exact regulatory mechanisms of action remain uncharacterized. Here we investigated genome wide dose-dependent epigenetic and transcriptome changes in response to HDACI largazole in a transformed and a non-transformed cell line. Exposure to low nanomolar largazole concentrations (

Asunto(s)
Depsipéptidos/farmacología , Elementos de Facilitación Genéticos , Código de Histonas/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Tiazoles/farmacología , Acetilación , Línea Celular , Línea Celular Transformada , Citostáticos/farmacología , Relación Dosis-Respuesta a Droga , Elementos de Facilitación Genéticos/efectos de los fármacos , Genoma , Histona Desacetilasas/fisiología , Histonas/metabolismo , Oncogenes , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , ARN Mensajero/metabolismo
20.
PLoS Genet ; 12(1): e1005746, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26751950

RESUMEN

Most genetic variants associated with disease occur within regulatory regions of the genome, underscoring the importance of defining the mechanisms underlying differences in regulation of gene expression between individuals. We discovered a pair of co-regulated, divergently oriented transcripts, AQY2 and ncFRE6, that are expressed in one strain of Saccharomyces cerevisiae, ∑1278b, but not in another, S288c. By combining classical genetics techniques with high-throughput sequencing, we identified a trans-acting single nucleotide polymorphism within the transcription factor RIM101 that causes the background-dependent expression of both transcripts. Subsequent RNA-seq experiments revealed that RIM101 regulates many more targets in S288c than in ∑1278b and that deletion of RIM101 in both backgrounds abrogates the majority of differential expression between the strains. Strikingly, only three transcripts undergo a significant change in expression after swapping RIM101 alleles between backgrounds, implying that the differences in the RIM101 allele lead to a remarkably focused transcriptional response. However, hundreds of RIM101-dependent targets undergo a subtle but consistent shift in expression in the S288c RIM101-swapped strain, but not its ∑1278b counterpart. We conclude that ∑1278b may harbor a variant(s) that buffers against widespread transcriptional dysregulation upon introduction of a non-native RIM101 allele, emphasizing the importance of accounting for genetic background when assessing the impact of a regulatory variant.


Asunto(s)
Proteínas Represoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Transcripción Genética , Alelos , Acuaporinas/biosíntesis , Acuaporinas/genética , Proteínas de Unión al ADN/genética , Regulación Fúngica de la Expresión Génica , Antecedentes Genéticos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Proteínas Represoras/biosíntesis , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/biosíntesis , Factores de Transcripción/biosíntesis
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