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Promoter-proximal RNA Pol II pausing is a critical step in transcriptional control. Pol II pausing has been predominantly studied in tissue culture systems. While Pol II pausing has been shown to be required for mammalian development, the phenotypic and mechanistic details of this requirement are unknown. Here, we found that loss of Pol II pausing stalls pluripotent state transitions within the epiblast of the early mouse embryo. Using Nelfb -/- mice and a NELFB degron mouse pluripotent stem cell model, we show that embryonic stem cells (ESCs) representing the naïve state of pluripotency successfully initiate a transition program but fail to balance levels of induced and repressed genes and enhancers in the absence of NELF. We found an increase in chromatin-associated NELF during transition from the naïve to later pluripotent states. Overall, our work defines the acute and long-term molecular consequences of NELF loss and reveals a role for Pol II pausing in the pluripotency continuum as a modulator of cell state transitions.
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BACKGROUND: The concentrations of distinct types of RNA in cells result from a dynamic equilibrium between RNA synthesis and decay. Despite the critical importance of RNA decay rates, current approaches for measuring them are generally labor-intensive, limited in sensitivity, and/or disruptive to normal cellular processes. Here, we introduce a simple method for estimating relative RNA half-lives that is based on two standard and widely available high-throughput assays: Precision Run-On sequencing (PRO-seq) and RNA sequencing (RNA-seq). RESULTS: Our method treats PRO-seq as a measure of transcription rate and RNA-seq as a measure of RNA concentration, and estimates the rate of RNA decay required for a steady-state equilibrium. We show that this approach can be used to assay relative RNA half-lives genome-wide, with good accuracy and sensitivity for both coding and noncoding transcription units. Using a structural equation model (SEM), we test several features of transcription units, nearby DNA sequences, and nearby epigenomic marks for associations with RNA stability after controlling for their effects on transcription. We find that RNA splicing-related features are positively correlated with RNA stability, whereas features related to miRNA binding and DNA methylation are negatively correlated with RNA stability. Furthermore, we find that a measure based on U1 binding and polyadenylation sites distinguishes between unstable noncoding and stable coding transcripts but is not predictive of relative stability within the mRNA or lincRNA classes. We also identify several histone modifications that are associated with RNA stability. CONCLUSION: We introduce an approach for estimating the relative half-lives of individual RNAs. Together, our estimation method and systematic analysis shed light on the pervasive impacts of RNA stability on cellular RNA concentrations.
Assuntos
Instabilidade Genômica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Estabilidade de RNA , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Humanos , RNA-Seq/métodosRESUMO
How enhancers control target gene expression over long genomic distances remains an important unsolved problem. Here we investigated enhancer-promoter communication by integrating data from nucleosome-resolution genomic contact maps, nascent transcription and perturbations affecting either RNA polymerase II (Pol II) dynamics or the activity of thousands of candidate enhancers. Integration of new Micro-C experiments with published CRISPRi data demonstrated that enhancers spend more time in close proximity to their target promoters in functional enhancer-promoter pairs compared to nonfunctional pairs, which can be attributed in part to factors unrelated to genomic position. Manipulation of the transcription cycle demonstrated a key role for Pol II in enhancer-promoter interactions. Notably, promoter-proximal paused Pol II itself partially stabilized interactions. We propose an updated model in which elements of transcriptional dynamics shape the duration or frequency of interactions to facilitate enhancer-promoter communication.
Assuntos
Elementos Facilitadores Genéticos , RNA Polimerase II , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Elementos Facilitadores Genéticos/genética , Regiões Promotoras Genéticas/genética , Transcrição GênicaRESUMO
Promoter-proximal pausing of RNA polymerase II (Pol II) is a key regulatory step during transcription. Despite the central role of pausing in gene regulation, we do not understand the evolutionary processes that led to the emergence of Pol II pausing or its transition to a rate-limiting step actively controlled by transcription factors. Here we analyzed transcription in species across the tree of life. We found that unicellular eukaryotes display a slow acceleration of Pol II near transcription start sites. This proto-paused-like state transitioned to a longer, focused pause in derived metazoans which coincided with the evolution of new subunits in the NELF and 7SK complexes. Depletion of NELF reverts the mammalian focal pause to a proto-pause-like state and compromises transcriptional activation for a set of heat shock genes. Collectively, this work details the evolutionary history of Pol II pausing and sheds light on how new transcriptional regulatory mechanisms evolve.
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Innate immune responses rely on inducible gene expression programmes which, in contrast to steady-state transcription, are highly dependent on cohesin. Here we address transcriptional parameters underlying this cohesin-dependence by single-molecule RNA-FISH and single-cell RNA-sequencing. We show that inducible innate immune genes are regulated predominantly by an increase in the probability of active transcription, and that probabilities of enhancer and promoter transcription are coordinated. Cohesin has no major impact on the fraction of transcribed inducible enhancers, or the number of mature mRNAs produced per transcribing cell. Cohesin is, however, required for coupling the probabilities of enhancer and promoter transcription. Enhancer-promoter coupling may not be explained by spatial proximity alone, and at the model locus Il12b can be disrupted by selective inhibition of the cohesinopathy-associated BET bromodomain BD2. Our data identify discrete steps in enhancer-mediated inducible gene expression that differ in cohesin-dependence, and suggest that cohesin and BD2 may act on shared pathways.
Assuntos
Proteínas Cromossômicas não Histona , Elementos Facilitadores Genéticos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Elementos Facilitadores Genéticos/genética , Probabilidade , RNA , CoesinasRESUMO
The role of histone modifications in transcription remains incompletely understood. Here, we examine the relationship between histone modifications and transcription using experimental perturbations combined with sensitive machine-learning tools. Transcription predicted the variation in active histone marks and complex chromatin states, like bivalent promoters, down to single-nucleosome resolution and at an accuracy that rivaled the correspondence between independent ChIP-seq experiments. Blocking transcription rapidly removed two punctate marks, H3K4me3 and H3K27ac, from chromatin indicating that transcription is required for active histone modifications. Transcription was also required for maintenance of H3K27me3, consistent with a role for RNA in recruiting PRC2. A subset of DNase-I-hypersensitive sites were refractory to prediction, precluding models where transcription initiates pervasively at any open chromatin. Our results, in combination with past literature, support a model in which active histone modifications serve a supportive, rather than an essential regulatory, role in transcription.
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Histonas , Processamento de Proteína Pós-Traducional , Cromatina/genética , Código das Histonas/genética , Histonas/genética , Histonas/metabolismo , Nucleossomos/genética , Processamento de Proteína Pós-Traducional/genéticaRESUMO
SETD2 is the sole histone methyltransferase responsible for H3K36me3, with roles in splicing, transcription initiation, and DNA damage response. Homozygous disruption of SETD2 yields a tumor suppressor effect in various cancers. However, SETD2 mutation is typically heterozygous in diffuse large B-cell lymphomas. Here we show that heterozygous Setd2 deficiency results in germinal center (GC) hyperplasia and increased competitive fitness, with reduced DNA damage checkpoint activity and apoptosis, resulting in accelerated lymphomagenesis. Impaired DNA damage sensing in Setd2-haploinsufficient germinal center B (GCB) and lymphoma cells associated with increased AICDA-induced somatic hypermutation, complex structural variants, and increased translocations including those activating MYC. DNA damage was selectively increased on the nontemplate strand, and H3K36me3 loss was associated with greater RNAPII processivity and mutational burden, suggesting that SETD2-mediated H3K36me3 is required for proper sensing of cytosine deamination. Hence, Setd2 haploinsufficiency delineates a novel GCB context-specific oncogenic pathway involving defective epigenetic surveillance of AICDA-mediated effects on transcribed genes. SIGNIFICANCE: Our findings define a B cell-specific oncogenic effect of SETD2 heterozygous mutation, which unleashes AICDA mutagenesis of nontemplate strand DNA in the GC reaction, resulting in lymphomas with heavy mutational burden. GC-derived lymphomas did not tolerate SETD2 homozygous deletion, pointing to a novel context-specific therapeutic vulnerability. This article is highlighted in the In This Issue feature, p. 1599.